Regulation of Cation Balance Saccharomyces cerevisiae · Potassium ions help balance negative charge inside cells and activate critical metabolic processes such as protein trans-lation

YEASTBOOK

CELL SIGNALING & DEVELOPMENT

Regulation of Cation Balancein Saccharomyces cerevisiaeMartha S. Cyert*,1 and Caroline C. Philpott†

*Department of Biology, Stanford University, Stanford, California 94305-5020, and yGenetics and Metabolism Section, National Institute of Diabetesand Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT All living organisms require nutrient minerals for growth and have developed mechanisms to acquire, utilize, and storenutrient minerals effectively. In the aqueous cellular environment, these elements exist as charged ions that, together with protons andhydroxide ions, facilitate biochemical reactions and establish the electrochemical gradients across membranes that drive cellularprocesses such as transport and ATP synthesis. Metal ions serve as essential enzyme cofactors and perform both structural andsignaling roles within cells. However, because these ions can also be toxic, cells have developed sophisticated homeostatic mechanismsto regulate their levels and avoid toxicity. Studies in Saccharomyces cerevisiae have characterized many of the gene products andprocesses responsible for acquiring, utilizing, storing, and regulating levels of these ions. Findings in this model organism have oftenallowed the corresponding machinery in humans to be identified and have provided insights into diseases that result from defects inion homeostasis. This review summarizes our current understanding of how cation balance is achieved and modulated in baker’s yeast.Control of intracellular pH is discussed, as well as uptake, storage, and efflux mechanisms for the alkali metal cations, Na+ and K+, thedivalent cations, Ca2+ and Mg2+, and the trace metal ions, Fe2+, Zn2+, Cu2+, and Mn2+. Signal transduction pathways that areregulated by pH and Ca2+ are reviewed, as well as the mechanisms that allow cells to maintain appropriate intracellular cationconcentrations when challenged by extreme conditions, i.e., either limited availability or toxic levels in the environment.

TABLE OF CONTENTS

Abstract 677

Introduction 678

Maintenance of Intracellular pH 679Pma1, the essential plasma membrane proton pump 679

Regulation of Pma1 679

Vacuolar ATPase acidifies the vacuole and secretory organelles 680

Regulation of V-ATPase activity 680

Coordination of Pma1 and V-ATPase 681

Effects of pH on transport across membranes 681

Do changes in intracellular pH serve a signaling function? 681

The Alkali Metals 681K+ entry into cells 682

Continued

Copyright © 2013 by the Genetics Society of Americadoi: 10.1534/genetics.112.147207Manuscript received October 26, 2012; accepted for publication December 4, 20121Corresponding author: Department of Biology, Stanford University, Stanford, CA 94305. E-mail: [email protected]

Genetics, Vol. 193, 677–713 March 2013 677

mailto:[email protected]

CONTENTS, continued

K+ efflux 683

Maintenance of intracellular K+ levels 683

Na+ entry into cells 683

Na+ efflux 683

Alkali metal cation transport in intracellular compartments 684Mitochondria: 684Vacuole, Golgi, and endosomes: 684

Divalent Cations: Ca2+ and Mg2+ 685Ca2+ ion homeostasis and regulation 685

Ca2+ entry into yeast cells 685

Ca2+ sequestration in intracellular compartments 686ER/Golgi/secretory pathway: 686Vacuole: 686

Ca2+ release from intracellular compartments 686Vacuolar Ca2+ release mediated by Yvc1: 686

Ca2+-dependent signaling pathways 687Responses to stress: 687Response to mating factor: 688Response to nutrients: 688Regulation of cell cycle and morphogenesis: 688

Mg2+ ion homeostasis and regulation 689

Biological Roles of Transition Metal Ions 689

Iron 690Transcriptional control through Aft1 and Aft2 690

The Aft1/Aft2 regulon 690

Reductive uptake of iron at the cell surface 691

Nonreductive uptake of siderophore–iron chelates 692

Mobilization of vacuolar iron stores 693

Other transporters 693

Metabolic adaptation to iron deficiency 693

Cellular iron utilization 694

Zinc 695Transcriptional regulation through Zap1 695

Transporters regulated by Zap1 696

Adaptation to zinc deficiency 697

Copper 698Transcriptional regulation through Mac1 and Ace1 698

Copper transport systems 699

Copper detoxification and utilization 699

Mitochondrial copper utilization 700

Manganese 700Uptake of manganese through Smf1 and Smf2 700

Phosphate-dependent manganese transport 701

Transport of manganese into the secretory pathway 701

Unanswered Questions 702

678 M. S. Cyert and C. C. Philpott

IN addition to the major components of organic molecules,i.e., carbon, nitrogen, hydrogen, and oxygen, living organ-

isms require multiple chemical elements, termed nutrientminerals, for growth. In the aqueous cellular environment,these elements exist as charged ions that, together withprotons and hydroxide ions, facilitate biochemical reactions.Charged ions, which cannot diffuse across lipid bilayers, alsoprovide the raw material to establish electrochemical gra-dients that drive cellular processes such as ATP synthesis.Potassium ions help balance negative charge inside cells andactivate critical metabolic processes such as protein trans-lation. Trace elements, such as zinc, copper, iron, and man-ganese, are critical determinants of protein structure andserve as essential enzyme cofactors. Calcium performs struc-tural, enzymatic, and signaling roles within cells. All of theseessential elements can also be toxic. Thus, cells must be ableto acquire, utilize, and store nutrient minerals effectively,but have also developed sophisticated homeostatic mecha-nisms to regulate their levels and avoid toxicity. Geneticstudies in yeast have identified key components responsiblefor acquiring, utilizing, storing, and regulating levels ofthese ions. Furthermore, because many of these proteinsare highly conserved, yeast serves as an excellent model toidentify the corresponding machinery in humans and under-stand diseases that result from defects in ion homeostasis. Agenome-wide study measured levels of 13 elements in.4000 yeast deletion strains grown in rich medium to es-tablish the yeast “ionome.” Relatively few mutations (212)were found to significantly perturb the ionome, revealingthat robust mechanisms exist to compensate for loss of a sin-gle component of ion homeostasis (Eide et al. 2005). How-ever, the vast majority of the 212 mutations identifiedaltered the level of more than one element, and subsets ofelements covaried, illustrating the cooperative nature of theregulatory networks that control intracellular ion levels.These studies also highlighted the critical role that intracel-lular organelles, particularly the vacuole and the mitochon-dria, play in ion regulation.

This chapter reviews our current understanding of howcation balance is achieved and regulated in baker’s yeast.Starting with monovalent cations and proceeding to divalentmetal ions, the role of each cation is briefly reviewed, withparticular emphasis on current knowledge of its uptake,storage, and efflux mechanisms. Where appropriate, rolesfor cation in signal transduction pathways are also discussed.

Maintenance of Intracellular pH

The concentration of protons (H+) in the cell, expressed asintracellular pH (pHi), dramatically influences every aspectof cellular biochemistry and must be carefully regulatedboth in the cytosol and in the lumen of intracellular organ-elles. In rapidly growing yeast cells, cytosolic pH is stable at7.2 and changes little over a broad range of extracellular pH(Martinez-Munoz and Kane 2008; Orij et al. 2009). How-ever, mounting evidence indicates that intracellular pH is

regulated and can serve a signaling function, in particular,to report nutrient availability (reviewed in Orij et al. 2011).Different methods have been used to assess pHi in Saccharo-myces cerevisiae including 31P NMR (Navon et al. 1979), pHsensitive dyes (Haworth et al. 1991), and more recently, ex-pression of pHlourin, a ratiometric pH-sensitive fluorescentprotein (Orij et al. 2009) that can be targeted to the cytosolor intracellular compartments to measure pH in living cells(Braun et al. 2010; Maresova et al. 2010). The concentrationof protons in the cytosol is largely determined by the activityof two proton pumps: Pma1, which resides in the plasmamembrane (PM), and a large protein complex, termed theV-ATPase, which acidifies the endomembrane system includ-ing the vacuole, Golgi, and endosomal compartments.

Pma1, the essential plasma membrane proton pump

Pma1, a P2-type ATPase, is made up of a single 100-kDasubunit that pumps one proton across the plasma membraneper ATP molecule hydrolyzed. This H+-ATPase is one of themost abundant cellular proteins, consuming at least 20% ofcellular ATP. Pma1 is essential and rate limiting for growth;mutations that compromise its activity decrease both cyto-solic pH and growth (McCusker et al. 1987; Portillo andSerrano 1989). Mutants with impaired Pma1 function areunable to grow in low pH media or in the presence of weakacids, reflecting their reduced ability to pump protons acrossthe plasma membrane. They are also resistant to a variety ofcationic drugs and ions, including the aminoglycoside,hygromycin B, because decreasing the proton-motive forceacross the plasma membrane leads to reduced cellular up-take of these compounds (Perlin et al. 1988). PMA2 encodesa closely related gene product that is normally present ata very low level, but can substitute for Pma1 whenexpressed from a strong promoter (Supply et al. 1993).

Regulation of Pma1

Pma1 is regulated by at least two distinct mechanisms: First,to maintain neutral pH in the cytosol, Pma1 is activatedwhen intracellular pH drops. This has been most clearlyshown in cells exposed to weak organic acids, which crossthe PM in their protonated state and become deprotonatedin the cell to lower pHi. Under these conditions, the Km ofPma1 is decreased through an unknown mechanism (Erasoand Gansedo 1987; Carmelo et al. 1997). A drop in internalpH also affects Pma1 activity indirectly by activating K+

uptake through plasma membrane transporters, Trk1/2; thisoffsets the electrogenic potential created by Pma1-mediatedproton pumping and facilitates its activation (Yenush et al.2005). Pma1 is also regulated post-translationally by phos-phoryation, especially in response to nutrients. When glu-cose is added back to starved cells, pHi decreases transiently(30 sec), followed by activation of Pma1, which promotesrecovery of pHi to pH 7.2 (Figure 1). Pma1 activation ismediated in part by phosphorylation of the autoinhibitoryC-terminal tail of Pma1, which increases its Vmax, affinity forATP, and pH optimum. Thr-912 of Pma1 is constitutively

Regulation of Ion Balance 679

http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000002976

















phosphorylated; a modification that is necessary but not suf-ficient for Pma1 activation (Portillo et al. 1991). Glucoseaddition results in further phosphorylation at Ser-911 toproduce a tandemly phosphorylated, activated ATPase (Serrano1983; Lecchi et al. 2007). Ptk2 and Hrk1, members of theNpr1 family of protein kinases, which regulates multiplemembrane transporters, are thought to phosphorylate Pma1

(Goossens et al. 2000; Eraso et al. 2006), and it may bedephosphorylated by a PP1-type phosphatase, containing theGlc7 catalytic subunit (Williams-Hart et al. 2002). Other stud-ies suggest that Ca2+-dependent signaling also contributes toactivation of Pma1, by unknown mechanisms, in response toglucose addition (Tropia et al. 2006; Bouillet et al. 2012).

Vacuolar ATPase acidifies the vacuole andsecretory organelles

The other major protein that impacts cytosolic pH is thevacuolar proton-translocating ATPase, or V-ATPase, whichpumps protons into the vacuole. The V-ATPase helps maintaincytosolic pH by removing protons from the cytosol; treatingcells with concanamycin A, a V-ATPase inhibitor, causes a rapiddrop in intracellular pH (Martinez-Munoz and Kane 2008).The V-ATPase is a large highly conserved protein complex re-lated to the mitochondrial F1/F0 ATPase. It is made up of 14different subunits (some in multiple copies per enzyme) thatare organized into two discrete subcomplexes: an integralmembrane complex, V0, which contains the proton pore, andan associated V1 complex, which is responsible for ATP hydro-lysis and is made up of peripheral membrane proteins (Kane2006). In addition to these structural components, several ad-ditional proteins are required for assembly of the V0 subcom-plex in the endoplasmic reticulum (ER) and its subsequenttransport/exit from the ER, but are not present in the matureenzyme (Graham et al. 2000; Malkus et al. 2004; Davis-Kaplanet al. 2006; Ryan et al. 2008). There are two different forms ofthe V-ATPase in cells; one, containing the Vph1 subunit of theV0 complex, resides on the vacuolar membrane and is respon-sible for acidifying that compartment (Tarsio et al. 2011). Theother contains Stv1 (similar to Vph1) instead of Vph1, and istransported to endosomes, the late Golgi, and secretoryvesicles whose lumens are also acidic (Braun et al. 2010).

The major function of the V-ATPase is to maintain acidicpH in the vacuole, which is approximately 5.6 in growingcells (Martinez-Munoz and Kane 2008). This acidic pH isessential for enzymatic processes that take place in the vac-uole, such as proteolysis, and creates a proton gradientacross the vacuolar membrane that enables proton exchang-ers (i.e., Ca2+/H+, K+/H+, and Na+/H+) to transport theirsubstrates into the vacuole. The V-ATPase is not essential forgrowth under standard laboratory conditions; however,vma2 mutants, which lack V-ATPase enzyme function, havegrowth defects on both low (,3) and high (.7) pH mediaand are sensitive to a variety of cations, including Ca2+, dueto decreased sequestration of ions in the vacuole. In cellslacking functional V-ATPase, the only mechanism for vacu-ole acidification is the uptake of protons from the media viaendocytosis (Munn and Riezman 1994).

Regulation of V-ATPase activity

One mode of V-ATPase regulation is through the reversibledissociation/association of its two subcomplexes. This phe-nomenon was first demonstrated in response to glucosedeprivation and readdition; during glucose starvation, when

Figure 1 During glucose starvation, changes in intracellular pH servea signaling function. (A) In the presence of glucose, Pma1 and the V-ATPase are active; cytosolic pH is 7.0–7.2 and vacuolar pH is 5.6. PKA isactive, and V-ATPase association is proposed to regulate its activity. Ino-sitol synthesis is active due to sequestration of the Opi1 repressor on thesurface of the ER. (B) In glucose-starved cells, cytosolic pH drops due todecreased activity of Pma1 and dissociation of the V-ATPase subcom-plexes. Opi1 is released from the ER due to protonation of phosphatadicacid (PA); Op1 entry into the nucleus represses expression of INO1 byIno2/4 transcriptonal activators. Bright colors in A indicate active proteins;dull colors in B indicate reduced activity. See text for details.













ATP is limited, the intact V1 subcomplex detaches from theintegral membrane V0 complex (Kane 2011), causing inac-tivation of both portions of the V-ATPase and loss of ATPhydrolysis (Figure 1). This regulation is only observed forV-ATPase enzymes residing on the vacuolar membrane(Kawasaki-Nishi et al. 2001).

Association of the V1 and V0 in response to glucose read-dition promotes vacuolar acidification and requires theRAVE complex (Smardon et al. 2002), PKA/Ras activity,and other factors (Smardon et al. 2002; Kane 2011). Thisrapid mode of V-ATPase regulation appears to be highlyconserved, as V-ATPase integrity is regulated in many organ-isms including mammals (Kane 2011) and responds to ad-ditional signals including pH; decreasing intracellular pH isnecessary and sufficient to trigger V-ATPase disassembly(Dechant et al. 2010), and increasing extracellular pH sup-presses dissociation and stimulates V-ATPase activity (Diakovand Kane 2010). Furthermore, in cells grown in poor nutri-ent conditions, the V-ATPase pool is partially disassociated,suggesting that this regulatory mechanism fine tunes theamount of V-ATPase activity to fit different growth condi-tions (Kane 2011).

Coordination of Pma1 and V-ATPase

In normal cells, cytosolic pH is maintained by the combinedefforts of Pma1 and the V-ATPase, and the activities of theseproton pumps are coordinated at multiple levels. For exam-ple, in vma2 mutants Pma1 is partially mislocalized to thevacuole and other compartments, which may help compen-sate for loss of the V-ATPase (Martinez-Munoz and Kane2008). Other conditions that alter vacuolar pH, such as in-cubation with concamycin A or deletion of VPH1, cause a re-duction in Pma1 activity without perturbing its localization;the mechanism that mediates this Pma1 modulation in re-sponse to changes in vacuolar pH is unknown (Martinez-Munoz and Kane 2008; Tarsio et al. 2011).

Effects of pH on transport across membranes

In addition to their effects on intracellular pH, the protongradients created by Pma1 and the V-ATPase are majordeterminants of membrane potential (Dc), which is thesum of all ionic gradients over a membrane. This electro-chemical gradient is harnessed by protein transporters todrive the uptake and efflux of ions and nutrients acrossthe membrane. The uptake of nutrients depends on mainte-nance of the plasma membrane proton gradient, as majornutrient permeases for glucose and other sugars as well asamino acids are H+ symporters. Furthermore, these proton-coupled transporters, together with the K+ transporters,Trk1 and Trk2, and the Na+/H+ antiporter, Nha1, influencecytosolic pH by consuming the proton gradient (Orij et al.2011). Similarly, the electrochemical gradient across thevacuolar membrane powers transport into and out of thevacuole. Ca2+ accumulates in the vacuole via the Ca2+/H+

antiporter, Vcx1. Similarly, transport of heavy metals(Ycf1 and Zrc1), amino acids (Avt3, Avt4, and Avt6),

and polyphosphate accumulation depend on the proton gra-dient across the vacuolar membrane (reviewed in Li andKane 2009).

Do changes in intracellular pH serve a signaling function?

When cells are actively growing, homeostatic mechanismsmaintain a relatively constant intracellular pH of 7.2(Martinez-Munoz and Kane 2008; Orij et al. 2009). In con-trast, glucose starvation causes a drop in cytosolic pH due todecreased Pma1 activity and V-ATPase disassembly (Figure1). Readdition of glucose to starved cells causes a furthertransient (30 sec) intracellular acidification, followed bya rise in intracellular pH mediated by activation of Pma1and association of the V-ATPase subcomplexes. Some obser-vations suggest that these rapid changes in pHi may be a sig-nal that couples nutrient status to downstream events. Inparticular, adding glucose back to starved cells results inrapid activation of PKA, which phosphorylates multiple tar-gets and signals high glucose availability. PKA is activated bya spike of cAMP, produced when the small GTPase, Ras,activates adenylate cyclase (Cyr1). Recent studies suggestthat the V-ATPase is required for this glucose-mediated stim-ulation of PKA and that cytosolic pH serves as a secondmessenger in this regulatory pathway (Dechant et al.2010) (Figure 1). However, glucose acts through a complexnetwork of signaling events, some of which, i.e., activationof Cyr 1 through negative regulation of Ras GAPs (Ira1 andIra2), are independent of changes in intracellular pH (Theveleinand de Winde 1999).

Changes in intracellular pH are also thought to regulatephospholipid metabolism. During normal growth, whencytosolic pH is close to neutral, the Opi1 transcriptional re-pressor is tethered to the surface of the ER by its interactionswith Scs2 and phosphatidic acid (PA), and as a result, ino-sitol-regulated genes are active (Loewen et al. 2004) (Figure1). However, when intracellular pH drops, as in glucose-starved cells, the phosphate head group of PA becomes pro-tonated, decreasing its affinity for Opi1 and releasing therepressor from the ER. This allows Opi1 to enter the nu-cleus, where it inhibits expression of genes includingINO1, which encodes the rate-limiting enzyme in inositolbiosynthesis (Young et al. 2010). Thus, changes in intracel-lular pH couple nutrient availability to the production of keyenzymes required for membrane biogenesis.

The Alkali Metals

Sodium and potassium, together with lithium, rubidium,cesium, and francium, make up the family of alkali metals,which share similar properties and atomic structures andreadily form monovalent cations. In cells, K+, plays manyimportant physiological roles; it is required for negativecharge compensation and activation of key metabolic pro-cesses, such as pyruvate synthesis and protein translation(Page and Di Cera 2006). In contrast, Na+ is toxic at highlevels, in part because it readily substitutes for K+ (Page and



























Di Cera 2006). Thus, despite the much greater abundance ofNa+ in the environment, yeast must maintain a high intra-cellular ratio of K+/Na+ (Figure 4A), and achieve this byselectively accumulating K+, while actively extruding Na+.K+ is critical for balancing charge across the plasma mem-brane, and thus contributes to maintaining both intracellularpH and membrane potential. K+ is continually taken up andextruded by cells, and both processes are important; mem-brane potential increases when potassium influx is crippledand decreases in cells defective for K+ efflux (Madrid et al.1998; Kinclova-Zimmermannova et al. 2006); furthermore,K+ efflux systems are regulated in response to changes inthe membrane potential (Zahradka and Sychrová 2012). K+

transport is also closely coordinated with H+-ATPase activ-ity, and Pma1 is activated when K+ uptake increases andalso under K+ starvation conditions (Seto-Young and Perlin1991; Kahm et al. 2012).

K+ entry into cells

Under ideal growth conditions, cellular K+ content is 200–300 mM (Figure 4A); however, yeast can grow over a widerange of external K+ concentrations (from 10 mM to 2.5 M),and must maintain a minimal amount of internal K+ (�30mM) to survive (Arino et al. 2010). Gene products respon-sible for high affinity K+ uptake, the closely related Trk1 andTrk2, were identified by screening for mutants unable togrow under K+-limiting conditions (Gaber et al. 1988; Koet al. 1990; Ko and Gaber 1991) (Figure 2). Of these, Trk1 isthe dominant determinant of K+ influx, due to its higherexpression level. However, trk1D trk2D cells require �10-fold higher levels of K+ supplementation for normal growththan trk1D, demonstrating the contribution of Trk2 to K+

uptake. Trk1 and Trk2 are large plasma membrane proteins,1235 and 889 amino acids long, respectively, each contain-ing four M1-P-M2 sequence motifs, where M1 and M2 de-note hydrophobic domains that are connected by ana-helical P segment. By analogy with the crystal structureof KcsA, a K+ channel from Streptomyces lividans, each pro-tein is proposed to fold into a symmetric array of four re-peating pairs of membrane-spanning domains that containa central K+-conducting pore (Durell and Guy 1999). EachTrk1 or Trk2 monomer is further suggested to associate intoa homotetramer. Trk1/2-mediated transport displays highaffinity for K+ and Rb+, and high velocity (Vmax = 30nmol/mg cells/min) that is driven by the negative electro-chemical potential established by the H+-APTase (Rodri-guez-Navarro and Ramos 1984). Each transporter has twobinding sites for cations, and normally cotransports twoidentical (K+) ions, in contrast to related transporters inplants that cotransport one K+ and one Na+ ion (Haroand Rodriguez-Navarro 2002). Surprisingly, these proteinsalso mediate efflux of anions, including halides (I2, Br2, andCl2) and nonhalide chaotropic anions (SCN2 and NO3

2), anactivity that is not coupled to K+ transport (Kuroda et al.2004; Rivetta et al. 2011). The physiological relevance ofthis efflux activity is not well understood, but may allow

cells to balance charges created by Pma1-mediated protonextrusion (Rivetta et al. 2011).

The activity and/or stability of Trk1/2 are altered byseveral protein kinases and phosphatases. The functionallyredundant kinases, Hal4 and Hal5, are required to stabilizeTrk1/2 at the plasma membrane, especially under condi-tions of low extracellular [K+] (Perez-Valle et al. 2007).Furthermore cells lacking calcineurin, the Ca2+/calmodu-lin-dependent protein phosphatase, display decreased K+ up-take, which is due in part to a defect in HAL5 expression,which is regulated by Crz1 (see below) and potentially todirect regulation of the transporters as well (Mendoza et al.1994; Casado et al. 2010). Sky1, an SR protein kinase, sonamed for its role in regulating SR-type splicing factors, mayalso regulate Trk1/2 and/or additional components of K+

homeostasis (Erez and Kahana 2002; Forment et al. 2002).Finally, Trk1 physically associates with the Ppz1 phospha-tase in membrane microdomains, and increased phosphory-lation of Trk1 in ppz1ppz2 mutants suggests that it is asubstrate for these phosphatases. Hal3 negatively regulatesPpz1 and associates with it in a pH-dependent manner, thusleading to the proposal that the Hal3–Ppz1 interactionallows intracellular levels of H+ and K+ to be adjusted co-ordinately through regulation of Trk1 activity (Yenush et al.2002, 2005).

In addition to high affinity K+/Rb+ transport, yeast dis-play a low-affinity mode, which is likely also mediated byTrk1/2 (Arino et al. 2010). However, trk1 trk2 cells displaya very low affinity K+ transport activity, with a Km in themillimolar range, for which the responsible protein(s) hasnot been identified (Madrid et al. 1998). NSC1 (for nonspe-cific cation channel), an ion conductance identified usingelectrophysiological methods whose molecular identity isunknown, mediates K+ currents that are blocked by Ca2+

Figure 2 The major transporters responsible for uptake, efflux, and in-tracellular transport of alkali metal ions in S. cerevisiae. Note that the V-ATPase also resides in endosomes and late Golgi, but was omitted fromthe figure due to space constraints.



































and other divalent cations in trk1 trk2 cells, and could beresponsible for the low affinity K+ transport observed (Bihleret al. 1998; Madrid et al. 1998). Thus, additional K+ trans-porters remain to be identified in yeast.

K+ efflux

Nha1 and Ena1, discussed below, play primary roles in Na+

transport, but also efflux K+. Tok1, an outwardly rectifyingK+ channel in the plasma membrane that is activated bymembrane depolarization, is the only K+-specific effluxmechanism in yeast cells (Gustin et al. 1986; Bertl et al.1993; Ketchum et al. 1995; Zhou et al. 1995) (Figure 2).Tok1 is a 691-amino-acid-long integral membrane proteinthat contains two pore domains, each of which conducts K+.It is the founding member of a conserved family of K+ chan-nels (2P) that contain two tandem pore domains (Enyediand Czirjak 2010). The cytosolic carboxy terminal tail ofTok1 participates in its regulation and gating by preventingchannel closure (Loukin and Saimi 2002). Despite extensiveelectrophysiological characterization of this channel, itsphysiological significance is somewhat unclear. Tok1-medi-ated release of cellular K+ at low membrane potentialshould boost the electrochemical potential across the plasmamembrane. In fact, deletion of TOK1 does lead to depolari-zation, and its overexpression hyperpolarizes cells (Maresovaet al. 2006), but tok1 mutants display no growth defects,changes in K+ content, or transport activity. This channel isthe target of the K1 viral killer toxin, which kills yeast bybinding to and activating Tok1 (Ahmed et al. 1999).

Maintenance of intracellular K+ levels

Although many components of K+ transport in yeast havebeen identified, a greater understanding of the ways inwhich these transport systems work together is needed. Acomputational model was recently developed to explain howcells maintain a minimal concentration of intracellular K+

under K+ starvation conditions. Surprisingly, these investiga-tions suggest that increased levels of Pma1 activity and bi-carbonate ion, rather than regulation of Trk1/2 or Nha1, arecritical under these conditions (Kahm et al. 2012). Intracel-lular CO2, produced in many enzymatic reactions, is con-verted by carbonic anhydrase (encoded by NCE103) tocarbonic acid, which dissociates into bicarbonate (HCO3

2)and protons. Protons are pumped out of the cell by Pma1;however, HCO3

2 accumulates inside the cell and becomesa negative-charge sink that increases K+ retention inside thecell. Experimental studies confirm that NCE103 expressionincreases under conditions of K+ starvation (Kahm et al.2012) and also show that while cells maintain a minimalintracellular K+ concentration, the amount of K+ in a yeastcell varies as a function of extracellular K+ concentration andis an example of nonperfect adaptation (Kahm et al. 2012).

Na+ entry into cells

Yeast cells do not actively accumulate Na+; cellular levels ofthis ion are low under standard growth conditions, but rise

when cells are challenged with a high Na+ environment. Forwild-type yeast, Na+ is a competitive inhibitor of K+ trans-port and has a lower affinity than K+ for Trk1 (Haro andRodriguez-Navarro 2002). In high Na+ environments, Trk1is thought to transport some Na+, although it is also modi-fied under these conditions, through an unknown mecha-nism, to become more selective for K+ (Mendoza et al.1994). Cells lacking high affinity K+ transport (trk1 trk2)have a higher Na+ content than wild-type cells (Gomez et al.1996), because the remaining, nonspecific cation transport-ers, such as NSC1, mediate influx of both K+ and Na+. Ingeneral, yeast transport mechanisms are biased toward min-imizing Na+ uptake and promoting its efflux. However,somewhat surprisingly, S. cerevisiae also depend on Na+

for phosphate assimilation by Pho89, a plasma protein con-taining 12 predicted membrane-spanning domains thatcotransports Na+ and inorganic phosphate (Figure 2).Pho89p has a Km for phosphate of 0.5 mM, is highly specificfor Na+, and is maximally active at alkaline pH (9.5) (Mar-tinez and Persson 1998). It is the only nutrient transporterin yeast known to require Na+ for its activity. Expression ofPho89 is induced by phosphate limitation and by high pH; itis one of two high-affinity phosphate transporters, the otherof which, Pho84, mediates proton-coupled phosphate trans-port and is active at low pH (Pattison-Granberg and Persson2000).

Na+ efflux

Two different mechanisms promote efflux of Na+, Li2, andto a lesser extent, K+; the Ena P-type ATPases and the Nha1antiporter both promote yeast growth in the presence oftoxic cations (Figure 2). Chromosome IV of S. cerevisiae con-tains a group of tandemly repeated genes, ENA1–5, each ofwhich encodes a highly related (.97% identical in aminoacid sequence) P-type ATPase of the fungal-specific P2D sub-type (Palmgren and Nissen 2011). These gene products lo-calize to the plasma membrane, where they use ATPhydrolysis to extrude Na+, Li+, and possibly K+ (Haroet al. 1991; Benito et al. 1997). The size of the ENA clustervaries; most laboratory strains contain three to five genes,with more Na+- and Li+-tolerant strains containing largerarrays (Arino et al. 2010). CEN.PK strains, which containa single atypical gene (ENA6), and deletion mutants lackingthe entire gene cluster are extremely sensitive to Na+ andLi+ (Haro et al. 1991; Daran-Lapujade et al. 2009) and dis-play growth defects under alkaline conditions (Haro et al.1991). Expression of ENA1 is induced under these growthconditions by a constellation of signaling pathways includ-ing the osmotic stress-responsive HOG pathway, the Ca2+-dependent calcineurin/Crz1 pathway that promotes survivalunder a variety of stress conditions, the Rim101 pathway,which modulates tolerance to NaCl and high pH as well asregulating meiotic gene transcription, and nutrient-depen-dent pathways regulated by Snf1 and TOR (reviewed inArino et al. 2010). At high pH, Ena-mediated Na+ and K+

efflux become essential, most likely because the Na+, K+/H+

































antiporter, Nha1 cannot function under these conditions(Haro et al. 1991).

Nha1 is a plasma membrane proton antiporter that medi-ates extrusion of Na+ and K+ with similar affinities andpromotes tolerance to alkali cations at acidic pH (Bañueloset al. 1998; Ohgaki et al. 2005). This protein functions asa dimer and is electrogenic, exchanging multiple protons foreach molecule of Na+ or K+ transported (Mitsui et al. 2005;Ohgaki et al. 2005). Nha1 is 985 amino acids in length; itsoverall structure is similar to other Na+/H+ exchangers(NHE) with a predicted short N-terminal cytosolic domain,followed by 12 membrane-spanning segments and a verylong (551 amino acids) C-terminal cytosolic domain. ThisC-terminal domain contains six regions that are conservedin other fungal NHEs, and includes sequences required forits targeting to the plasma membrane, activity, substratespecificity, and regulation, such as its interaction with 14-3-3 proteins (Kinclova et al. 2001; Simon et al. 2001; Mitsuiet al. 2004; Zahradka et al. 2012). In addition to its role inNa+ detoxification, Nha1-mediated K+ efflux contributes tothe constitutive K+ uptake and efflux that is used to regulatethe membrane potential of yeast cells (Zahradka and Syc-hrová 2012). It also promotes rapid adaptations to alkalineand osmotic stress (Bañuelos et al. 1998; Kinclova et al.2001; Proft and Struhl 2004), and may contribute to cell-cycle regulation, as Nha1 overexpression suppresses the G1arrest of a phosphatase-deficient (sit4 hal3) strain (Simonet al. 2001, 2003).

Alkali metal cation transport in intracellular compartments

Mitochondria: K+/H+ exchange (KHE) is carried out bymitochondria, and mutations that compromise this ex-change alter mitochondrial K+ content, cause defects in re-spiratory chain assembly, and disrupt mitochondrialmorphology and volume homeostasis (Nowikovsky et al.2012). In S. cerevisiae, Mdm38 and Mrs7, members of theLETM1 protein family, are required for KHE (Figure 2); how-ever, LETM1 proteins contain a single trans-membrane do-main, suggesting that they modulate transport rather thandirectly conducting ion exchange. In support of this hypoth-esis, both Mdm38 and Mrs7 are components of larger pro-tein complexes (Nowikovsky et al. 2012). Human LETM1family members contain EF hand calcium-binding domainsthat are not present in the yeast proteins, and Letm1 hasbeen proposed to participate in mitochondrial Ca2+ uptake,although this issue is controversial. Human LETM1 is asso-ciated with Wolf-Hirschhorn syndrome (WHS), a pleiotropicneurological disorder, which is caused by a partial deletionof chromosome 4; loss of Letm1 occurs in a subset of WHScases and is linked to the occurrence of seizures (Nowikovskyet al. 2012).

Vacuole, Golgi, and endosomes: Yeast cells accumulate Na+

and K+ in the vacuole and other organelles of the secretorysystem, i.e., the Golgi and endosomes, via proton-coupledantiport, with the V-type ATPase generating the proton gradient

that drives accumulation of the alkali cation. Vnx1, whichwas identified using reverse genetics, mediates the majorityof this transport in the vacuole (Cagnac et al. 2007) (Figure2). Vnx1 encodes a 102-kDa protein with 13 predicted mem-brane-spanning domains and is a member of the calciumexchanger (CAX) superfamily, although it lacks several se-quence motifs thought to mediate Ca2+ binding and doesnot transport Ca2+. Vnx1 localizes to the vacuole, and vnx1Dyeast display increased sensitivity to NaCl and to the posi-tively charged aminoglycoside, hygromycin B (Cagnac et al.2007). Vacuolar vesicles isolated from vnx1D cells aregreatly deficient in Na+ and K+/H+ exchange activity(Cagnac et al. 2007, 2010). Surprisingly, the vacuolar Ca2+/H+ exchanger, Vcx1 was shown to mediate a smallamount of residual vacuolar K+/H+ exchange observed invnx1D cells (Cagnac et al. 2010).

Kha1 encodes a Golgi-localized, 97-kDa protein with 12predicted transmembrane domains that is related to bacte-rial K+ and Na+ transporters (Maresova and Sychrová2005). The transport activity of Kha1 has not been exam-ined biochemically; however, phenotypic characterizationsuggests that it carries out K+/H+ transport. kha1D strainsdisplay sensitivity to hygromycin B and a growth defect un-der alkaline conditions that can be suppressed by addition ofK+ to the media (Maresova and Sychrová 2005). Further-more, studies of a mammalian chloride channel, CIC-2,showed that this channel was able to substitute for a yeastchloride channel, Gef1, only in cells that also overexpressedKha1. This suggests that increased levels of Kha1 improvesCIC-2 function by bringing the pH of the yeast Golgi closerto the CIC-2 optimum (Flis et al. 2005). Thus, Kha1 isthought to play roles in both K+ and pH homeostasis bycarrying out K+/H+ exchange.

Nhx1 encodes an endosomal Na+/H+ antiporter that isa member of the NHE superfamily and localizes mainly tothe late endosome, as well as other secretory organelles(Nass and Rao 1998; Kojima et al. 2012) (Figure 2). A rolefor Nhx1 in sequestration of Na+ and K+ into secretoryorganelles has been demonstrated, for which it utilizes theproton gradient established by the V-type ATPase (Nass et al.1997; Brett et al. 2005). nxh1D cells display growth defectsin high salt or low pH conditions, and a decrease in intra-cellular pH (Nass et al. 1997; Brett et al. 2005). However,Nhx1 was also identified in a screen for mutants with vac-uolar protein sorting defects (Bowers et al. 2000), and itsrole in determining the pH of endosomal compartments iscritical to protein trafficking, where it may regulate the fu-sion of these compartments (Qiu and Fratti 2010; Kallayet al. 2011; Kojima et al. 2012).

Proton-independent transport may also contribute to K+

accumulation in the vacuole. Vhc1, a member of the cation-Cl2 cotransporter (CCC) family of transporters, is thought tofunction as a vacuolar K+-Cl2 cotransporter (Figure 2)(Andre and Scherens 1995; Petrezselyova et al. 2012). De-letion of VHC1 improves growth of a trk1D trk2D strain un-der low potassium conditions, but does not alter cytosolic


































pH, suggesting that this protein sequesters K+ in the vacuolewithout transporting protons (Petrezselyova et al. 2012).Like the related K+-Cl2 cotransporter, NKCC1, in humans,Vhc1 contributes to cellular volume control. When exposedto hyperosmotic shock, vacuoles in vhc1D cells shrink lessthan those in wild-type cells, and the mutants show reducedsurvival (Petrezselyova et al. 2012). Although Vhc1 trans-port activity has not been demonstrated directly, this phys-iological characterization is consistent with the proposal thatit mediates K+-Cl2 cotransport into the vacuole.

Divalent Cations: Ca2+ and Mg2+

Ca2+ ion homeostasis and regulation

Ca2+ is ubiquitous in the environment and serves a variety ofsignaling and structural functions in all eukaryotes. This cat-ion is required for growth of S. cerevisae. Yeast undergo G2arrest in growth medium that has been depleted of metalions, but resume cell cycle progression upon readdition ofZn2+, together with either Ca2+ or Mn2+ (Loukin and Kung1995). Calcium is abundant in yeast cells (Figure 4A). How-ever, for Ca2+ to serve its signaling function, a low cytosolicconcentration of this ion (50–200 nM) must be maintained(Miseta et al. 1999); this is achieved by Ca2+ pumps andexchangers that actively sequester Ca2+ in intracellular com-partments. Under specific conditions, such as exposure toenvironmental stress or mating pheromone, cytosolic Ca2+

transiently increases and activates downstream events. Thesesignals are generated by Ca2+ entry through plasma mem-brane ion channels and/or release from intracellular organ-elles. The primary Ca2+ store in yeast cells is the vacuole,where .90% of all cellular Ca2+ resides in association withpolyphosphate (total concentration, 2 mM) (Dunn et al.1994). Intracellular Ca2+ stores in yeast differ significantlyfrom those in mammalian cells: Yeast mitochondria do notaccumulate Ca2+ and do not express the Ca2+ uniporterfound in their mammalian counterparts (Carafoli et al.1970; Baughman et al. 2011; De Stefani et al. 2011). Also,the endoplasmic reticulum is not a significant Ca2+ store inyeast cells, containing 10 mM Ca2+, compared to 250–600mM observed in the ER of mammalian cells (Demaurex andFrieden 2003). However, there are direct parallels betweenthe main components of Ca2+ homeostasis in yeast cells andcardiac myocytes (described in Cui et al. 2009b).

Ca2+ entry into yeast cells

Yeast cells possess both high and low affinity mechanismsfor Ca2+ influx across the plasma membrane. The high af-finity, low capacity, Ca2+ system (HACS) requires at leastthree proteins: Mid1, Cch1, and Ecm7 (Cunningham2011). Mid1 and Cch1 are trans-membrane proteins thatinteract with each other (Iida et al. 1994; Fischer et al.1997; Paidhungat and Garrett 1997; Locke et al. 2000).Mid1 has four predicted trans-membrane domains and isbroadly conserved in yeast and fungi. Cch1 is a large protein

that is related to the alpha subunit of mammalian voltage-gated calcium channels (VGCCs) and is partially inhibited bythe L-type VGCC blockers nifedipine and verapamil (Tenget al. 2008). However, key residues that are required forvoltage sensing in mammalian channels are lacking inCch1 (Teng et al. 2008). Ecm7, the third protein proposedto be a component of the channel, is related to gamma sub-units of VGCCs and is a member of the claudin superfamilyof proteins. In cells lacking Mid1, Ecm7 is destabilized, sug-gesting that it associates with Mid1, although this interac-tion has yet to be demonstrated (Martin et al. 2011). Cellslacking either Mid1 or Cch1 or both have similar pheno-types, such as low Ca2+ uptake activity and loss of viabilityduring prolonged exposure to mating pheromone (Iida et al.1994; Fischer et al. 1997; Paidhungat and Garrett 1997),consistent with their contributing to a single functional com-plex. The electrophysiological properties of this Ca2+ chan-nel and its mechanism of activation are unclear. Expressionof Mid1 in mammalian cells resulted in a new stretch-acti-vated Ca2+ channel activity, and the endogenous yeast chan-nel may be similarly mechanosensitive. Studies of theanalogous channel in the fungal pathogen, Cryptococcus neo-formans, indicate that the channel is activated by the de-pletion of intracellular Ca2+ stores (Hong et al. 2010).Several physiological conditions that activate influx throughHACS have been identified, although the molecular detailsof this regulation have not been worked out (see below).

A second, less well-defined Ca2+ influx activity in yeasthas been termed LACS for low affinity Ca2+ system. LACS isactivated when cells grown in rich medium are exposed tohigh concentrations of pheromone. Genome-wide screensfor mutants defective in LACS identified components ofthe polarisome (Bni1, Spa2, and Pea2), a protein complexthat localizes to the tip of the mating projection and is in-volved in organizing and polymerizing actin (Howell andLew 2012), as well as other actin-binding proteins(Rvs167) and gene products required for cell fusion duringmating (Fig1, Fus1, and Fus2) (Muller et al. 2003, 2009).Pheromone induces expression of Fig1, a member of theclaudin superfamily of proteins that contains four predictedtransmembrane domains (Erdman et al. 1998), and localizesto sites of cell fusion during mating (Aguilar et al. 2007).Cells lacking Fig1 fail to induce LACS and have fusiondefects during mating that can be suppressed by additionof Ca2+ to the medium. This suggests that Ca2+ influxthrough LACS is intimately coupled to the cell fusion processand may represent a Ca2+-dependent wound repair path-way that is triggered by membrane holes induced duringthe fusion process (Engel and Walter 2008).

In addition to HACS and LACS, yeast cells possessadditional Ca2+ influx pathways that have not yet beencharacterized at the molecular level. Mathematical model-ing of Ca2+ transients predicts the presence of additionalinflux pathways that are subject to rapid feedback inhibitionby Ca2+ (Cui et al. 2009a). Subsequent experimental anal-yses indicated that these additional influx pathways are































inhibited by Mg2+ (Cui et al. 2009a), which is consistentwith observations of rapid Ca2+ influx in cells grown inMg2+-deficient medium (Wiesenberger et al. 2007). Ca2+

influx is also stimulated when glucose is added back to glu-cose-starved cells, and this pathway requires the Gpr1plasma membrane receptor coupled to Gpa2, a Ga subunit,and phospholipase C (see below). For cells grown in mini-mal medium, Mid1/Cch1 mediates this Ca2+ influx. How-ever, cells grown in rich medium show a large Ca2+ signalupon glucose addition that is retained in mutants lackingMid1, Cch1, or Fig1. This new activity, termed glucose-induced calcium (GIC) system, displays unique characteristics,including sensitivity to magnesium, gadolinium, and nifedi-pine and resistance to the related L-type VGCC inhibitor,verapamil (Groppi et al. 2011). Thus, GIC likely repre-sents one of the previously identified Mg2+-sensitive in-flux pathways.

Ca2+ sequestration in intracellular compartments

ER/Golgi/secretory pathway: As mentioned, the ER-lu-menal Ca2+ concentration is significantly lower in yeast thanin mammalian cells. However, the secretory pathway con-tains many Ca2+-dependent enzymes, and the lumen of theER and other secretory organelles contains a much higherconcentration of Ca2+ than the cytosol (�10 mM in the ERlumen compared to 125 nM in the cytosol). This Ca2+ gra-dient is achieved primarily by the activity of Pmr1, a con-served P-type ATPase that pumps Ca2+ and Mn2+, whosehuman homolog is hSPCA1 (for secretory pathway Ca2+-ATPase 1). Mutations in the gene that encodes hSPCA1 re-sult in a blistering skin disorder termed Hailey-Hailey dis-ease (HHD) (Hu et al. 2000; Sudbrak et al. 2000). Bothpmr1D yeast and human HHD cells have secretory vesiclesand organelles that are deficient in Ca2+ and Mn2+, whichresults in defects in the folding, modification, and sorting ofproteins that pass through the secretory pathway (Antebiand Fink 1992; Durr et al. 1998; Hu et al. 2000; Sudbraket al. 2000). Both proteins localize primarily to the Golgi(Burk et al. 1989; Antebi and Fink 1992); however, pmr1Dyeast are also significantly depleted for Ca2+ in the ER.Thus, the minor pool of Pmr1 passing through the ER playsa major role in concentrating Ca2+ and Mn2+ in this organ-elle (Strayle et al. 1999). In contrast, ER-localized SERCA-type Ca2+-ATPases fulfill this role in mammalian cells.

Spf1/Cod1, an ER-localized P-type ATPase that is a mem-ber of the evolutionarily conserved, but poorly understoodp5 subfamily of P-type ATPases (Palmgren and Nissen2011), also contributes to Ca2+ homeostasis in the ER andGolgi. spf1D and pmr1D exhibit similar defects; however,double mutants spf1D pmr1D are more severely compro-mised for Ca2+/Mn2+-dependent glycosylation events, andconstitutively induce the unfolded protein response (Croninet al. 2002; Vashist et al. 2002). Thus, although the sub-strate it transports remains unidentified, Spf1/Cod1 clearlyoverlaps functionally with Pmr1 to maintain proper Ca2+

and Mn2+ homeostasis in the ER and Golgi of yeast cells.

Vacuole: Yeast cells contain one or more vacuoles: dynamic,acidic, storage organelles akin to mammalian lysosomes andplant tonoplasts, that undergo fusion and fission during thecell cycle and in response to environmental stimuli (Baarset al. 2007). The vacuole is the primary Ca2+ storage organ-elle in yeast, containing.90% of total cellular Ca2+, and theprimary mechanism for clearing Ca2+ from the yeast cytosolis its rapid delivery to the vacuole. This vacuolar Ca2+ se-questration is carried out by the combined action of Vcx1,a H+/Ca2+ exchanger and member of the type II calciumexchanger family (CAX) and Pmc1, a P-type Ca2+-ATPase.

Vcx1 utilizes the steep proton gradient across the vacuo-lar membrane (generated by the V-type H+-ATPase) to cou-ple Ca2+ entry into the vacuole with H+ efflux into thecytosol (Cunningham and Fink 1996; Pozos et al. 1996;Pozos 1998). When wild-type yeast are exposed to a suddenincrease in extracellular Ca2+, cytosolic Ca2+ levels rise,peaking within seconds, and then sharply decrease within30 sec to restore a low steady state level of cytosolic Ca2+

(Miseta et al. 1999). Vcx1 is primarily responsible for thisrapid Ca2+ flux into the vacuole, and vcx1D mutants displaya greater increase and significantly slower decrease in cyto-solic Ca2+ under these same conditions. However, vcx1Dcells do not exhibit lower amounts of steady state vacuolarCa2+ and these cells are as tolerant as wild type to growthon media containing high levels of Ca2+ (Cunningham andFink 1996; Pozos et al. 1996; Pozos 1998).

In contrast, pmc1D cells display Ca2+ flux dynamics thatare identical to wild-type cells (Miseta et al. 1999), butexhibit Ca2+-sensitive growth and reduced vacuolar content(Cunningham and Fink 1994); both phenotypes are exacer-bated in pmc1D vcx1D cells (Cunningham and Fink 1996;Pozos et al. 1996; Pozos 1998). Thus, Vcx1 and Pmc1 worktogether to sequester Ca2+ into the yeast vacuole. Vcx1 rap-idly handles a large volume of Ca2+ and plays the dominantrole in short-term Ca2+ dynamics, and the slower, ATP-de-pendent action of Pmc1 establishes the extremely steepCa2+ concentration gradient across the vacuolar membranethat exists in wild-type cells. A mathematical model, devel-oped to explain the key characteristics of yeast Ca2+ tran-sients, predicted the existence of an additional regulatoryelement, i.e., rapid Ca2+-dependent negative feedback ofCa2+ influx pathways, which has not yet been demonstratedexperimentally (Cui et al. 2009a). Calcineurin, the Ca2+/cal-modulin-dependent phosphatase regulates Ca2+ homeosta-sis, but on a slower time scale. Calcineurin increasesexpression of PMC1 and PMR1 by activating the Crz1 tran-scription factor, and negatively regulates Vcx1 activitythrough an undetermined mechanism (Cunningham andFink 1994; Cunningham and Fink 1996; Matheos et al.1997; Stathopoulos and Cyert 1997; Pittman et al. 2004)(see below).

Ca2+ release from intracellular compartments

Vacuolar Ca2+ release mediated by Yvc1: Ca2+ stored inthe vacuole can be released into the cytosol through the







































Yvc1 Ca2+ channel, also called TRPY1 (Figure 3). The Yvc1channel was first identified by electrophysiological studies ofion conductances in isolated yeast vacuoles (Bertl and Slay-man 1990; Saimi et al. 1992), which showed that it wascation selective and inhibited by vacuolar Ca2+ (mM) orlow pH (,5). Many years later, the gene product responsiblefor this conductance was identified as the product of theYvc1 gene, which is a member of the large family of TRPchannels (transient receptor potential), whose mammalianmembers are responsible for sensory phenomenon such asdetection of heat, cold, and pain and are associated withdiseases including retinal degeneration and polycystic kid-ney disease (Palmer et al. 2001; Nilius and Owsianik 2011).Yeast strains lacking Yvc1 display no growth defects, butoverexpression of the gene confers Ca2+ sensitivity, suggest-ing a role for this protein in Ca2+ homeostasis (Denis andCyert 2002). Indeed, Yvc1 mediates vacuolar Ca2+ releasewhen yeast are exposed to osmotic shock (Denis and Cyert2002), a condition also reported to trigger Ca2+ influxthrough Mid1/Cch1 (Matsumoto et al. 2002). Further stud-ies showed that the Yvc1-encoded channel is mechanosen-sitive, suggesting that vacuolar shrinkage during osmoticshock may directly activate the channel (Zhou et al. 2003).Yvc1 is also thought to contribute to Ca2+ transients observedafter glucose readdition to starved cells (Bouillet et al. 2012),although the mechanism of its activation is unknown.

Ca2+-dependent signaling pathways

Responses to stress: Ca2+ is widely used in prokaryotic andeukaryotic cells as a second messenger that triggers down-stream signaling events. Ca2+ signals are extremely dynamicand are controlled both temporally and spatially to conferspecific cellular responses (Clapham 2007). In yeast, cal-modulin is a major target of Ca2+ which, in its Ca2+-boundform, binds to and activates protein kinases (Cmk1 andCmk2) and calcineurin, the conserved Ca2+/calmodulin-dependent phosphatase (Cyert 2001) (Figure 3). Thesekinases and phosphatases are often stimulated by the samephysiological conditions (Moser et al. 1996; Dudgeon et al.2008), however only calcineurin-dependent pathways,which are better understood, will be described here. Calci-neurin is a heterodimer composed of a catalytic and a regu-latory subunit (encoded by CNA1, CNA2, and CNB1,respectively). Calcineurin deficiency can by induced by mu-tation (cna1D cna2D or cnb1D) or by incubating yeast withthe calcineurin inhibitors, cyclosporine or FK506 (Foor et al.1992). Calcineurin is not essential for growth under typicallaboratory conditions, i.e., when [Ca2+]cyto is low and theenzyme exhibits little activity. However, exposure of yeast toany of a number of environmental stress conditions, includ-ing high pH, cell wall damage, and high concentrations ofcations (Mn2+, Na+, and Li+) activates calcineurin, and thephosphatase is required for cell survival under these condi-tions (Cyert 2003; Garcia et al. 2004; Viladevall et al. 2004;Zakrzewska et al. 2005) (Figure 3). This Ca2+-mediatedstress response is conserved in pathogenic fungi, where it

promotes survival in the host and is required for pathogen-esis (Bastidas et al. 2008). Calcineurin dephosphorylatesa range of protein targets in yeast, including the Crz1 tran-scription factor, which rapidly translocates from the cytosolto the nucleus upon dephosphorylation to express genesencoding cell wall biosynthetic enzymes (Fks2), ion pumps(Pmc1, Pmr1, and Ena1), signaling enzymes (Cmk2), com-ponents of vesicle trafficking (Gyp7 and Ypt53), and regu-lators of calcineurin (Rcn1) (Yoshimoto et al. 2002; Cyert2003; Heath et al. 2004; Viladevall et al. 2004; Bultyncket al. 2006). In addition to Crz1, calcineurin dephosphory-lates proteins involved in ER translocation (Hph1) (Pinaet al. 2011), TORC2 effectors (Slm1 and Slm2) (Bultyncket al. 2006; Berchtold et al. 2012; Niles et al. 2012), itsregulator (Rcn1) (Hilioti et al. 2004), and as mentioned,may regulate K+ transporters (Mendoza et al. 1994) (Figure3). In response to excess amino acids, calcineurin also acti-vates endocytosis of the Dip5 amino acid transporter bydephosphorylating the a-arrestin trafficking adaptor, Aly1/Art6 (A. F. O’Donnell, unpublished results). Calcineurin-dependent signaling is activated by the influx of extracellularCa2+, rather than Ca2+ release from intracellular stores. Insome cases, such as the responses to alkaline and ER stress,Ca2+ influx is mediated by the Mid1/Cch1 plasma mem-brane channel (Bonilla et al. 2002; Viladevall et al. 2004);however, for many calcineurin-activating conditions, the ef-fect of mid1 or cch1 mutations on signaling has not beentested. Although the mechanism of Mid1/Cch1 activationhas not yet been clearly defined, the channel is suggestedto be mechanosensitive (Kanzaki et al. 1999), which couldexplain the link between environmental stress and calci-neurin activation. Crz1/calcineurin signaling is active whenprotein secretion is compromised, i.e., in pmr1D cells, inmany mutants with defects in vesicle-mediated transport,

Figure 3 Environmental stress induces Ca2+ influx into S. cerevisiae viathe Mid1/Cch1 high affinity Ca2+ channel and activation of the calci-neurin phosphatase (CN), which dephosphorylates substrates Crz1,Aly1, Slm1/2, and Hph1. See text for details. Note that the V-ATPase alsoresides in endosomes and late Golgi, but was omitted from the figure dueto space constraints.













































and in cells experiencing ER stress caused by the accumula-tion of unfolded proteins (Locke et al. 2000; Bonilla et al.2002; Martin et al. 2011). All of these conditions causealterations in cell wall composition, and as yeast cells areunder constant turgor pressure, could result in activation ofCa2+ influx through a mechanosensitive ion channel in theplasma membrane. Furthermore, many conditions that acti-vate the protein kinase C-regulated cell wall integrity path-way, which responds to cell wall damage caused by chemicalagents, heat, mutation in cell wall biosynthetic enzymes, orhypotonic shock, also activate calcineurin-mediated stressresponses (Levin 2011). During heat stress, for example,both Ca2+/calcineurin and the cell wall integrity pathwayare activated and work together to regulate expression ofa cell wall biosynthetic enzyme, Fks2 (Zhao et al. 1998a).Thus, plasma membrane/cell wall stretch may activate thecell wall integrity pathway through its upstream sensors,Wsc1-3, Mid2, and Mtl1 (Levin 2011), while simultaneouslycausing Ca2+ influx via activation of Mid1/Cch1. Surpris-ingly, Ca2+ influx stimulated by hypotonic shock requiresCch1 but not Mid1 (Groppi 2011).

Response to mating factor: Exposure of haploidMATa yeastcells to a-factor mating pheromone activates a MAPK signal-ing pathway to induce cell-cycle arrest and morphologicalchanges, such as cell polarization and production of cellsurface agglutinins, that promote interaction and, ulti-mately, fusion with a mating partner (Dohlman and Slessar-eva 2006). Changes in Ca2+ ion accumulation also play animportant signaling role in this process, as cells incubatedwith pheromone for extensive periods die unless Ca2+ ispresent in the growth medium (Iida et al. 1990; Cunning-ham 2011). Indeed, exposure to mating factor stimulatesthe Mid1/Cch1 or HACS Ca2+ channel, inducing a rise in[Ca2+]cyto that activates Ca2+/calmodulin-dependent phos-phatases, Cna1/2, and kinases, Cmk1/2. Cells lacking any ofthe gene products in this signaling pathway behave like cellsdeprived of extracellular Ca2+ and lose viability during pro-longed exposure to pheromone (Iida et al. 1994; Moser et al.1996; Fischer et al. 1997; Paidhungat and Garrett 1997;Withee et al. 1997). Calcineurin activates gene expressionin pheromone-treated cells through regulation of Crz1, andlike calcineurin mutants, crz1D cells exhibit decreased sur-vival when incubated with pheromone (Stathopoulos andCyert 1997; Yoshimoto et al. 2002). The extensive cell wallremodeling that occurs as cells extend mating projectionsmay cause cell wall damage, and thus explain the require-ment for both calcineurin and PKC-mediated stressresponses for survival under these conditions (Levin2011). Once Ca2+ influx is activated by pheromone, calci-neurin negatively regulates this pathway by inhibitingHACS-mediated Ca2+ influx, possibly by dephosphorylatingCch1 (Locke et al. 2000). Ca2+ also seems to facilitate cellfusion during mating. At higher pheromone concentrationsthan those required for HACS activation, Ca2+ influxthrough LACS is stimulated, and transcription of Fig1, which

encodes a component of LACS is induced (Erdman et al.1998). Mutants that disrupt LACS, including fig1D, havedefects in cell fusion during mating that can be alleviatedby addition of high extracellular Ca2+ (Muller et al. 2003;Aguilar et al. 2007).

Response to nutrients: Yeast respond to the presence ofglucose, sucrose, and other sugars using a variety ofintracellular signals including cAMP and pHi (as discussedearlier) as well as Ca2+ (Gancedo 2008). Addition of glu-cose to yeast cells deprived of sugar results in a rapid influxof extracellular Ca2+ (Nakajima-Shimada et al. 1991) thatrequires glucose phosphorylation and Gpr1 (Tisi et al.2002), a plasma membrane protein that is a member ofa large class of G protein-coupled receptors containing sevenmembrane-spanning domains (Xue et al. 2008). Althoughsuch proteins typically interact with a heterotrimeric GTP-binding, or G protein, composed of a, b, and g subunits,Gpr1 interacts with and activates the Ga-related Gpa2, inthe absence of a traditional b or g subunit (Gancedo 2008).Sugars, i.e., glucose or sucrose, likely bind to Gpr1 (althoughthis has not been directly demonstrated), causing activationof Gpa2, which activates adenylcyclase to produce cAMP(Kraakman et al. 1999; Lemaire et al. 2004; Peeters et al.2006) and Plc1, a phospholipase C-type g that cleavesPI4,5P2 to create diacylglycerol and inositol-(1,4,5)-trisphos-phate (IP3) (Ansari et al. 1999; Tisi et al. 2002). It is IP3 thatis required for Ca2+ influx, which occurs through two dif-ferent influx pathways: Mid1/Cch1 and GIC (discussed ear-lier) (Tisi et al. 2004). The mechanism by which IP3stimulates these influx pathways has not yet been deter-mined; however, the Ca2+ signal generated may regulatePma1 (Tropia et al. 2006; Bouillet et al. 2012) and is suffi-cient to activate calcineurin/Crz1 signaling, suggesting a rolefor this pathway, which up-regulates the expression of sev-eral genes encoding carbohydrate-metabolizing enzymes, inthe glucose response (Ruiz et al. 2008; Groppi et al. 2011).

Regulation of cell cycle and morphogenesis: An earlyapproach to studying the role of Ca2+ in yeast was the iden-tification of Ca2+-sensitive mutants in which V-ATPase activ-ity was compromised and a budding-defective mutant, cls4,which is an allele of Cdc24, the essential guanine nucleotideexchange factor for Cdc42, a GTPase that is critical for yeastcell polarity and budding (Ohya et al. 1986a,b; Howell andLew 2012). The mechanism by which Ca2+ prevents bud-ding in cls4 cells is still not understood; however, recentwork identified additional Ca2+-sensitive mutants andshowed multiple effects of this ion on the morphology ofboth mutant and wild-type yeast cells (Ohnuki et al.2007). This suggests that our knowledge of Ca2+-dependentpathways regulating morphogenesis is quite deficient, per-haps due to regulatory redundancy. For example, study ofzds1 mutants, which lack a regulatory subunit of the PP2Aphosphatase (Rossio and Yoshida 2011), uncovered Ca2+

and calcineurin-dependent regulation of the morphogenesis


































checkpoint, which controls entry into mitosis (Miyakawaand Mizunuma 2007; Howell and Lew 2012).

Mg2+ ion homeostasis and regulation

Magnesium is abundant in the environment, serves as anessential cofactor for many cellular enzymes, and is requiredfor cell growth and proliferation (Wolf and Trapani 2008).Yeast cells actively accumulate this ion and regulate its con-centration in the cytoplasm and intracellular organelles. Al-though Mg2+ is stored in the vacuole and mitochondria,when grown under Mg2+-deficient conditions, yeast cellseventually stop dividing when the cellular Mg2+ contentdrops below �15 nmol/106 cells (Beeler et al. 1997; Pisatet al. 2009). Four yeast proteins that contribute to Mg2+

homoeostasis belong to the CorA or MIT (metal ion trans-porter) superfamily of Mg2+ transporters that are found inprokaryotes and eukaryotes and function as oligomers(Maguire 2006). Mrs2, a member of one subfamily, is re-sponsible for Mg2+ uptake into mitochondria (Schindlet al. 2007), and Alr1 and Alr2, closely related proteins thatbelong to a distinct branch of CorA-related proteins, mediateMg2+ uptake across the plasma membrane. Alr1-deficientcells display reduced Mg2+ content and a growth defect thatcan be suppressed by high extracellular Mg2+ or overexpres-sion of Alr2, which is normally present at low levels (Limet al. 2011). A third member of the MIT superfamily, Mnr2,localizes to the vacuolar membrane and is proposed to func-tion in utilization of Mg2+ stores. The vacuole accumulatesMg2+, and this is thought to occur via a yet-to-be-identifiedMg2+/H+ exchanger (Borrelly et al. 2001). mnr2 mutantsexhibit increased cellular Mg2+ content together witha Mg2+-suppressible growth defect indicative of low cyto-plasmic [Mg2+]. Wild-type cells grown under Mg2+-repleteconditions are able to survive for several generations in theabsence of extracellular Mg2+, and this requires Mnr2-mediated release of Mg2+ from the vacuole (Pisat et al.2009). Thus, Mg2+-limitation results in utilization of intra-cellular Mg2+ stores, as well as stimulation of Mg2+ uptakethrough Alr1, whose activity may be directly regulated bythis ion (Lim et al. 2011).

Biological Roles of Transition Metal Ions

Eukaryotic cells contain hundreds of proteins that arefunctionally dependent on bound transition metal cofac-tors. These metalloproteins perform critical functions invirtually every cellular process. Proteins lacking their metalcofactors are typically inactive; therefore, metal ionsconstitute essential nutrients for all organisms. The exactnumber of metalloproteins in the yeast proteome is notknown, as the presence of metal centers in known proteinscontinues to be discovered (Cvetkovic et al. 2010). An es-timate of the prevalence of metalloproteins can be obtainedby examining the metal content of enzymes for whichthree-dimensional structures have been solved. Using thisapproach, 9% of proteins contain zinc, 8% contain iron, 6%

contain manganese, and 1% contains copper (Andreiniet al. 2008). While the presence of a metal ion in a crystalstructure strongly suggests that a metal ion is present in theprotein in vivo, the identity of the metal ion in the solvedstructure may not correspond to that of the metal ion pres-ent in the endogenous protein. Bioinformatic approachesbased on the number of known metal binding domainsidentified in sequenced genomes have also been used toestimate the prevalence of metalloproteins. This approachindicates that zinc metalloproteins are the most abundantand comprise �10% of the yeast proteome (Andreini et al.2009). Iron proteins containing mononuclear and diironcenters, iron-sulfur clusters, and heme cofactors are alsopredicted to be very abundant, comprising �2% of theyeast proteome. Copper and manganese are consideredtrace nutrients, with cells containing many fewer copperand manganese metalloproteins and far lower levels ofthese elements. Nickel, cobalt, molybdenum, and cadmiumare present in trace amounts in yeast but are not known tobe incorporated into metalloenzymes and are not consid-ered nutrient metals (Bleackley and Macgillivray 2011;Zhang and Gladyshev 2011). The abundance of transitionmetals and other ions in yeast cells grown in rich mediumhas been measured and roughly parallels the estimates ofmetalloprotein abundance (Figure 4) (Eide et al. 2005).

This reliance on metal ions exacts a toll on cells. Iron,manganese, and copper are redox-active metals that occupymultiple valence states in biological systems. This propensityto readily pick up or lose electrons renders these metalsextremely useful in enzymatic reactions involving the trans-fer of electrons; however, the presence of reduced iron orcopper in cells that also contain oxygen can lead to theformation of reactive oxygen species and consequent oxida-tive damage to cellular components. Metals can also causetoxicity by binding to noncognate sites in metalloproteins(Waldron et al. 2009). This binding of the “wrong”metal ioncan preclude the binding of the correct metal ion and typi-cally inactivates metalloenzymes. For these reasons, cellularsystems involved in the uptake and utilization of metal ionsare precisely regulated according to the availability of andthe cellular requirement for the ion.

Uptake systems for individual metals are homeostaticallyregulated in S. cerevisiae, with expression of uptake systemsat high levels during periods of metal scarcity and at lowlevels when metals are abundant (reviewed in Philpott andProtchenko 2008; Eide 2009; Reddi et al. 2009a; Nevitt et al.2012). Yeast cells are also capable of adjusting metal uptaketo accommodate different metabolic states. For example,respiratory growth is accompanied by expansion of mito-chondria rich in respiratory complexes that contain numer-ous iron and copper cofactors (Stevens 1977). Respiratorygrowth is also accompanied by increases in iron and copperuptake and expansion of intracellular pools of these metals.Conversely, anaerobic growth is associated with down-regulationof oxygen-requiring processes and is associated with largereductions in iron uptake (Hassett et al. 1998a).











Here we will discuss the major nutrient metals: iron, zinc,copper, and manganese. For each metal we first discuss theregulation of systems involved in uptake, utilization, anddetoxification, and then the functions of the protein compo-nents of these systems and the metabolic adaptations associatedwith scarcity or excess of metals.

Iron

Transcriptional control through Aft1 and Aft2

Iron homeostasis is largely achieved through the transcrip-tional regulation of genes involved in iron uptake. Thesegenes are controlled by the major iron-dependent transcrip-tional activator, Aft1 (Yamaguchi-Iwai et al. 1995, 1996;Shakoury-Elizeh et al. 2004), and, to a lesser extent, by itsparalog, Aft2 (Blaiseau et al. 2001; Rutherford et al. 2001,2003; Courel et al. 2005), which bind DNA through a con-sensus upstream activation sequence (PyPuCACCC). Aft1 isconstitutively expressed and shuttles in and out of the nu-cleus (Yamaguchi-Iwai et al. 2002). When cytosolic iron lev-els fall, Aft1 accumulates in the nucleus, where it binds DNAand activates transcription. Elevated intracellular iron levelstrigger the nuclear export and inactivation of Aft1, a processthat requires several proteins involved in the assembly andtransport of iron–sulfur clusters (ISCs) (Chen et al. 2004;Rutherford et al. 2005).

ISCs are inorganic cofactors of iron and sulfide, fre-quently in the form of a simple 2Fe–2S cluster or a cubane

4Fe–4S cluster, which are directly coordinated by cysteine orhistidine residues in recipient proteins (reviewed in Lill andMuhlenhoff 2008). In yeast, ISCs are assembled in the mi-tochondrial matrix and in the cytosol. Cells lacking Yfh1(a mitochondrial iron carrier and component of the ISC as-sembly machinery) (Chen et al. 2004), Grx5 (the mitochon-drial monothiol glutaredoxin) (Belli et al. 2004), orglutathione (Rutherford et al. 2005) exhibit impaired mito-chondrial ISC assembly as well as defects in iron-mediatedinactivation of Aft1. An as yet undefined product of themitochondrial ISC machinery is exported to the cytosol,where it interacts with proteins of the cytosolic ISC assemblymachinery to deliver ISCs to target enzymes of the cytosoland nucleus (Kispal et al. 1999; Lange et al. 2000; Li et al.2001a). Most of the cytosolic ISC machinery is not requiredfor the inactivation of Aft1, however. Instead, a complexconsisting of monothiol glutaredoxins, Grx3 or Grx4, a BolA-like protein, Fra2, and a third protein, Fra1, is required for ironsensing through Aft1 (Ojeda et al. 2006; Pujol-Carrion et al.2006; Kumanovics et al. 2008; Li et al. 2009).

While the exact mechanism of iron-induced inactivationof Aft1 is not yet clear, several observations, considered to-gether, suggest a possible model. Grx3/4, Fra2, and Fra1can be isolated in a complex with Aft1 in vivo. Grx3 andFra2 form a heterodimer that, along with glutathione, con-tains a bridging 2Fe–2S cluster in vitro. Aft1 undergoes aniron-induced dimerization when it is inactivated, and Aft1contains a conserved Cys-Xaa-Cys motif that is required forboth iron-dependent inactivation and dimerization (Uetaet al. 2007). Together, these observations suggest a modelin which the 2Fe–2S cluster bound to a Grx3/4-Fra2 heter-odimer could be transferred to the Cys-Xaa-Cys motifs inAft1 to promote the formation of a transcriptionally inactiveAft1 homodimer. Thus, ISC-bound Grx3/4-Fra2 hetero-dimers may constitute the iron signal that leads to Aft1inactivation.

Remarkably, only Saccharomyces and related species ofyeast rely on Aft1-like transcription factors to control ironhomeostasis. Other fungal species, such as Schizosaccharo-myces pombe, Candida albicans, and Aspergillus spp. rely oniron-regulated transcriptional repressors of the GATA andCCAAT-box binding families (Labbe et al. 2007; Schrettland Haas 2011). The evolutionary forces that led to thisdiversity of iron regulators are not clear; however, themonothiol glutaredoxins also appear to have a conservedrole as iron sensors in S. pombe (Jbel et al. 2011).

The Aft1/Aft2 regulon

Aft1 and Aft2 activate the transcription of a set of genesinvolved in the uptake of iron from the environment, themobilization of iron from sites of intracellular storage, andthe adaptation to an iron-limited metabolism (Philpott andProtchenko 2008). Table 1 contains a list of the Aft1/2 tar-get genes along with their function and cellular location (seealso Figure 5). The majority of these genes are involved ei-ther directly or indirectly in the uptake of iron at the plasma

Figure 4 Abundance of common elements in S. cerevisiae. StrainBY4741 was grown in rich (YPD) medium and ionic content was mea-sured using inductively coupled plasma-atomic emission spectroscopy. (A)Data expressed as parts per million (ppm). (B) Abundance of metals, withdata expressed as parts per billion (ppb). Data from Eide et al. (2005).




































membrane. Most of the iron in the aerobic extracellular mi-lieu is present as poorly soluble ferric oxyhydroxides. To solvethis bioavailability problem, unicellular organisms enhancethe solubility of iron through (1) acidification of the environ-ment, (2) reduction of ferric iron to the more soluble ferrousform, and (3) secretion of soluble iron-chelating molecules. S.cerevisiae relies on all three strategies to varying extents.Many unicellular organisms and some plants synthesize andsecrete siderophores, which are a heterogeneous group oforganic compounds that bind ferric iron with extremely highaffinity and specificity. These compounds are secreted in theiriron-free form to the extracellular environment, where theybind and solublize ferric iron. The iron–siderophore com-plexes can then be taken up by specific transport systems.While S. cerevisiae does not synthesize or secrete sidero-phores, it can take up and utilize the iron bound to sidero-phores secreted by a variety of other species.

Before any iron compound can be taken up by a yeastcell, the iron must first traverse the cell wall. The yeast cellwall is a dynamic structure and contains a layer of manno-proteins covalently attached to a latticework of glucans andchitin. The most highly expressed genes of the Aft1 regulonare three cell wall mannoproteins, Fit1, Fit2, and Fit3(Protchenko et al. 2001). These proteins enhance the re-tention of siderophore in the cell wall and increase theuptake of siderophore–iron at the cell surface, although

the precise mechanisms by which these proteins functionis not known.

Reductive uptake of iron at the cell surface

Aft1 controls the expression of two genetically separate sys-tems of iron uptake, one that requires the external reductionof ferric iron to ferrous before uptake (the reductive system)and one that takes up intact ferric–siderophore chelates (thenonreductive system). Reductive uptake of iron is a two-stepprocess in which ferric salts and ferric chelates are first re-duced to the ferrous form by members of the FRE family ofmetalloreductases, then the ferrous ion is transferred to thecytosol by a high-affinity, ferrous-specific transport complex(reviewed in Philpott 2006). FRE1 and FRE2 encode flavo-cytochromes that constitute the majority (.90%) of the cellsurface reductase activity (Dancis et al. 1990, 1992; Geor-gatsou and Alexandraki 1994; Lesuisse and Labbe 1994;Hassett and Kosman 1995; Georgatsou et al. 1997). Theyare required for growth on media containing low concentra-tions of ferric salts and can catalyze the reduction of ferric–siderophore chelates (Yun et al. 2001). Because sidero-phores have low affinity for ferrous iron, reduction of theferric–siderophore chelate results in the release of ferrousiron, which can be taken up by ferrous-specific transporters.Additional family members (Fre3 and Fre4) have weak ac-tivity against ferric–siderophore chelates. Fre5 has been

Table 1 Genes activated by Aft1 and Aft2

ORF name Gene name Location Function

Uptake of iron at the cell surfaceYDR534C FIT1 Cell wall Siderophore binding/uptake

YOR382W FIT2 Cell wall Siderophore binding/uptakeYOR383C FIT3 Cell wall Siderophore binding/uptakeYLR214W FRE1 Plasma membrane MetalloreductaseYKL220C FRE2 Plasma membrane MetalloreductaseYOR381W FRE3 Plasma membrane Siderophore reductaseYNR060W FRE4 Unknown Putative reductaseYMR058W FET3 Plasma membrane Multicopper oxidase, Fe(II) uptakeYER145C FTR1 Plasma membrane Permease, Fe(II) uptakeYNL259C ATX1 Cytosol Cu chaperone, deliver Cu to Ccc2pYDR270W CCC2 Post-Golgi vesicle Cu transport into vesiclesYHL040C ARN1 Endosome, plasma membrane Ferrichrome transportYHL047C ARN2/TAF1 Unknown TAFC transportYEL065W ARN3/SIT1 Endosome, plasma membrane Hydroxamate siderophore transportYOL158C ARN4/ENB1 Plasma membrane Enterobactin transport

Efflux of iron from vacuole to cytosolYLL051C FRE6 Vacuole MetalloreductaseYLR034C SMF3a Vacuole Fe(II) transportYFL041W FET5 Vacuole Multicopper oxidase, Fe(II) transportYBR207W FTH1 Vacuole Permease, Fe(II) transport

Other transportersYGR065C VHT1 Plasma membrane Biotin transporterYOR316C COT1 Vacuole Zn, Co storage/detoxificationYKR052C MRS4a Mitochondria Mitochondrial iron importYOR384W FRE5 Mitochondria Putative reductase

Metabolic adaptation to low ironYLR205C HMX1 Endoplasmic reticulum Heme oxygenaseYLR136C CTH2/TIS11 Cytosol mRNA degradation

a Predominately regulated by Aft2p. All others predominately regulated by Aft1p.












detected in purified mitochondria (Sickmann et al. 2003),where its function is unknown, and Fre6 is expressed in thevacuole (see below) (Rees and Thiele 2007; Singh et al.2007). The FRE reductases exhibit specificity for oxidizedforms of both iron and copper and can enzymatically reducea variety of nonmetallic compounds that act as one-electronacceptors (Lesuisse et al. 1987).

Reduced iron is taken up via a high-affinity transportcomplex that consists of a copper-dependent ferrous oxidase(Fet3) (Askwith et al. 1994) and an iron permease (Ftr1)(Stearman et al. 1996). Fet3 oxidizes ferrous iron to ferricbefore transferring the ferric iron directly to Ftr1 for trans-port across the plasma membrane (de Silva et al. 1995; deSilva et al. 1997; Hassett et al. 1998a,b). Why the cell cou-ples the oxidation of iron to its transport is not known, butthe coupled process may permit the transport complex todistinguish between ferrous iron and other transition met-als. Because Fet3 requires four copper ions for activity andbecause oxygen is a cosubstrate for the enzyme (Hassettet al. 1998b), iron uptake through the Fet3–Ftr1 complexis both a copper- and oxygen-dependent process (Yuan et al.1995). Two genes activated by Aft1 under iron-deficientconditions are dedicated to the post-translational deliveryof copper to Fet3. CCC2 encodes a P-type ATPase that trans-ports copper ions from the cytosol to the lumen of post-Golgivesicles (Yuan et al. 1995), where the copper is inserted intoFet3 (Dancis et al. 1994b). ATX1 encodes a copper chaper-one that binds cytosolic copper and delivers it to the Ccc2transporter (Lin et al. 1997).

When iron is abundant, Aft1 is inactive, the high-affinityuptake systems are not expressed, and yeast rely on broadspecificity metal transporters for iron uptake. Yeast expressthree members of the Nramp family of divalent metal trans-porters, Smf1, Smf2, and Smf3 (Portnoy et al. 2000). Smf1and Smf2 are primarily manganese transporters, but they

can efficiently transport ferrous iron, as well, and cells over-expressing Smf1 accumulate higher levels of intracellulariron (Cohen et al. 2000). Fet3 cannot function under anaer-obic conditions and cells grown in reduced oxygen inducethe expression of a low-affinity, low-specificity ferrous irontransporter, Fet4 (Dix et al. 1994; Hassett et al. 1998a; Jensenand Culotta 2002). Fet4 also exhibits transport activity forzinc, copper, and cadmium (Hassett et al. 2000; Portnoyet al. 2001; Waters and Eide 2002), but may be the primarysystem for iron uptake in hypoxic environments.

Nonreductive uptake of siderophore–iron chelates

Even though S. cerevisiae does not synthesize or secrete side-rophores, this yeast expresses four siderophore–iron trans-porters of the ARN/SIT family that specifically take upsiderophore–iron chelates produced by other species offungi and bacteria (Lesuisse et al. 1998; Heymann et al.1999, 2000; Yun et al. 2000a,b). These transporters aremembers of the major facilitator superfamily, have 14 pre-dicted membrane-spanning domains, and are thought to beenergized by proton symport. Each transporter exhibitsspecificity for a subset of fungal or bacterial siderophores(Table 2). Two of these transporters, Arn1 and Arn3/Sit1,are regulated post-translationally through their traffickingthrough the late secretory pathway.

Under conditions of iron deficiency, Aft1 directs thetranscription of the ARN/SIT family of siderophore trans-porters and after translation the proteins proceed throughthe secretory pathway to the trans-Golgi network (TGN).When siderophore substrates for Arn1 and Arn3 are notavailable outside the cell, the transporters at the TGN aresorted into vesicles destined for degradation in the vacuolarlumen, bypassing the plasma membrane (Kim et al. 2002;Froissard et al. 2007). This sorting process requires recogni-tion by clathrin adaptor proteins at the TGN, followed by

Figure 5 Iron homeostasis in S. cerevisiae. Protein prod-ucts of Aft1- and Aft2-regulated genes are shown in theirrespective subcellular locations. Red spheres are Fe(III); or-ange spheres are Fe(II). Ccc1 and mitochondrial proteinsinvolved in the heme synthesis, the TCA cycle, biotin syn-thesis, and glutamate synthesis are down-regulated byiron deficiency. Reproduced with permission (Philpottand Smith 2013).





























ubiquitination and sorting into luminal vesicles of the multi-vesicular body, which are then degraded in the lumen of thevacuole (Kim et al. 2007; Erpapazoglou et al. 2008; Denget al. 2009). When siderophore substrates for Arn1 or Arn3are present outside the cell, even in very low concentrations,the substrates entering the endosome through fluid phaseendocytosis bind to a receptor domain on the transporter,which redirects the transporter into vesicles destined for theplasma membrane (Moore et al. 2003; Kim et al. 2005;Froissard et al. 2007; Erpapazoglou et al. 2008; Deng et al.2009). Thus the Arn1 and Arn3 transporters are notexpressed on the surface of yeast unless the cells are bothiron deficient and the specific siderophore substrates areavailable for uptake.

Mobilization of vacuolar iron stores

When yeast are grown in media containing adequateamounts of iron, iron transported into the cytosol that isnot immediately required for metabolic purposes is stored inthe vacuole. CCC1 encodes an iron and manganese trans-porter that localizes to the vacuolar membrane and is re-quired for the transfer of iron from the cytosol to thevacuole (Lapinskas et al. 1996; Chen and Kaplan 2000; Liet al. 2001b). CCC1 is actively transcribed under conditionsof iron excess by the transcription factor Yap5 (Li et al. 2008).Cells lacking Ccc1 are sensitive to the toxic effects of iron.When cells are grown in iron-poor medium, Ccc1 expressionis turned off and Aft1 and Aft2 direct the transcription of a setof genes that effect the transfer of vacuolar iron back to thecytosol. These genes largely duplicate on the vacuolar mem-brane the reductive iron uptake system of the plasma mem-brane. Fre6, a paralog of the plasma membrane ferric andcupric reductases (Martins et al. 1998), is expressed exclu-sively on the vacuolar membrane where it is required for thereduction of vacuolar iron and copper (Rees and Thiele 2007;Singh et al. 2007). Reduced vacuolar iron is transported intothe cytosol via the Fet5/Fth1 oxidase/permease complex thatis homologous to the plasma membrane Fet3/Ftr1 complex(Spizzo et al. 1997; Urbanowski and Piper 1999). Smf3 isa vacuolar iron transporter of the Nramp family that is tran-scriptionally activated by Aft2 in response to iron deficiency(Portnoy et al. 2002; Courel et al. 2005). Both of these ferrous-

specific transporters contribute to the mobilization of storediron when cells are iron deficient.

Other transporters

Iron-deficient yeast activate the expression of a low-specificitytransporter, Cot1, that mediates vacuolar accumulation ofzinc and cobalt (Conklin et al. 1992; MaDiarmid et al.2000; Shakoury-Elizeh et al. 2004). Because iron-deficientcells are sensitive to the toxic effects of other metals, acti-vation of this transporter suggests that the sequestration ofother metals in the vacuole is an adaptive response duringiron deficiency (Li and Kaplan 1998). Iron-deficient cellsalso activate the expression of Fre5, a metalloreductase lo-calized to mitochondrial membranes (Sickmann et al. 2003),and Mrs4, a protein of the mitochondrial carrier family that,along with Mrs3, is involved in the uptake of ferrous iron(and possibly copper) across the mitochondrial inner mem-brane (Foury and Roganti 2002; Muhlenhoff et al. 2003b;Froschauer et al. 2009). As the mitochondrial intermem-brane space is a relatively oxidizing environment, Fre5may serve to supply Mrs3 and Mrs4 with reduced iron foruptake. Iron deficiency also triggers the expression of Vht1,the high-affinity biotin transporter, and Bio5, a transporterof biotin biosynthetic intermediates (Phalip et al. 1999; Stolzet al. 1999; Belli et al. 2004; Shakoury-Elizeh et al. 2004).Although yeast are formally auxotrophic for biotin, theyexpress enzymes for the terminal three steps of the biosyn-thetic pathway and can synthesize biotin from intermedi-ates. The ultimate step in biotin synthesis is catalyzed byBio2, an Fe–S protein (Ugulava et al. 2001), and the entiresynthetic pathway is transcriptionally down-regulated dur-ing iron deficiency. This shift from biosynthesis to uptake ofbiotin during iron deficiency allows yeast to conserve iron,a pattern that is replicated in other Fe–S- and heme-dependentpathways.

Metabolic adaptation to iron deficiency

Budding yeast can survive in environments that contain verylow or very high concentrations of iron. Survival in low ironenvironments involves metabolic adaptations that reducethe cell’s dependence on iron-containing proteins. Metabolicadaptation to iron deficiency includes shifting from iron-de-pendent biosynthetic pathways to iron-independent path-ways. As mentioned above, iron-deficient yeast shut downthe iron-requiring biosynthetic pathway for biotin and up-regulate uptake systems for biotin and biotin precursors.Another example of the shift to iron-independent pathwaysis glutamate synthesis. Yeast express two genetically sepa-rate systems for the synthesis of glutamate. One requires theglutamate dehydrogenases Gdh1 or Gdh3. The other re-quires glutamine synthetase, Gln1, and glutamate synthase,Glt1, an iron-sulfur cluster enzyme. GLT1 is transcriptionallyshut off in iron-deficient yeast while GDH3 transcripts increase,thereby shifting glutamate synthesis to an iron-independentpathway (Shakoury-Elizeh et al. 2004). Similarly, yeast altercarbon source utilization according to the availability of iron.

Table 2 Specificity and kinetics of ARN Transporters

Transporter Siderophore substrates Km for transport (mM)

Arn1 Ferrichromesa 0.9Coprogen(Triacetylfusarinine C)b

Arn2/Taf1 Triacetylfusarinine C 1.6Arn3/Sit1 Ferrioxamine B 0.5

Ferrichromesa 2.3Coprogen(Triacetylfusarinine C)b

Arn4/Enb1 Enterobactin 1.9a Arn1 and Arn3 exhibit specificity for multiple members of the ferrichrome family ofsiderophores. Km determined for ferrichrome.

b Arn1 and Arn3 exhibit a trace amount of transport activity for triacetylfusarinine C.
































Growth on nonfermentable carbon sources is accompaniedby an expansion of mitochondria containing iron-rich respi-ratory complexes, and yeast cannot grow on media contain-ing nonfermentable carbon sources that are also low in iron.Yeast grown on glucose media metabolize glucose largelythrough fermentation, with only a small amount of respira-tion. When yeast are shifted to iron-poor media, levels oftranscripts involved in respiration fall and metabolite anal-ysis indicates increased flux through the glycolytic pathway.These studies suggest that iron-deficient yeast shut downrespiration, thereby allowing the cell to reuse the iron scav-enged from respiratory complexes for other metabolic purpo-ses (Shakoury-Elizeh et al. 2010). These metabolic adaptationsoccur through multiple mechanisms. Some are transcriptionaland depend on Aft1 and Aft2 or the heme-dependent ac-tivator Hap1 or Hap4. Some occur post-transcriptionallyand depend on the mRNA-destabilizing proteins Cth1 andCth2.

Under conditions of iron deficiency, Aft1 activates thetranscription of HMX1, which encodes the yeast heme oxy-genase (Protchenko and Philpott 2003). Hmx1 degradesheme, releasing the heme iron for other metabolic uses.Growth in iron-poor medium is associated with large reduc-tions in cellular heme levels. This loss of heme is due, inpart, to the activity of Hmx1, in part to less iron availabilityfor insertion into heme, and in part to lower expressionlevels of heme biosynthetic enzymes (Lesuisse et al. 2003).In addition to its role as an enzyme cofactor, heme is alsoa regulatory molecule that is required for the activation ofthe transcription factor Hap1 (Guarante 1992; Kwast et al.1998; Lee and Zhang 2009). Hap1 activates the transcrip-tion of many genes involved in respiration and aerobicgrowth. These genes are down-regulated in iron-deficientcells due, in part, to loss of heme-dependent activation ofHap1. Many proteins involved in the tricarboxylic acid cycleand respiration require heme and Fe–S cluster cofactors;thus, down-regulation of these pathways reduces expressionof proteins that may ultimately lack required cofactors andallows the limited iron that may have been used in thesepathways to be redirected to other pathways.

CTH2 and, to a lesser extent, CTH1 are Aft1 targets thatare actively transcribed under iron deficiency and are involvedin mediating the metabolic adaptation to iron deficiency (Puiget al. 2005, 2008). Cth1 and Cth2 are mRNA-binding proteinsthat recognize and bind to AU-rich sequences in the 39-UTR ofa number of transcripts that are down-regulated during irondeficiency. Binding of Cth1 or Cth2 to the AU-rich elementsleads to destabilization and degradation of the transcripts.Many of the transcripts that are putative targets of Cth1 orCth2 encode proteins that either contain iron cofactors orfunction in pathways with other iron-dependent enzymes.These pathways include cellular respiration, heme and Fe–Scluster biosynthesis, iron homeostasis, fatty acid and ergos-terol metabolism, and amino acid metabolism. Strains lack-ing both Cth1 and Cth2 exhibit slow growth on iron-poormedium, indicating that the reduced expression of the set of

Cth1 and Cth2 targets offers an adaptive advantage to iron-deficient yeast. Although the amount of mRNA degradationthat can be attributed to Cth1 or Cth2 is relatively small,usually twofold or less, the net effect represents a significant“iron savings” for the cell because of the relatively largenumber of genes targeted.

Cellular iron utilization

Cellular iron is used for the synthesis of heme and ISCs or itcan be directly coordinated by enzymes in the form ofmononuclear or diiron centers. In the cytosol, the monothiolglutaredoxins, Grx3 and Grx4, have roles in iron deliverythat go beyond the regulation of Aft1 (Ojeda et al. 2006;Pujol-Carrion et al. 2006). Grx3 can form a homodimer thatcoordinates a bridging 2Fe–2S cluster in vitro and Grx3/4that is affinity purified from yeast also contains bound iron(Li et al. 2009). Yeast strains lacking Grx3 and Grx4 exhibitdefects in heme and ISC synthesis as well as defects in themetallation and activity of ribonucleotide reductase, a cyto-solic diiron enzyme (Muhlenhoff et al. 2010). These obser-vations suggest that Grx3/4 is involved in the delivery ofiron to mitochondria, the cytosolic ISC assembly complex,and to at least one cytosolic diiron protein. Direct physicalinteractions between Grx3/4 and these iron recipients havenot yet been demonstrated.

Iron is imported from the cytosol into the mitochondrialmatrix for the synthesis of heme and Fe–S clusters. Themitochondrial carrier proteins Mrs3 and Mrs4 facilitate thetransport of iron across the mitochondrial inner membrane,but additional pathways of mitochondrial iron import exist,as strains deleted for both MRS3 and MRS4 can maintainheme and Fe–S cluster synthesis when grown in iron-richmedia (Foury and Roganti 2002; Muhlenhoff et al. 2003b).The mitochondrial pyrimidine exchanger Rim2, also amember of the mitochondrial carrier family, was identifiedin a screen for mutations synthetically lethal with amrs3Dmrs4D strain (Yoon et al. 2011). Rim2 contributesto mitochondrial iron utilization, but appears not to di-rectly facilitate the uptake of iron.

Heme biosynthesis is a highly conserved eight-steppathway with individual steps occurring in both the mito-chondria and cytosol (reviewed in Labbe-Bois and Labbe1989). The initial step of heme synthesis occurs in the mi-tochondrial matrix where glycine and succinyl-coA condenseto form d-aminolevulinic acid (ALA). ALA is exported to thecytosol, where the next five steps occur, then protoporphyri-nogen IX moves from the cytosol to the mitochondria, whereit is converted to protoporphyrin IX. The final step in hemesynthesis is the insertion of iron into protoporphyrin IX,which is catalyzed by ferrocheletase and occurs in the mito-chondrial matrix. Heme is then distributed to proteins locatedin the mitochondria, cytosol, and organelles throughout thecell. Proteins involved in the intracellular trafficking of hemehave not been identified.

ISC assembly is a highly conserved process in both pro-karyotes and eukaryotes (reviewed in Lill and Muhlenhoff















































2008). While protein complexes devoted to the assembly ofISCs are located in both the mitochondria and cytosol, prod-ucts of mitochondrial ISC synthesis are required for cytosolicISC assembly. The core ISC biosynthetic complex consists ofthe cysteine desulfurase Nfs1 (Kispal et al. 1999), whichsupplies sulfide, an accessory protein Isd11 (Adam et al.2006; Wiedemann et al. 2006), the yeast frataxin homologYfh1, which supplies iron (Chen et al. 2002; Gerber et al.2003; Muhlenhoff et al. 2003a; Ramazzotti et al. 2004) andallosterically activates Nfs1 (Tsai and Barondeau 2010),and Isu1 or Isu2, which serves as a scaffold for assembly(Muhlenhoff et al. 2003a). Additional components providereducing equivalents, and protein chaperones, along with amonothiol glutaredoxin, facilitate the transfer of nascentISCs to recipient proteins. Some product of the ISC assemblycomplex is exported to the cytosolic ISC assembly complex.This product has not been molecularly characterized, but itmust include sulfide, as there is no alternative source ofsulfide in the cytosol. The mitochondrial ABC transporterAtm1 was shown to be required for cytosolic ISC assembly,presumably by exporting a product of the mitochondrial ISCassembly complex (Kispal et al. 1999). However, a more re-cent study found that Atm1 was not involved in cytosolicFe–S cluster assembly in yeast (Bedekovics et al. 2011). Asulfhydryl oxidase, Erv1, located in the intermembranespace (Lange et al. 2001), and mitochondrial glutathioneare also required for export of the unknown product.

Assembly of ISCs in the cytosol requires a genetically andbiochemically distinct set of proteins from that of themitochondria (reviewed in Lill 2009). Cytosolic ISCs appearto be assembled in a two-step manner. The first step involvesthe transient assembly of a pair of bridging 4Fe–4S clusterson a scaffold composed of the P-loop NTPases Cfd1 andNbp35 arranged in a heterotetramer (Roy et al. 2003; Haus-mann et al. 2005; Netz et al. 2007). An electron transfercomplex consisting of the flavoprotein Tah18 and the Fe–Sprotein Dre2 are also required for this early step of ISCassembly (Zhang et al. 2008; Netz et al. 2010). Clustersare then transferred to a complex consisting of Nar1, similarto iron-only hydrogenases of bacteria, and Cia1, a WD40repeat protein (Balk et al. 2004, 2005). From this complexISCs are then transferred to recipient proteins in the cytosoland nucleus.

Zinc

Based on the detection of known zinc-binding domains inthe S. cerevisiae proteome, budding yeast are estimated tocontain �476 different zinc proteins (Andreini et al. 2006).Zinc can have a catalytic function, as in the active site of anenzyme, or a structural function, as in a zinc finger. The useof zinc as a catalytic cofactor is widespread. Of the zinc-dependent enzymes, more than half are hydrolases (Andreiniand Bertini 2011). Structural zinc-binding domains are verycommon in trancriptional regulators and represent approxi-mately one-third of all zinc proteins in eukaryotes. By far, the

most common structural zinc-binding domains are zinc fin-gers of the Cys4 or Cys2His2 type (Andreini et al. 2006).

Transcriptional regulation through Zap1

Zinc homeostasis is largely achieved through transcriptionalcontrol of gene expression (Eide 2009). Zap1 is the zinc-sensing transcription factor that controls the response tozinc deficiency in yeast (Zhao and Eide 1997). Under con-ditions of zinc deficiency, Zap1 activates the transcription of�80 genes, while also repressing the transcription of a smallnumber (Lyons et al. 2000; Yuan 2000; Higgins et al. 2003;De Nicola et al. 2007; Wu et al. 2008). Zap1 target genes arelargely involved in zinc homeostasis and in the metabolicadaptation to zinc deficiency (Figure 6). In zinc-replete cells,Zap1 is inactivated. The protein contains both a DNA-bind-ing domain and multiple transcriptional activation domains,all of which bind zinc (Figure 6A). The DNA binding domainis located in the carboxy-terminal third of the protein andconsists of five zinc fingers of the C2H2 type, each of which isrequired for full DNA binding activity (Bird et al. 2000a,b;Evans-Galea et al. 2003). The high-affinity, structural zinc-binding sites formed by the zinc finger motifs are occupiedby zinc ions whether cells are zinc deficient or zinc replete.However, in zinc-replete cells, Zap1 does not bind DNA,which is likely due to a post-translational modification thatinterferes with DNA binding (Frey et al. 2011). In zinc-deficient cells, Zap1 binds as a monomer to a palindromicconsensus DNA sequence, ACCTTNAAGGT, known as a zinc-responsive element (ZRE) (Zhao et al. 1998b). Considerablevariation from this sequence has been observed in functionalZap1 binding sites, but Zap1-regulated promoters typicallycontain one or a few recognizable ZREs. The ZAP1 geneitself contains a ZRE and is activated by Zap1, thereby in-creasing the amount of Zap1 protein during zinc deficiency.Some Zap1 targets are transcribed during mild zinc defi-ciency, while others are activated only when zinc deficiencybecomes severe. ZREs with the highest binding affinity forZap1 tend to appear in genes activated by mild iron defi-ciency, which suggests that Zap1 occupies lower-affinitysites only when the Zap1 levels rise, as they do in severezinc deficiency. The autoactivation of ZAP1 is thus a mecha-nism by which cells can achieve a graded response to differ-ing levels of zinc deficiency (Wu et al. 2008).

Zap1’s two activation domains, known as AD1 and AD2,function independently in zinc sensing and activation (Birdet al. 2000b). AD1 is part of a larger zinc-regulatory domainlocated in the amino-terminal half of the protein and it con-tains two clusters of potential zinc-binding residues at eitherend of the domain (Herbig et al. 2005). Zinc binding in thisdomain has been demonstrated in vitro, and mutation ofpotential zinc-binding residues results in constitutive ac-tivity of AD1. Thus, AD1 likely senses zinc through directbinding of the metal, which results in loss of transcrip-tional activation.

AD2 of Zap1 consists of two C2H2-type zinc fingers lo-cated in the middle of the protein. Neither of these zinc



































fingers is involved in DNA binding, but together they canconfer zinc-regulated transcriptional activation (Bird et al.2003). The zinc-binding sites within AD2 are of lower affin-ity than those of most C2H2 zinc fingers, as would be appro-priate for a zinc sensor. In vitro studies indicate that zincbinding by the two zinc fingers promotes a stable interactionbetween the fingers (Wang et al. 2006). Presumably thisconformation of AD2 prevents the recruitment of transcrip-tional coactivators.

Most Zap1 target genes can be fully activated by a mu-tated Zap1 containing only AD1 (Frey and Eide 2011). Al-though some genes respond to both AD1 and AD2, onlya few genes require AD2 for full induction. AD2 appears tobe more important for Zap1 activity when zinc deficiency iscombined with other environmental stresses.

Transporters regulated by Zap1

The primary response to zinc deficiency is the Zap1-medi-ated expression of zinc transporters that serve to transferzinc from the extracellular milieu and the vacuolar lumento the cytosol. Zrt1 and Zrt2 are Zn-specific transporters ofthe ZIP family, a large, ancient family of zinc transporterswith representatives in most species of prokaryotes andeukaryotes. Zrt1 provides essentially all of the high-affinity

zinc uptake activity (Zhao and Eide 1996a); deletion ofZRT1 results in loss of high-affinity zinc uptake and slowgrowth in zinc-limited medium. Zrt2 exhibits a moderatedegree of sequence identity (44%) with Zrt1 and functionsas a low-affinity zinc transporter (Zhao and Eide 1996b).Strains deleted for both ZRT1 and ZRT2 can grow on mediacontaining moderately reduced levels of zinc, indicating thepresence of additional low-affinity zinc-uptake systems. Fet4is a low-affinity, broad-specificity metal transporter, the ex-pression of which is induced by Zap1 under zinc-limitingconditions (Waters and Eide 2002). Expression of Fet4 ina zrt1Dzrt2D strain was associated with improved growth inlow zinc medium. Yeast grown in a very zinc-restricted en-vironment depend on Zrt1 for uptake, while Zrt2 and Fet4provide uptake activity when zinc levels are normal ormildly reduced.

Surprisingly, Zrt2 is repressed by Zap1 under conditionsof severe zinc deficiency (Bird et al. 2004). This repression ismediated through a lower-affinity ZRE located upstream ofZRT2. Transcriptional activation of ZRT2 is mediated byZap1 binding to high-affinity ZREs located upstream of theTATA box. Severe zinc deficiency increases the levels ofZap1 through autoactivation at the ZAP1 promoter. In thesetting of these higher levels, Zap1 also binds to a lower

Figure 6 Zinc homeostasis. (A) Schematic of Zap1.Regions representing zinc fingers are filled andnumbered. Activation domains (AD) in black areembedded within zinc-regulatory domains (ZRD)in orange. Reproduced with permission (Frey andEide 2011). (B) Transcriptional response to zinc de-ficiency. Proteins involved in zinc homeostasis areon the left, proteins involved in metabolic adapta-tion to zinc deficiency on the right. Yellow spheresindicate up-regulation, blue spheres indicate down-regulation, gray spheres indicate no zinc regula-tion. Subcellular localizations are shown. PM,plasma membrane; MITO, mitochondria; ORF,open reading frame. Reproduced with permission(Eide 2009).






























affinity ZRE located near the transcription start site of ZRT2,thereby inhibiting transcription. Thus, under conditionswhere zinc levels are too low for Zrt2 to function, expressionis repressed.

As is the case with iron, yeast grown in zinc-replete medastore excess zinc in the vacuole, both to prevent toxicity andto meet metabolic need during zinc deficiency. Zinc storageis mediated by two unrelated transporters located on thevacuolar membrane, Cot1 and Zrc1 (Kamizono et al. 1989;MacDiarmid et al. 2000). Under conditions of zinc defi-ciency, vacuolar zinc stores are mobilized through the activ-ity of Zrt3, a third yeast member of the ZIP family oftransporters that is expressed on vacuolar membranes.Strains deleted for ZRT3 exhibit high levels of intracelluarzinc, which is not effectively mobilized for use during zincdeficiency (MacDiarmid et al. 2000).

Although Zrc1 is involved in zinc storage when yeast aregrown in zinc-replete media, somewhat counterintuitively,this transporter is also induced by Zap1 during zinc defi-ciency. An explanation for this expression pattern came fromstudies examining the response of zinc-deficient yeast toa sudden increase in extracellular zinc (MacDiarmid et al.2003). Because zinc-deficient yeast express uptake systemsat high levels, acute exposure of these yeast to high concen-trations of zinc results in a sudden influx of zinc to thecytosol. The presence of Zrc1 on the vacuole exerts a pro-tective effect for the yeast exposed to this influx of zinc, aszinc-deficient strains lacking ZRC1 exhibited increased sen-sitivity to zinc.

Eukaryotic cells have a requirement for zinc within thelumen of the ER. This requirement is due to the presence ofseveral zinc metalloenzymes (metalloproteases, proteinchaperones, and a transferase of glycosylphosphatidylinosi-tol anchor synthesis) in the lumen of the ER (Ellis et al.2004, 2005). Zinc is transferred from the cytosol to the ERlumen by the activity of a heterodimeric transporter com-posed of Msc2 and Zrg17, both of which are members of thecation diffusion facilitator family of efflux pumps. Althoughboth peptide components of this pump are required forzinc transport, only one, Zrg17, is zinc-regulated directlythrough Zap1 (Wu et al. 2011).

Adaptation to zinc deficiency

Zap1 also controls the expression of several genes involvedin the adaptation to zinc deficiency. One of the most highlyzinc-regulated genes is ADH1 (Lyons et al. 2000), whichencodes a very abundant, zinc-binding alcohol dehydroge-nase (Leskovac et al. 2002). Adh1 is repressed by Zap1 inzinc-limited cells. This repression is mediated by the Zap1-dependent transcription of a noncoding RNA, termed ZRR1,from a site just upstream of the ADH1 promoter (Bird et al.2006). Transcription of ZRR1 leads to the displacement ofRap1, the transcriptional activator for ADH1, from the pro-moter, thus inhibiting ADH1 transcription. A mitochondriallytargeted paralog of Adh1, Adh3, is regulated in a similarmanner. Zinc deficiency also induces the Zap1-mediated

transcription of ADH4, which encodes another zinc-depen-dent alcohol dehydrogenase (Drewke and Ciriacy 1988).The adaptive advantage of the shift from Adh1 and Adh3to Adh4 during zinc limitation is not entirely clear, but likelyinvolves the overall conservation of zinc. Structural studiesof Adh1 indicate that it binds two zinc ions per monomer.Adh4 is closely related to the iron-dependent alcohol dehy-drogenase of Zymomonas mobilis, which binds a single metalion per monomer (Neale et al. 1986).

Zinc deficiency is accompanied by significant changes inthe lipid composition of the cell, namely a large increase inphophatidylinositol (PI) and a large decrease in phosphati-dylethanolamine (Iwanyshyn et al. 2004). The major phos-pholipids of the cell are primarily synthesized in a stepwisemanner from the precursor cytodine diphosphate (CDP)–diacylglycerol (Henry et al. 2012). A secondary pathway,termed the Kennedy pathway, can also supply phospholipidsfrom the precursors ethanolamine and choline. Under zinclimitation, Zap1 increases the transcription of PIS1, the PIsynthase, PAH1, the phosphatidate (PA) phosphatase, andEKI1 and CKI1, two genes of the Kennedy pathway. In-creased PI synthase leads to greater synthesis of PI, andincreased PA phosphatase leads to depletion of PA levels(Soto-Cardalda et al. 2012). Reduced PA triggers the releaseof the transcriptional repressor Opi1, which results in down-regulation of multiple genes of the CDP–diacylglycerol path-way. Thus zinc deficiency leads to a shift in phospholipidsynthesis to the secondary Kennedy pathway and to a majorchange in the phospholipid composition of the cell. Theadaptive value of these changes is not known, but one couldspeculate that the activity or trafficking of the zinc transporterscould be affected. Alternatively, reliance on the Kennedy path-way would reduce the requirement for S-adenosyl methio-nine (possibly limiting in zinc deficiency, see below), whichis consumed in the synthesis of phospholipids via the CDP–diacylglycerol pathway.

Zinc deficiency in yeast is also associated with a decreasein sulfate assimilation and reduced levels of free intracellu-lar cysteine and methionine, which can again be traced totranscriptional events under the control of Zap1 (Wu et al.2009). Diminished sulfate assimilation is due to transcrip-tional repression of MET3, MET14, and MET16, the threegenes required for the stepwise reduction of intracellularsulfate to sulfite, which is used in the synthesis of sulfur-containing amino acids. These genes are under the controlof Met4, a transcriptional activator that is, in turn, post-translationally regulated through ubiquitin-mediated degra-dation (Rouillon et al. 2000; Smothers et al. 2000). Met30,an F-box protein and component of the SCF(Met30) ubiq-uitin ligase complex, targets Met4 for degradation. In zinc-deficient cells, Zap1 activates the transcription of MET30,which leads to the degradation of Met4 and reduced expres-sion of genes of sulfate assimilation. Permeases for the up-take of S-adenosyl methionine (Sam3) and cysteine andmethionine (Mup1) are also transcriptionally activated byZap1. Although the reduced availability of cysteine and






















































methionine that results from these regulatory changeswould appear to be maladaptive, this regulatory patternpartially protects cells from the oxidative stress associatedwith zinc deficiency. The source of the increased oxidativestress that occurs with zinc deficiency is not known, but cellsgrown in zinc-limited medium exhibit increased sensitivityto exogenous H2O2 and produce increased amounts of re-active oxygen species (Wu et al. 2007, 2009). The relation-ship between zinc deficiency, sulfate assimilation, andoxidative stress may be traced to intracellular pools ofNADPH. Sulfate assimilation involves the consumption ofrelatively large amounts of reducing equivalents in the formof NADPH. Zinc deficiency triggers the Zap1-mediated tran-scription of TSA1, which encodes the major peroxiredoxin ofyeast and protects cells against oxidative damage. Thedown-regulation of sulfate assimilation in zinc deficiencywould make more NADPH available for the regenerationof reduced peroxiredoxin and glutathione in the setting ofoxidative stress (Eide 2009).

Copper

Transcriptional regulation through Mac1 and Ace1

Although copper ions and copper proteins are present inyeast cells at far lower levels than iron or zinc, copperproteins play a role in multiple critical cellular processes.Similar to iron, copper ions can occupy multiple valencestates in cells and can thus participate in redox reactionsthat generate toxic reactive oxygen species. Similar to zinc,copper ions have an affinity for thiolate ligands and can thuscompete with zinc or iron-sulfur clusters for cysteine-richmetal binding sites. Yeast cells therefore tightly regulatecopper uptake and utilization. This regulation is largelyaccomplished at the level of transcription by a pair ofreciprocally regulated, copper-sensing transcriptional acti-vators, Mac1 and Ace1 (also called Cup2) (Figure 7).

Mac1 is a constitutively expressed transcriptional activa-tor that is stably localized to the nucleus in both copper-deficient and copper-replete cells (Jungmann et al. 1993;Graden and Winge 1997; Jensen and Winge 1998; Serpeet al. 1999; Keller et al. 2005). Under conditions of copperdeficiency, Mac1 binds to the consensus sequence TTTGC(T/G)C(A/G) located in the promoters of the Mac1-regulatedgenes (Labbe et al. 1997; Yamaguchi-Iwai et al. 1997).These promoter elements are present in multiple copies aseither direct or inverted repeats, and Mac1 binds as a dimerto these cis-acting elements (Joshi et al. 1999). When in-tracellular copper levels rise, Mac1 becomes transcription-ally inactive and dissociates from promoter elements.Structurally, Mac1 contains an amino-terminal DNA bindingdomain and a carboxy-terminal activation domain that alsocontains two cysteine-rich, copper-binding motifs (Jensenet al. 1998; Brown et al. 2002). These two motifs can bindup to eight copper ions as a copper-thiolate cluster and arethought to function as direct copper sensors. In the presence

of copper, Mac1 inactivation is accomplished through anintramolecular interaction between the amino-terminalDNA binding domain and the first of the two cysteine-richmotifs, which results in the loss of both DNA binding andtransactivation (Graden and Winge 1997; Labbe et al. 1997;Jensen and Winge 1998). Copper binding in the secondcysteine-rich motif also plays a role in the loss of transacti-vation (Keller et al. 2005). Mac1 is phosphorylated in vivoand treatment with phosphatase disrupts DNA bindingin vitro (Heredia et al. 2001). The copper-dependent regu-lation of Mac1 suggests that cells contain a nuclear pool ofcopper that can bind to and inactivate Mac1. The molecularform of this copper pool and whether it is coordinated bya copper-binding metallochaperone is not known.

Yeast are protected from the toxic effects of excess copperthrough the activity of Ace1 (Thiele 1988). When cells areexposed to excess copper, this transcription factor activatesa group of genes involved in the detoxification of copper.Ace1 is also thought to be a direct sensor of nuclear copperlevels and has some structural features similar to those ofMac1. Ace1 contains an amino-terminal DNA-binding domain

Figure 7 Copper homeostasis. Proteins involved in the regulation, up-take, distribution, and utilization of copper. Subcellular localizations areindicated. Reproduced with permission (Nevitt et al. 2012).





















that is also the site of regulatory copper binding (Buchmanet al. 1989; Huibregtse et al. 1989; Szczypka and Thiele1989). Both Mac1 and Ace1 contain a single zinc-bindingdomain that is involved in DNA minor groove interactions(Thorvaldsen et al. 1994; Farrell et al. 1996). Ace1 containsfour pairs of cysteine residues that bind four atoms of copperin a polycopper cluster that is structurally similar to those ofMac1 (Dameron et al. 1991; Graden et al. 1996; Brown et al.2002). Copper-binding by Ace1 induces a conformationalchange in the protein that allows it to bind to and activatetranscription at specific copper regulatory sites with the se-quence TTX2GCTG (Huibregtse et al. 1989; Evans et al.1990; Pachkov et al. 2007). Ace1 binds DNA as a monomerand is present in the nucleus at both high and low copperlevels. Affinity measurements for the copper bindingdomains of both Ace1 and Mac1 indicate that Mac1 hasa higher affinity for copper (Kd = 9.7 · 10220) than doesAce1 (Kd = 4.7 · 10218). These extremely high bindingaffinities would indicate that the copper binding sites ofboth sensors are fully occupied at concentrations equatingto less than a single free copper ion in the cell. However,these measurements are obtained under equilibrium condi-tions in which the metal fully dissociates from the proteinand is fully hydrated in aqueous solution. In living cells,metal ions are surrounded by metal buffers, consisting ofsmall molecules and proteins that constitute labile ligands.In this situation, metal exhange between ligands of thebuffer and ligands of the sensors is likely to occur with muchmore labile kinetics. These measured affinities should beregarded as reference values, which suggest that, as intra-cellular copper levels rise, Mac1 is inactivated by copperbinding at concentrations below those that activate Ace1by copper binding (Wegner et al. 2011) and well belowone fully hydrated (“free”) copper ion per cell.

Copper transport systems

Similar to iron, extracellular copper is largely present in theoxidized state, Cu(II), and must undergo reduction to Cu(I)before uptake. Under conditions of copper scarcity, Mac1activates the transcription of two members of the FRE familyof metalloreductases, FRE1 and FRE7 (Yamaguchi-Iwaiet al. 1997; Martins et al. 1998; Georgatsou and Alexandraki1999). Fre1 catalyzes the majority of Cu(II) reduction at theplasma membrane while Fre2 catalyzes a lesser amount(Dancis et al. 1992; Georgatsou and Alexandraki 1994;Georgatsou et al. 1997). Overexpression studies of Fre7 sug-gest it also functions as a metalloreductase on the plasmamembrane (Rees and Thiele 2007). Mac1 also activates thetranscription of three high-affinity copper transporters. Twoof these, Ctr1 and Ctr3, are expressed on the plasma mem-brane and mediate the uptake of reduced copper (Danciset al. 1994a,b; Knight et al. 1996). A third Ctr paralog,Ctr2, is expressed on the vacuolar membrane and, alongwith Fre6, mediates the reduction and transfer of copperfrom the lumen of the vacuole to the cytosol (Portnoyet al. 2001; Rees and Thiele 2007). Ctr-type transporters

contain extramembranous copper-binding domains at boththe amino and carboxy termini. They also contain threecentral membrane-spanning domains, with copper-bindingmotifs in the second transmembrane domain (Puig et al.2002; Xiao et al. 2004). Structural studies of human Ctr1indicate that the functional unit is a trimer, with an exter-nally oriented amino terminus and an internally orientedcarboxy terminus that cap a central, funnel-shaped pore(Aller and Unger 2006; De Feo et al. 2009). Ctr1 has beenproposed to function by a series of sequential copper ex-change reactions between the copper-binding sites that arecoupled to sequential changes in the conformation of thetransporter. In copper-replete media, high-affinity coppertransport is shut off, Ctr1 is degraded (Ooi et al. 1996),and copper uptake is mediated by low-affinity, broad-speci-ficity transporters, such as Fet4 and Smf1 (Cohen et al.2000; Portnoy et al. 2001). Mac1 also activates the tran-scription of IRC7, a beta-lyase involved in the productionof thiols (Roncoroni et al. 2011). Its role in the responseto copper deficiency is not yet clear.

Intracellular copper is present at exceedingly low con-centrations (Rae et al. 1999), and yeast rely on cytosoliccopper-binding proteins called copper chaperones for thetransfer of copper from import proteins (such as Ctr1) toenzymes (such as Cu,Zn superoxide dismutase, Sod1, andthe copper exporter, Ccc1). It is unclear whether copperchaperones pick up their metal ligands directly or indirectlyfrom copper transporters, although rapid exchange of cop-per ligands between copper-binding domains of transportersand chaperones has been demonstrated in vitro (Xiao et al.2004). Copper chaperones deliver copper to target enzymesvia a metal-enhanced, protein–protein interaction that facil-itates a ligand exchange reaction for the copper ion. Ccs1 isthe chaperone for Sod1 (Culotta et al. 1997) and Atx1 isrequired for the delivery of copper to Ccc2 (Lin et al.1997; Pufahl et al. 1997). Ccc2 is a P-type ATPase that cou-ples the hydrolysis of ATP to the transport of Cu(I) from thecytosol to the lumen of post-Golgi vesicles (Yuan et al.1995). Luminal copper binds to and activates the multicop-per oxidase Fet3 (Askwith et al. 1994), which is required forhigh-affinity iron uptake. Thus, iron uptake is a copper-dependent process in yeast.

Copper detoxification and utilization

When extracellular copper concentrations rise, yeast areprotected from the influx of copper to the cytosol by theexpression of copper-binding proteins called metallothio-neins. Ace1 directs the transcription of two metallothioneingenes, CUP1 (often present in multiple copies) and CRS5,which encode cysteine-rich proteins that bind and detoxifycytosolic copper (Butt et al. 1984; Winge et al. 1985; Culottaet al. 1994). Ace1 also activates the transcription of Sod1under conditions of copper excess (Gralla et al. 1991). Sod1is expressed at basal levels in copper-deficient cells. In-creased expression under conditions of copper excess mayoffer cells additional protection against oxidative damage in















































the presence of this redox-active metal. Sod1 and its chap-erone, Ccs1, are primarily located in the cytosol, but a smallamount of both are also located in the mitochondrial inter-membrane space (Sturtz et al. 2001) and in the nucleus(Wood and Thiele 2009). Surprisingly, both the nuclear lo-calization and enzymatic activity of Sod1 are required forthe Mac1 response to low copper.

Mitochondrial copper utilization

Copper plays an essential role in respiration through itsactivation of cytochrome c oxidase (CcO). CcO is a multi-subunit enzyme (also known as complex IV), located in themitochondrial inner membrane, which catalyzes the finalstep of the electron transport chain of cellular respiration.Cox1 (subunit 1) contains a single copper ion bound ina heterobimetallic active site (termed the CuB site) andCox2 (subunit 2) contains two copper ions bound in a dinu-clear center termed the CuA site. In addition to the copperions, CcO contains two heme a cofactors plus zinc, magne-sium, and sodium ions. Assembly of functional CcO is a com-plex process and requires the assistance of copper chaperonesspecific for CcO, namely Cox17, Cox11, and Sco1 (Carr andWinge 2003; Robinson and Winge 2010). Mitochondrialcopper is largely present in the matrix associated witha low molecular weight ligand and must be transferred tothe intermembrane space for incorporation into CcO(Cobine et al. 2004). Cox17 is a soluble protein with local-ization to both the cytosol and the mitochondrial intermem-brane space (Beers et al. 1997). Yeast lacking functionalCox17 exhibit respiratory and CcO deficiency that can berescued with high concentrations of copper (Glerum et al.1996a). Cox17 binds three copper ions per monomer of pro-tein and likely binds copper after it translocates to the in-termembrane space, where it can specifically interact withand donate copper to Sco1 and Cox11 (Horng et al. 2004).

Sco1 is a mitochondrial copper-binding protein that isrequired for the formation of the CuA site in Cox2. Sco1was initially linked to Cox17 when it was isolated asa high-copy suppressor of the respiratory defect of a Cox17mutant (Glerum et al. 1996b). Cells lacking Sco1 exhibit re-spiratory deficiency and defects in CcO biogenesis (Schulzeand Rodel 1988; Krummeck and Rodel 1990). Sco1 is anchoredin the mitochondrial inner membrane by a single amino-terminal transmembrane helix. The carboxy-terminal domainextends into the intermembrane space and binds a single Cu(I) ion (Rentzsch et al. 1999; Nittis et al. 2001). Interactionsbetween Sco1 and Cox2 are likely mediated by electrostaticinteractions on a surface distinct from the copper-binding site(Rigby et al. 2008).

Formation of the CuB site in Cox1 requires Cox11, an-other copper-binding protein anchored in the mitochondrialinner membrane. Although yeast lacking Cox11 also exhibitrespiratory deficiency and reduced levels of CcO (Tzagoloffet al. 1990), the specific function of Cox11 came from stud-ies in the aerobic bacterium, Rhodobacter sphaeroides (Hiseret al. 2000). CcO isolated from strains lacking Cox11 contained

both heme centers and the CuA site, but lacked the spectro-scopic evidence for a CuB site. The CuB site of CcO is buriedin the inner membrane in the mature enzyme, and copper isthought to be inserted into Cox1 via a channel-like cavitythat is transiently occupied by Cu-bound Cox11 (Khalimonchuket al. 2007).

Manganese

Manganese is a redox-active metal that can occupy multiplevalence states in biological systems. Similar to iron, Mn(II)and Mn(III) [and to a lesser extent, Mn(IV)] are frequentlyfound in biological systems. In vitro, Mn(II) can substitutefor a number of divalent cations (such as Mg2+ and Ca2+) inthe activation of enzymes; thus the endogenous, in vivometal ligand for a protein is not always clear. It may differin different species or depend on the relative concentrationsof cations in the cellular compartment where the enzyme islocated. Manganese-dependent enzymes include oxidore-ductases, dehydrogenases, transferases, and hydrolases(Crowley et al. 2000). In yeast, particular attention has beengiven to the manganese-containing superoxide dismutase(Sod2) of the mitochondria and the manganese-dependentsugar transferases of the secretory pathway. Similar to othernutrient metals, uptake of manganese is homeostaticallyregulated. Unlike other nutrient metals, the regulation ofmanganese uptake appears to occur exclusively throughpost-translational mechanisms.

Uptake of manganese through Smf1 and Smf2

As is the case with other nutrient metals, under conditions ofmanganese deficiency, yeast express high-affinity manga-nese transporters (Figure 8). Two homologous transportersof the Nramp family, Smf1 and Smf2, account for the ma-jority of the high-affinity transport of manganese into thecytosol (Supek et al. 1996; Liu et al. 1997; Cohen et al.2000). Both transporters exhibit proton-coupled metaltransport when expressed in xenopus oocytes (Chen et al.1999), and, similar to other members of the Nramp family,can transport Mn(II), Fe(II), Cu(II), and Zn(II), as well asother divalent metal ions (Chen et al. 1999; Cohen et al.2000). In cells grown in media containing sufficient concen-trations of manganese, both Smf1 and Smf2 have relativelyshort half-lives and the transporters are sorted directly fromthe Golgi compartment to the vacuole for degradation (Liuet al. 1997; Liu and Culotta 1999; Portnoy et al. 2000,2002). When manganese levels are low, both proteins be-come relatively more stable, and Smf1 is predominatelyexpressed on the plasma membrane while Smf2 is predom-inately localized to endomembranes that likely correspondto the Golgi or endosomal compartment. Deletion of eitherSMF1 or SMF2 leads to slow growth in metal-depleted me-dium (Supek et al. 1996; Cohen et al. 2000), but the relativecontributions of these transporters to cellular manganesehomeostasis are not equal. In rich medium, a strain lackingSMF1 exhibits nearly wild-type levels of intracellular manganese,










































while a strain lacking SMF2 exhibits a marked reduction inintracellular manganese as well as a loss of Mn-SOD activityand defects in glycosylation (Luk and Culotta 2001). Ele-vated intracellular levels of manganese can protect cellsfrom oxidative stress, and Smf1 is required for the intracel-lular accumulation of manganese when high levels of exog-enous manganese are used to rescue the oxidative sensitivityof a sod1D strain (Reddi et al. 2009b).

The phenotypic differences between the smf1D and thesmf2D strains may be due to the distinct subcellular local-izations of Smf1 and Smf2. Overall manganese uptake inyeast is relatively low compared to iron or zinc uptake,and only a small number of transporters on the cell surfacewould be required to account for the measured levels ofuptake (Stimpson et al. 2006). In contrast, levels of manga-nese within the lumen of the secretory pathway are rela-tively high due to the activity of the Golgi-localized, Ca2+/Mn2+ efflux pump, Pmr1 (see below) (Lapinskas et al. 1995;Durr et al. 1998). Recovery of intraluminal manganese bySmf2 in the late secretory pathway may represent the ma-jority of manganese influx to the cytosol.

The activities of Smf1 and Smf2 are controlled post-translationally by both intracellular trafficking and regu-lated degradation pathways. Yeast grown in manganese-suf-ficient medium rapidly degrade newly synthesized Smf1 andSmf2. This degradation occurs by sorting Smfs from the lateGolgi compartment into the multivesicular body of the latesecretory pathway with subsequent degradation in the vac-uole. Similar to other transporters, degradation of Smf1 andSmf2 is dependent on ubiquitination by the ubiquitin ligaseRsp5 (Sullivan et al. 2007), but also requires several adaptorproteins that are involved in recruiting Rsp5 to the trans-porters. These include Bsd2 and Tre1/Tre2 (Liu et al. 1997;Liu and Culotta 1999; Stimpson et al. 2006). In yeast grownin manganese-depleted medium, Smf1 and Smf2 escape rec-ognition by Bsd2 and Tre1/Tre2 and are stably expressed onthe plasma membrane and endosomes, respectively. Mecha-nistically, it is not clear how Smf1 and Smf2 escape detection

or what controls their sorting to different membranes. Intra-luminal manganese appears to be required for recognitionby Bsd2 and Tre1/Tre2, however, because a pmr1D strainthat fails to pump manganese into the lumen of Golgivesicles also exhibits impaired degradation of Smf1 in man-ganese-sufficient medium (Jensen et al. 2009). These obser-vations have led to a model in which it is the conformationalchanges associated with transport activity that lead to re-cruitment of Rsp5 and subsequent degradation (Stimpsonet al. 2006). Under conditions of manganese toxicity, Smf1degradation becomes independent of Bsd2 and Tre1/2 andis only partially dependent on Rsp5. Elevated cytosolic levelsof manganese trigger this pathway of degradation and thepresence of manganese in the Golgi lumen is no longer re-quired (Jensen et al. 2009).

Phosphate-dependent manganese transport

When yeast are grown in medium containing elevated levelsof manganese, uptake can occur through the high-affinityphosphate transporter, Pho84. The transport substrate ofPho84 is a complex of phosphate anion and divalent metalcation (Fristedt et al. 1999). In standard laboratory media,magnesium concentrations are relatively high, and the sub-strate for Pho84 is likely a MgHPO4 complex. However,when extracellular manganese levels are high, Pho84 canalso transport MnHPO4, and strains lacking PHO84 exhibitresistance to manganese toxicity (Jensen et al. 2003). Pho84is strongly regulated at the transcriptional level by intracel-lular phosphate (Johnston and Carlson 1992), but no evi-dence exists for regulation by manganese. Thus, uptake ofmanganese through Pho84 can significantly contribute tomanganese toxicity.

Transport of manganese into the secretory pathway

Yeast require substantial concentrations of manganese inGolgi compartments to correctly mannosylate proteins of thesecretory pathway that have been modified with O- andN-glucosylation in the endoplasmic reticulum. Yeast mannosyl

Figure 8 Manganese homeostasis. Proteins involvedin the transport and utilization of manganese areshown. Subcellular localization of proteins observedunder manganese-deficient conditions is depicted.














































transferases, similar to the oligosaccharide transferases ofbacteria, plants, and animals, are dependent on bound man-ganese cofactors for activity. Full activation of these enzymesin vitro requires manganese at.1 mM concentrations (Crowleyet al. 2000). It is inferred from these observations that theintraluminal concentration of manganese must be substantialand similar to the concentration of Ca2+ (1–2 mM) (Pezzatiet al. 1997). A single transporter in yeast, Pmr1, catalyzes theATP-dependent transfer of both Ca2+ and Mn2+ into the lu-men of the Golgi (Rudolph et al. 1989; Antebi and Fink 1992;Lapinskas et al. 1995; Durr et al. 1998) (see also DivalentCations: Ca2+ and Mg2+, Ca2+ sequestration in intracellularcompartments, above). Strains lacking Pmr1 exhibit multiplephenotypes attributable to defects in Golgi and endoplasmicreticulum function. While the protein sorting defects arelargely attributable to luminal Ca2+ deficiency, the glycosyl-ation defects are rescued only by manganese supplementa-tion. Strains lacking Pmr1 also exhibit increased intracellularmanganese accumulation.

Yeast can also store manganese in the vacuolar lumen viathe efflux pump Ccc1. Although primarily an iron trans-porter, overexpression of Ccc1 is associated with increasedvacuolar manganese and can partially suppress the Mn2+

hypersensitivity of a pmr1D strain (Lapinskas et al. 1996;Li et al. 2001b).

Unanswered Questions

While considerable progress has been made in dissecting themolecular details of ion homeostasis in S. cerevisiae, severalareas remain mysterious. Several activities that mediatetransport of the alkali metal ions, Na+ and K+, as well asthe cation Ca2+, have been described using electrophysiol-ogy, but remain to be identified at the molecular level. Sim-ilarly, the regulation of transporters, including the Trk1/2potassium transporters and the Ca2+ channel made up ofMid1, Cch1, and Ecm7, must be characterized at the molec-ular level. Many components of signal transduction path-ways that are regulated by these ions must be elucidated,and methods for directly identifying and analyzing changesin intracellular ions, particularly Ca2+, within yeast cellsshould be further developed, as these tools have been in-valuable for studying Ca2+-dependent signaling in mamma-lian cells. Recent advancements in the design and use ofgenetically encoded calcium sensors offer hope that suchmethods may be successfully applied to the study of Ca2+-dependent signaling in yeast (Mehta and Zhang 2011).

Metal trafficking within the cell is another significantarea that needs exploration. All of the nutrient metals arerequired for activation of mitochondrial proteins, but onlysome of the transporters that control the uptake and effluxof iron in that organelle have been identified. The regulatorymechanisms that control the amount of metal taken up bymitochondria also remain to be identified. Copper, andpossibly other metals, is present in mitochondria as a complexwith a low-molecular-weight ligand that is uncharacterized.

Whether mitochondrial metal chaperones in addition to theidentified copper chaperones are required for protein metal-lation there has not been determined.

Heme and some product of the mitochondrial ISCmachinery must exit the mitochondria and carrier proteinsand/or molecules are likely involved, but how this occurs isunknown (in the case of heme) or only beginning to beunderstood (in the case of ISCs). With the exception of Atx1and Ccs1, the existence or identity of cytosolic metal chap-erones for the metallation of iron, zinc, or manganese pro-teins is unknown. ISCs are very likely involved in signalingcellular iron status to the Aft1 and Aft2 regulators, but themolecular details of regulation have not been described.Copper must access the nucleus in some form to bind tothe Mac1 and Ace1 regulators, but how this occurs is notknown. The number of proteins known to comprise the yeastmetalloproteome continues to expand, as does our appreci-ation of the importance of nutrient metals in the function ofcells. Studies of cat regulation in S. cerevisiae have alreadyprovided many insights into these processes in larger eukar-yotes, and will likely continue to uncover conserved compo-nents of these fundamental cellular networks.

Acknowledgments

The authors would like to thank the anonymous reviewersfor their helpful comments. M.S.C. is funded the NationalInstitutes of Health (NIH) grant R01GM48729 from theNational Institute of General Medicine. C.C.P. is supportedby the NIH Intramural Research Program of the NationalInstitue of Diabetes and Digestive and Kidney Disease.

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Communicating editor: J. Thorner


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