Genetic Analysis of Calmodulin and Its Targets in Saccharomyces cerevisiae

19 Oct 2001 17:20 AR ar144p-22.tex ar144p-22.sgm ARv2(2001/05/10)P1: GJB

Annu. Rev. Genet. 2001. 35:647–72Copyright c© 2001 by Annual Reviews. All rights reserved

GENETIC ANALYSIS OF CALMODULIN AND ITS

TARGETS IN SACCHAROMYCES CEREVISIAE

Martha S. CyertDepartment of Biological Sciences, Stanford University, Stanford,California 94305-5020; e-mail: [email protected]

Key Words calcium, signal transduction, calcineurin, myosin, spindle pole body

■ Abstract Calmodulin, a small, ubiquitous Ca2+-binding protein, regulates a widevariety of proteins and processes in all eukaryotes.CMD1, the single gene encodingcalmodulin inS. cerevisiae, is essential, and this review discusses studies that identifiedmany of calmodulin’s physiological targets and their functions in yeast cells. Calmod-ulin performs essential roles in mitosis, through its regulation of Nuf1p/Spc110p, acomponent of the spindle pole body, and in bud growth, by binding Myo2p, an un-conventional class V myosin required for polarized secretion. Surprisingly, mutantcalmodulins that fail to bind Ca2+ can perform these essential functions. Calmod-ulin is also required for endocytosis in yeast and participates in Ca2+-dependent,stress-activated signaling pathways through its regulation of a protein phosphatase,calcineurin, and the protein kinases, Cmk1p and Cmk2p. Thus, calmodulin performsimportant physiological functions in yeast cells in both its Ca2+-bound and Ca2+-freeform.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648Calmodulin Structure and Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648Identification and Characterization ofCalmodulin fromS. cerevisiae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650

GENETIC ANALYSIS OF CALMODULIN FUNCTION . . . . . . . . . . . . . . . . . . . . . 650Analysis of Conditional Mutants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650Analysis of Ca2+-Binding–Defective Mutants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651

TARGETS OF CALMODULIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652Ca2+-Independent Targets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652Ca2+-Dependent Targets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 656Other Potential Targets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661

PERSPECTIVES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662

0066-4197/01/1215-0647$14.00 647

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INTRODUCTION

Calmodulin Structure and Function

Regulated changes in the concentration of cytosolic Ca2+ control such diversebiological processes as muscle contraction, fertilization, secretion, cell prolifer-ation, and apoptosis. Calmodulin, a small Ca2+-binding protein, is found in alleukaryotic organisms and is highly conserved. Calmodulin serves as a major in-tracellular Ca2+ receptor and mediates many of the effects of this ion. At restinglevels of Ca2+, calmodulin exists in the Ca2+-free, or apo-calmodulin form. Inresponse to a Ca2+ signal, calmodulin binds Ca2+ and consequently undergoesa conformational change that allows it to bind to and activate a host of targetenzymes. Since its discovery in 1970 (14), the mechanism of Ca2+/calmodulin-dependent regulation of target enzymes has been characterized extensively throughin vitro biochemical and structural analyses. More recently, studies of calmodulinin several genetically tractable organisms established that calmodulin is requiredfor viability (25, 123, 142). Genetic dissection of calmodulin function in the yeastSaccharomyces cerevisiaehas added significantly to our understanding of thisregulator by identifying physiologically relevant targets of calmodulin (Table 1),and by establishing the functional significance of both Ca2+-bound and Ca2+-freecalmodulin in vivo.

Calmodulin contains four copies of a Ca2+-binding motif known as an EF-hand,each of which binds one Ca2+ ion. An EF-hand is made up of a 12-residue Ca2+-binding loop flanked by twoα-helices (60). Within the loop, Ca2+ is coordinated byoxygens on six different amino acid residues. Calmodulin is one member of a largeclass of EF-hand–containing Ca2+-binding proteins (98).S. cerevisiaecontainsfive calmodulin-related proteins, each of which contains four EF-hand motifs:Cdc31p, is a component of the yeast microtubule organizing center and the yeasthomologue of centractin (5). Mlc1p and Mlc2p are myosin light chains, whichregulate distinct yeast myosins (6, 136). Cnb1p encodes the regulatory subunit ofthe Ca2+/calmodulin-regulated phosphatase, calcineurin (22, 62). Frq1p encodesthe regulatory subunit of a phosphatidylinositol-4-OH kinase and is a homologueof frequenin, a protein found in vertebrate neurons (46).

EF-hand–containing proteins typically undergo a structural change upon bind-ing Ca2+; however, this conformational change differs substantially for each classof EF-hand protein (155). Structural analyses have established that calmodulinis a dumbbell-shaped molecule with two similar domains, each containing twoEF-hand Ca2+-binding motifs, connected by a short flexible linker. In the absenceof Ca2+, the EF-hands are in a “closed” conformation. This Ca2+-free form ofcalmodulin is able to bind to a subset of target proteins. Ca2+ binding causes achange to an “open” conformation, which also results in exposure of two hydro-phobic surfaces that allow calmodulin to bind to its Ca2+-dependent target proteins(reviewed in 155). Binding sites for calmodulin share limited sequence homology,but are similar in structure, and are typically regions 20 amino acids long that

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YEAST CALMODULIN AND ITS TARGETS 649TA

BLE

1C

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odul

inta

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amm

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ence

s

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MT

sto

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drin

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ized

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3,65

,79

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190

124,

129)

Myo

4pN

oP

utat

ive

mR

NA

loca

lizat

ion

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136,

144,

145)

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5pN

oI;

IQm

otifs

:E

ndoc

ytos

isA

ctin

patc

hes

Cla

ssIm

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ns(4

1)72

5–75

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inor

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48)

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Cm

k2p

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mk1

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3–34

0S

igna

ling,

stre

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a Cal

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ulin

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depe

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min

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ndca

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ulin

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alm

odul

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Ca2+

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sis

invi

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Ca

2+in

depe

nden

t(se

ete

xt).

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650 CYERT

form basic amphipathicα-helices. Several types of calmodulin-binding sites canbe distinguished based on the spacing of particular bulky residues and the tendencyto bind apo-calmodulin (18, 125).

Identification and Characterizationof Calmodulin from S. cerevisiae

Calmodulin was purified fromS. cerevisiaebased on the similarity of its physi-cal properties to those of vertebrate calmodulin (25, 72, 107). Once purified, par-tial amino acid sequence was determined and used to synthesize oligonucleotideprobes to identify the gene,CMD1, from a yeast genomic library (25).S. cerevisiaecontains a single calmodulin gene that is required for viability and encodes a pro-tein 60% identical to vertebrate calmodulins (25). The primary structure of yeastcalmodulin is like its vertebrate counterpart, having four predicted helix-loop-helix EF-hand domains distributed similarly in the protein sequence. However,there are significant differences in the structure and Ca2+-binding properties ofyeast and vertebrate calmodulins. Vertebrate calmodulin binds four molecules ofCa2+ per molecule of calmodulin, whereas yeast calmodulin binds a maximumof three molecules of Ca2+ (72, 76, 133). The most C-terminal EF-hand in yeastcalmodulin (site IV) has a deletion of one residue in the Ca2+-binding loop andalso contains a substitution of glutamine for a highly conserved glutamate at posi-tion 12. Thus, while this region of the protein still maintains the helix-loop-helixconformation found in other calmodulins, site IV is defective for Ca2+ binding(77, 133). The other EF-hands in yeast calmodulin bind Ca2+ with high affinity(Kd= 2−5 × 10−6 M), although the exchange rate of Ca2+ for these sites is slowerthan that observed for vertebrate calmodulins (133). In its Ca2+-bound form, yeastcalmodulin exists in a more compact form than do its vertebrate counterparts (157),owing to interactions between the N-terminal and C-terminal domains (63). De-spite these differences in biochemical properties, vertebrate calmodulin is able tocomplement the essential function of calmodulin in yeast (24, 45, 103).

GENETIC ANALYSIS OF CALMODULIN FUNCTION

Analysis of Conditional Mutants

The identification and characterization of calmodulin inS. cerevisiaemade possi-ble an extensive genetic dissection of calmodulin function. Examination of condi-tional calmodulin mutants identified multiple distinct essential functions for thisprotein in vivo. Initially, two temperature-sensitive calmodulin mutants were stud-ied: cmd1-1, which contains two amino acid substitutions (23) andcmd1-101,which contains an allele engineered in vitro to allow overexpression of a truncatedcalmodulin lacking its N-terminal half in the yeast genome (139). Temperature-shift experiments with both strains revealed a requirement for calmodulin primarilyduring nuclear division; at the nonpermissive temperature, cells with a duplicated

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YEAST CALMODULIN AND ITS TARGETS 651

DNA content accumulated, and further analysis of these cells identified abnormal-ities in spindle morphology (23, 139). A defect in bud growth was also observedfor cmd1-1. The consequences of depleting calmodulin in vivo, i.e., shutting offexpression ofCMD1 driven by a galactose-regulated promoter, were similar tothe effects observed in the temperature-sensitive calmodulin mutant strains (104).Thus, the first essential role demonstrated for calmodulin was in nuclear division.

Further genetic analysis by Ohya & Botstein established that calmodulin hasseveral additional essential functions. A panel of conditional mutations was gen-erated by changing conserved phenylalanine residues of calmodulin to alanine(105). The resulting collection of mutants fall into four distinct phenotypic groups,cmd1A–D, that also display intragenic complementation. Members of one of thegroups (cmd1C) exhibit a defect in mitosis similar to that described forcmd1-1andcmd1-101. Other mutants reveal an essential role for calmodulin in bud emer-gence (cmd1D) and actin localization (cmd1A) (105). In the final group of mutants(cmd1B), the characteristic pattern of calmodulin localization is disrupted (105).Calmodulin localizes to sites of bud formation, bud tips, and the bud neck invivo (10, 138). This cellular distribution overlaps in part with that of actin patchesand reflects calmodulin’s role in polarized growth. Incmd1Bmutants, however,calmodulin is distributed diffusely throughout the cell.

The distinct nature of the four phenotypic groups ofcmd1mutants and theirability to complement each other suggested that each group was compromised foractivation of different essential target(s) in vivo. Further analyses have confirmedthatcmd1Cmutants are specifically compromised for regulation of Nuf1p. How-ever, other mutant groups may be deficient for activation of more than one target(see section on Ca2+-Independent Targets). Nonetheless, this panel of mutants hasbeen a powerful tool for analyzing and characterizing the diverse functions ofcalmodulin in vivo.

Analysis of Ca2+-Binding–Defective Mutants

The essential role of calmodulin in vivo was expected to depend on its ability to bindCa2+, because the role of this protein as an intracellular Ca2+ sensor was so wellestablished. However, this notion was challenged by the finding that mutant allelesof CMD1that are completely defective for Ca2+-binding support yeast growth (39).Ca2+-binding–defective alleles were constructed by directed substitution of aminoacid residues required for Ca2+ ion coordination, and in vitro analyses confirmedthat the resulting proteins (cmd1–3p, cmd1–6p) were deficient for Ca2+ binding.Surprisingly, yeast cells whose only source of calmodulin are these Ca2+-binding-defective–mutant proteins show minimal disruptions in growth and morphologyunder standard culture conditions (39). The intracellular localization of the mutantproteins is also indistinguishable from that of wild-type calmodulin (10, 92). Al-though Ca2+-independent binding of mammalian calmodulin to several proteinshad been demonstrated previously, the physiological relevance of these interac-tions was not fully appreciated. The studies inS. cerevisiaeclearly established that

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652 CYERT

for this organism the essential functions of calmodulin do not depend on its abilityto bind Ca2+, and later identification of the essential targets, Nuf1p/Spc110p andMyo2p, have confirmed their Ca2+-independent interaction with calmodulin (seesection on Targets of Calmodulin).

Unfortunately, these findings are often misinterpreted as indicating that thereare no Ca2+-dependent functions for calmodulin inS. cerevisiaeand that this yeastis devoid of Ca2+-dependent signaling pathways. However, as discussed below,the activation by calmodulin of at least two different target proteins, calcineurin,the Ca2+/calmodulin-dependent phosphatase, and calmodulin-regulated kinases(Cmk1p and Cmk2p), is Ca2+ dependent, and the Ca2+-binding–defective calmod-ulin mutants fail to activate these targets in vivo (20, 93). However, under standardlaboratory conditions, neither the Ca2+/calmodulin-dependent phosphatase nor thekinase is required for viability (21, 69, 106, 111).

TARGETS OF CALMODULIN

Ca2+-Independent Targets

Nuf1p/Spc110p Calmodulin localizes to the spindle pole body (SPB), the yeastmicrotubule organizing center (MTOC), throughout the cell cycle, and the essen-tial target of calmodulin in mitosis, Nuf1/Spc110p, is a component of the SPB(38, 92, 137). The SPB is embedded in the yeast nuclear envelope and is the soleorganelle responsible for nucleating nuclear and cytoplasmic microtubules. Thus,the SPB is equivalent in function to the centrosome of animal cells and thereis substantial similarity among the protein components of these two organelles(reviewed in 35).

Nuf1p was identified as a target of calmodulin by genetic selections as wellas by direct screening of expression libraries for calmodulin-binding proteins(38, 137). Dominant mutations ofNUF1 were selected as extragenic suppressorsof the temperature-sensitive allele,cmd1-1. These mutations all result in trunca-tion of the C terminus of Nuf1p (see below) and suppress thecmd1-1allele butnot a deletion allele ofcmd1(cmd/1) (38). NUF1 was independently identifiedthrough a two-hybrid screen using the Ca2+-binding defectivecmd1-6as the bait(38). The calmodulin-binding site on Nuf1p, which is in the C-terminal portion ofthe protein, was defined through two-hybrid, biochemical, and mutational analy-ses (38, 137). Further studies demonstrated that a peptide derived from this region(aa. 897–917) binds calmodulin in vitro (37).

Nuf1p contains a central region predicted to form an extended coil-coil, andforms the spacer region of the SPB that lies between the “inner plaque” and the“central plaque” (57, 86). The inner plaque of the SPB lies inside the nuclear enve-lope and is made up of theγ -tubulin-containing microtubule nucleation complex.The central plaque, which contains the additional SPB components Spc29p andSpc42p, is embedded in the nuclear envelope and forms the core structural com-ponent of the SPB (reviewed in 35). Calmodulin binds to Nuf1p at the central

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plaque of the SPB (132, 141), and this interaction seems to anchor Nuf1p to thespindle pole during mitosis. Disruption of the Cmd1p-Nuf1p interaction throughmutation of either Cmd1p or Nuf1p results in defective spindle formation and a lossof microtubule attachment to the spindle pole (58, 141). Thus, calmodulin forms anessential connection between microtubules and the SPB. However,NUF1 allelesthat encode a C-terminally truncated protein completely lacking the calmodulin-binding site are dominant suppressors ofcmd1-1(38). Together with the findingthat Nuf1p binds to Spc29p only in the calmodulin-bound form (28), these observa-tions suggest that calmodulin binding to Nuf1p relieves intramolecular inhibitionin Nuf1p to promote its binding to Spc29p and consequent association with the SPBcentral plaque. Also, calmodulin binding stabilizes Nuf1p levels in vivo (137). Ge-netic interactions are consistent with the physical associations observed betweenthese gene products. Synthetic lethality is observed betweennuf1and eitherspc29or cmd1, whilenuf1conditional mutants are suppressed byCMD1overexpressionbut exacerbated bySPC29overexpression (28, 137, 140).

In contrast toS. cerevisiae, in Schizosaccharomyces pombeCa2+ binding tocalmodulin is required for its essential function, and a mutant that is compromisedfor Ca2+ binding exhibits defects in spindle function during mitosis (92, 94). Thisfinding suggests that the same regulatory function that is Ca2+ independent inS. cerevisiaemay be Ca2+dependent inS. pombe, although the target of calmodulinat theS. pombeSPB has not yet been identified. Calmodulin also localizes to theMTOC of vertebrate cells, and kendrin, a component of human centrosomes thatis structurally similar to Nuf1p, binds calmodulin through a site that is related tothe calmodulin-binding site of Nuf1p (34).

Myo2p The second essential, Ca2+-independent target of calmodulin is Myo2p.MYO2is an essential gene originally identified in a screen for conditional mutantsthat were defective for bud emergence but continued to increase in mass at restric-tive temperature (54).MYO2encodes the heavy chain for a non-muscle myosin thatis designated as a class V myosin. Class V myosins are associated with Griscellisyndrome and deafness in humans and have been implicated in vesicle trafficking(reviewed in 124). Several other such myosins bind calmodulin (124). Myo2p isrequired for polarized secretion and vacuole inheritance in yeast (47).

Myo2p interacts with two different EF-hand proteins, Cmd1p and Mlc1p, bothof which bind to six tandemly repeated IQ sites in its neck region. IQ sites arewell established as sites of calmodulin binding and usually bind the Ca2+ freeform of calmodulin (125). Cmd1p binds directly to the Myo2p IQ sites in vitro,and can be co-immunoprecipitated with Myo2p from extracts (11, 129). Myo2plocalizes to the bud tip and bud neck of yeast cells (11, 66). Calmodulin localizationto these same sites is dependent on Myo2p-binding, as polarized localization ofcalmodulin is disrupted in cells that contain Myo2p lacking the IQ sites (136).Surprisingly, yeast cells containing Myo2p devoid of IQ sites are viable, indicatingthat calmodulin binding per se is not essential for Myo2p function (136). Sincestudies of other myosins show that the size of the neck domain correlates with

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the distance the myosin head moves along actin filaments in each cycle of ATPhydrolysis, this observation also suggests that processive movement may not berequired for Myo2p’s essential function (124).

Genetic interactions betweenCMD1 andMYO2are consistent with the inter-actions of their protein products. Conditional mutations incmd1, including thosein the cmd1Asubclass defined by Ohya & Botstein, display allele-specific syn-thetic lethality withmyo2-66, and fail to bind to Myo2p in vitro (11, 105, 129).In contrast,myo2-66phenotypes are not exacerbated in strains containing Ca2+-binding–deficient calmodulin as the sole source of calmodulin (11). These geneticobservations indicate that calmodulin’s role in Myo2p function is Ca2+ indepen-dent, and are consistent with the finding that Cmd1p binds to Myo2p in the absenceof Ca2+ (11).

Myo2p is required for the polarized growth of yeast cells and the inheritanceof the vacuole by the daughter cell during mitosis (13, 47, 54). Myo2p is thoughtto direct polarized growth by attaching to secretory vesicles and transporting themalong actin cables to the bud tip. Movement of Myo2p or secretory vesicles alongactin cables has not been shown directly. However, both Myo2p and actin cablesare required for polarized secretion and disruption of either leads to accumulationof vesicles in mother cells (43, 56, 120, 128). Some mutations in the C-terminaltail of Myo2p selectively disrupt either polarized secretion or vacuole inheritance,indicating that distinct regions within this domain mediate Myo2p attachment todifferent cargoes (12).

In addition to Myo2p, yeast contain a second class V myosin heavy chain,encoded byMYO4. Myo4p function is distinct from that of Myo2p: it is not essentialand is involved in transport/localization of specific mRNAs (7, 52, 143–145). LikeMyo2p, Myo4p contains six IQ sites, which could bind calmodulin and/or lightchains. Calmodulin binding to Myo4p has not been demonstrated; however, incells containing Myo2p that lack IQ sites, calmodulin localization is disrupted evenfurther whenmyo4is deleted, suggesting that Cmd1p and Myo4p interact (136).

Calmodulin and endocytosis: Myo5p and Arc35p Genetic analysis of endocy-tosis in yeast identified a role for calmodulin that seems to involve at least twodifferent target proteins: the unconventional type I myosin, Myo5p, and Arc35p,a component of the Arp2/3 complex.cmd1mutants were identified in a searchfor endocytosis-defective mutants. Its role in endocytosis was shown to be Ca2+-independent, as thecmd1-3Ca2+-binding–defective mutant is competent for en-docytosis (61). Further genetic analysis, using the collection ofcmd1mutantsgenerated by Ohya & Botstein (105), identified several calmodulin alleles that aredefective for endocytosis (cmd1-226, cmd1-247, andcmd1-228). Intragenic com-plementation for the endocytosis phenotype was observed betweencmd1-247andcmd1-228, suggesting that at least two distinct calmodulin targets are required forthis process (41).

Myo5p, an unconventional myosin type I heavy chain, is one target for calmod-ulin in endocytosis.myo51 cells are defective for internalization of the plasmamembrane mating factor receptor (Ste2p) at high temperature, and Myo5p contains

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two IQ sites that are both necessary and sufficient for its Ca2+-independent inter-action with calmodulin. The requirement for calmodulin in endocytosis is partiallyovercome in cells containing Myo5p that lacks the IQ sites (41).cmd1-226andcmd1-247both display defects in endocytosis and the mutant calmodulins fail tointeract physically with Myo5p (41).

Yeast cells contain an additional type I myosin, Myo3p. Myo5p and Myo3pboth localize to actin patches, andmyo3andmyo5are synthetically lethal. ThusMyo3p and Myo5p are redundant in executing their essential function of actinorganization (42). However,myo3cells are not defective for endocytosis (40).Myo3p also contains two IQ sites, but has not been shown to bind calmodulin.

A second function for calmodulin in endocytosis requires an essential protein,Arc35p. Arc35p encodes the 35-kD subunit of the highly conserved Arp2/3 com-plex, which has been purified from several different sources and shown to stimulateactin polymerization (151).arc35mutants display defects in endocytosis and or-ganization of the actin cytoskeleton (96, 152). These defects can be suppressed byoverexpression of calmodulin (126, 127). However, overexpression of two mutantalleles,cmd1-226andcmd1-228, fails to suppress thearc35-1endocytosis defect(126, 127). These observations, together with the intragenic complementation ofendocytosis defects betweencmd1-247andcmd1-228(41), suggest thatcmd1-247compromises calmodulin’s interaction with Myo5p,cmd1-228compromisescalmodulin’s interaction with Arc35p, and cmd1-226p interacts with neither tar-get. Other findings also indicate that calmodulin and Arc35p physically associate.Calmodulin localization to bud tips is disrupted at high temperature in anarc 35-1mutant, and interaction of Arc35p with calmodulin has been demonstrated both bytwo-hybrid analysis and by co-immunoprecipitation (126). Consistent with geneticfindings, cmd1-226p and cmd1-228p fail to interact with Arc35p by these meth-ods (126). However, it is not yet clear whether the association of these proteins isdirect or is mediated by additional components. Another complication is that incontrast to genetic analyses that indicate a Ca2+-independent role for calmodulinin endocytosis, co-immunoprecipitation of calmodulin and Arc35p does requireCa2+. It is possible that Ca2+ is required in vitro to stabilize the calmodulin-Arc35pinteraction, whereas a lower-affinity, Ca2+-independent interaction is sufficient forfunction in vivo. Finally, although the biochemical function of Arc35p in endo-cytosis is unclear, it may well involve its interaction with the Arp2/3 complex asarp2 mutants are also endocytosis defective (126). Arp2/3-mediated actin poly-merization may be needed to push endocytic vesicles away from the cell surfaceinto the cytosol (95).

Surprisingly, studies ofarc35-1revealed that in addition to defects in the actincytoskeleton, these mutants also exhibit a defect in metaphase spindle formationthat results in cell cycle arrest (127). This cell cycle/spindle defect can also besuppressed by overexpression ofCMD1; however, expression of mutant calmod-ulins that fail to suppress thearc35endocytosis defect (cmd1-226 and cmd1-228)do suppress the spindle formation defect. In contrast, overexpression ofcmd1-239suppresses the endocytosis defect but not the cell cycle defect.cmd1-239falls intothecmd1Cphenotypic class described by Ohya & Botstein that is compromised

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for interaction with Nuf1p/Spc110p, a component of the spindle pole body (seeabove). Thus, Arc35p may somehow affect Nuf1p/spindle pole body function. Al-ternatively, a distinct calmodulin-dependent function that affects SPB function isdisrupted in botharc35andcmd1-239.

Ca2+-Dependent Targets

CALCINEURIN Calcineurin, or PP2B, is a highly conserved, Ca2+ calmodulin-dependent phosphoserine/phosphothreonine-specific phosphatase (reviewed in3, 59). In mammals, calcineurin regulates many processes including NMDA sig-naling (64), Na+/K+ ATPase function (2), cardiac development and hypertrophy(26, 90, 121), learning and memory (74), T-cell activation (15, 100), and angiogen-esis (44). For many of these functions, including T-cell activation, the critical targetof calcineurin is the NF-AT family of transcription factors (reviewed in 17, 122).NF-AT resides in the cytosol when phosphorylated and translocates to the nucleusupon dephosphorylation by calcineurin. Thus, calcineurin regulates the activity ofthese transcription factors primarily through regulating their localization. Highlyspecific inhibitors of calcineurin, FK506 and cyclosporin A, bind to an intracellu-lar binding protein (FKBP or cyclophilin, respectively), and form a drug-proteincomplex, which then binds to and inhibits calcineurin (reviewed in 68). Thesecompounds inhibit calcineurin in a wide variety of organisms, including yeast andhumans. In humans, inhibition of calcineurin by FK506 and cyclosporin A rendersthese compounds powerful immunosuppressants.

Calcineurin is a heterodimer composed of a catalytic A subunit and an essen-tial regulatory or B subunit, which is an EF-hand–containing protein related tocalmodulin. Under resting Ca2+ levels, the A and B subunits remain associated,but the enzyme is inactive due to an autoinhibitory domain at the C terminus of theA subunit. Upon elevation of [Ca2+], Ca2+-bound calmodulin binds to the A sub-unit and displaces the autoinhibitory domain, thus activating phosphatase activity(reviewed in 59).

In S. cerevisiae, calcineurin is encoded by three genes:CNA1andCNA2/CMP2encode functionally redundant catalytic subunits, andCNB1encodes the regulatorysubunit (21, 22, 62, 69). Complete disruption of calcineurin activity in vivo canbe achieved by mutation of both catalytic subunits (cna1 cna2), mutation of theregulatory subunit (cnb1), addition of calcineurin inhibitors (FK506, cyclosporinA, or related compounds), or expression of Ca2+-binding–defectivecmd1alleles(cmd1-3, cmd1-6) as the sole source of calmodulin (20–22, 62, 69, 93). Expressionof a C-terminally truncated allele of calcineurin,CNA1/21C, that removes theC-terminal autoinhibitory domain, leads to constitutive phosphatase activity in theabsence of Ca2+/calmodulin (84, 153, 158).

Yeast calcineurin carries out at least three different functions in yeast, regulatinga stress-activated transcriptional pathway, Ca2+ homeostasis, and the G2 to Mtransition of the cell cycle. These different functions reflect the activities of distinctcalcineurin substrates.

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ROLE OF CALCINEURIN IN STRESS RESPONSE Calcineurin regulates a signal trans-duction pathway inS. cerevisiaethat is activated by intracellular Ca2+and results inincreased expression of a specific set of calcineurin-dependent genes. Under stan-dard laboratory growth conditions, this calcineurin-dependent pathway is “off”and calcineurin is dispensable for growth. However, under specific environmentalconditions, including exposure to high concentrations of ions (Ca2+, OH−, Mn2+,Na+/Li+), mating pheromone (α-factor) and high temperature, and in mutants inwhich cell wall structure is compromised, calcineurin-mediated gene expression isactivated (19, 75, 80, 83, 85, 134, 158). Yeast cells lacking calcineurin activity aresensitive to high pH, Mn2+, Na+/Li+, and lose viability during prolonged exposureto mating pheromone (21, 31, 85, 93, 97, 153). Calcineurin mutants also displaysynthetic lethality withfks1, mpk1, pkc1, and several other mutations that com-promise cell wall integrity (29, 36, 110). Thus, under most conditions in whichcalcineurin-dependent transcription is activated, calcineurin is essential for cellsurvival.

Ca2+/calcineurin-dependent transcription is mediated by a zinc-finger trans-cription factor, encoded byCRZ1/TCN1/HAL8, that activates the expression ofthe structural genes for several P-type ATPases (PMC1, ENA1, PMR1), cell wallbiosynthetic enzymes (FKS2) (75, 83, 134), and many other genes (H. Yoshimoto& M. Cyert, unpublished observations). Dissection of theFKS2 promoter de-fined the CDRE (calcineurin dependent response element), a 24-bp DNA elementthat is necessary and sufficient to mediate Ca2+-induced, calcineurin-dependentactivation of gene expression.CRZ1/TCN1 was identified as a multicopy sup-pressor that restored expression of a CDRE-lacZ reporter gene in a calcineurinmutant strain (134). Independently, loss-of-function alleles ofCRZ1/TCN1wereidentified as mutations that eliminated Ca2+-induced calcineurin-dependent geneexpression of aPMC1-lacZ reporter gene (75).crz1/tcn1mutants are viable understandard laboratory growth conditions but display growth defects in the presenceof high concentrations of OH−, Mn2+, or Na+/Li+ and lose viability during pro-longed incubation with mating pheromone (α-factor) (75, 83, 134). Thus, in largepart, the phenotypes of acrz11 strain are similar to, but not as severe as, thoseof a strain lacking calcineurin activity. For these phenotypes, thecnb11 crz11double mutants display identical growth properties as acnb11 mutant, suggest-ing that the sole mode of Crz1p regulation is calcineurin dependent. However,calcineurin mutants have additional phenotypes not shared bycrz11 (see be-low), suggesting that there are additional roles for calcineurin distinct from Crz1pregulation. Calcineurin dephosphorylates Crz1p in vitro, establishing Crz1p asa direct substrate of the phosphatase (135). Analysis of Crz1p localization invivo reveals that, like NF-AT, Crz1p rapidly relocalizes from the cytosol to thenucleus in a Ca2+-induced, calcineurin-dependent manner (135). Recent stud-ies have defined the mechanism of this relocalization and have established thatboth nuclear import and export of Crz1p depend on its phosphorylation stateand are regulated by calcineurin (117; L. Boustany & M. Cyert, unpublishedobservations).

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Activation of calcineurin in vivo requires a rise in cytosolic Ca2+. The mech-anisms underlying Ca2+ signal generation are not well characterized inS. cere-visiaebut are best understood during the response to mating factor. When haploida cells are incubated in the presence of theα-mating pheromone, Ca2+ uptakeincreases after approximately 45 minutes, resulting in an increase in cytosolic[Ca2+] (51, 102). Generation of this Ca2+ signal requires a sufficient concentra-tion of Ca2+ in the extracellular medium, and in media lacking Ca2+, yeast cellsexposed to pheromone die (51, 102). Thus, activation of calcineurin and otherCa2+-regulated processes is dependent on Ca2+ entry (153), which is mediatedduring pheromone treatment by a plasma membrane Ca2+ channel encoded by theMID1 andCCH1genes (32, 49, 70, 109). While the mechanism of Mid1p/Cch1pactivation has not been definitively demonstrated, expression of Mid1p in mam-malian cells generates a novel Ca2+ channel that is activated by membrane stretch(55). This finding suggests that physical perturbation of the yeast cell surface maygenerate a Ca2+ signal directly through the Mid1p/Cch1p channel and activateCa2+-dependent signaling pathways. It is not yet known if Mid1p and Cch1p arerequired for Ca2+ signaling under other environmental conditions. In contrast tothe well-characterized, Ca2+-dependent signaling pathways in mammalian cells,it is unclear whether Ca2+ release from intracellular stores is required during Ca2+

signaling inS. cerevisiae. Yeast possess phospholipase C (Plc1p), which generatesIP3 from hydrolysis of PIP2 (33, 112, 156). However, no IP3-regulated Ca2+ releasechannel has been identified in yeast, and calcineurin/Crz1p-dependent signalingis not disrupted inplc1mutants (M. Cyert, unpublished observations).

Regulation of Ca2+ homeostasis by calcineurin More than 95% of Ca2+ in yeastcells is stored in the vacuole (27), and this organelle plays a critical role in reg-ulating Ca2+ homeostasis in yeast. Ca2+ enters the vacuole through two knownroutes. First, the V-type ATPase encoded by theVMA and VPH1 genes acid-ifies the vacuole and generates an H+ gradient across the vacuolar membrane(reviewed in 1). This proton gradient powers a Ca2+/H+ exchanger encoded byVCX1/HUM1, allowing rapid entry of Ca2+ into the vacuole (19, 101, 119). In ad-dition to the Ca2+/H+ exchanger, yeast vacuoles contain a P-type ATPase, encodedby PMC1, that pumps Ca2+ into the vacuole against a concentration gradient (20).

Calcineurin regulates Ca2+ homeostasis in yeast, although its effects are notcompletely understood. First, calcineurin activatesPMC1expression through theCrz1p transcription factor and thus promotes vacuolar Ca2+ sequestration.crz1mutants fail to grow on media containing high concentrations of Ca2+ due to thisdecrease in Pmc1p. However, calcineurin mutants (cnb1) are Ca2+ tolerant, i.e.,they grow better than wild-type cells on Ca2+-containing media, andcnb1 crz1double mutants display growth on Ca2+ that is indistinguishable from that of thecnb1mutant. Thus, the Ca2+ tolerance ofcnb1mutants must reflect calcineurin-dependent regulation of substrate(s) other than Crz1p.

A series of observations suggest that calcineurin may regulate Vcx1p.pmc1mutants are Ca2+ sensitive, and mutations in calcineurin (cnb1) suppress this Ca2+

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sensitivity (20). However,vcx1 pmc1cells are more Ca2+ sensitive thanpmc1mu-tants, andcnb1does not suppressvcx1 pmc1sensitivity. These findings suggestthatcnb1may promote Ca2+ tolerance through regulation of Vcx1p, and that cal-cineurin decreases Vcx1p activity (19). Consistent with this idea,cnb1mutationscause increased vacuolar Ca2+ accumulation inVCX1cells but not invcx1cells(19, 153). However, Vcx1p levels are unchanged incnb1cells, and vacuolar vesi-cles isolated from wild-type and calcineurin-deficient cells display the same levelof Ca2+/H+ exchange activity in vitro (19, 118). Therefore, there is no evidencethat Vcx1p is a direct substrate of calcineurin.

Other studies indicate that calcineurin’s effects on Ca2+ homeostasis cannotbe explained solely by regulation of Vcx1p. In mutants that lack the vacuolarH+-ATPase, no H+ gradient is generated across the vacuolar membrane, and con-sequently H+/Ca2+ exchange is severely compromised. Inhibiting calcineurin inthese cells decreases cytosolic [Ca2+] (147), suggesting that calcineurin can affectCa2+ homeostasis independently of Vcx1p. Also, in calcineurin mutants the rate ofCa2+ uptake at the plasma membrane is increased, and the activity of the plasmamembrane H+-ATPase, Pma1p, which impacts Ca2+ homeostasis, is decreased(153, 154). Thus, calcineurin-dependent regulation of Ca2+ homeostasis may becomplex, and understanding its role in this process will require identification ofsubstrates.

Calcineurin-dependent regulation of the cell cycle Calcineurin also participatesin G2/M cell cycle regulation. Mutations inZDS1, which encodes a protein ofunknown biochemical function, result in increased expression ofSWE1, whichencodes a kinase that phosphorylates and negatively regulates Cdc28p (the majorcyclin-dependent kinase inS. cerevisiae) at G2/M (8). Addition of Ca2+ causes adelay in the onset of mitosis inzds11 cells due to high, sustained levels ofSWE1transcription (88). This delay is relieved by mutational inactivation ofSWE1, cal-cineurin (CNB1), orMPK1, the MAP kinase that acts downstream of protein kinaseC (PKC1) (88). Incnb11 zds11mutants, a normal pattern ofSWE1transcription isrestored; thus calcineurin is required for the Ca2+-induced increase inSWE1tran-scription inzds11mutants (88). The mechanism of this transcriptional regulationis not known and does not require Crz1p (T. Miyakawa, personal communication).Furthermore, calcineurin also seems to regulate Swe1p at a posttranslational level.Calcineurin promotes Ca2+-induced degradation of Hsl1p, a kinase that inhibitsSwe1p (73, 89, 146). Calcineurin co-immunoprecipitates with Hsl1p, and the phos-phorylation state of Hsl1p in vivo is calcineurin dependent (89). Thus, Hsl1p maybe a direct substrate of calcineurin, with dephosphorylation of Hsl1p leading to itsdegradation and consequently to increased Swe1p activity and slowed progressionfrom G2 to M.

CALMODULIN-STIMULATED PROTEIN KINASES Calmodulin regulates a number ofdifferent protein kinases in a variety of organisms, including myosin light chainkinase (MLCK), CamK I, II, and IV and CamKK. These kinases have distinct

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structural features and participate in different biological processes (reviewed in 81).In S. cerevisiae, there are only two protein kinases, the products ofCMK1 andCMK2, that are regulated by calmodulin (106, 111). These kinases are closelyrelated to each other, showing 60% amino acid identity and 90% similarity. A thirdkinase, the product of theCMK3/CLK1/RCK2gene, has some sequence similarityto calmodulin-regulated kinases, but it fails to bind calmodulin, and its activity isnot stimulated by calmodulin in vitro. Therefore, it should not be classified as acalmodulin-dependent kinase (82).

Cmk1p and Cmk2p most resemble the multifunctional calmodulin kinase type IIfrom mammalian cells. In mammals this kinase has broad substrate specificity anddisplays a characteristic pattern of regulation. It assembles into a large oligomer(10–12 subunits) and its initial activity is dependent on Ca2+/calmodulin. However,activation of the enzyme leads to its autophosphorylation on individual subunits,which results in camodulin-independent kinase activity. Mammalian CamK II haswell-documented roles in learning and memory (reviewed in 81).

Cmk1p and Cmk2p display some biochemical characteristics that are simi-lar to mammalian Cam kinase II: Although no physiological substrates for theseenzymes have been documented, in the presence of Ca2+ and calmodulin theyphosphorylate a number of different substrates in vitro and also become autophos-phorylated (71, 106, 111). Cmk2p activity becomes Ca2+/calmodulin independentafter autophosphorylation; however, this is not observed for Cmk1p (87, 106, 111).Unlike mammalian Cam kinase II, the yeast kinases do not appear to form largeoligomers, although the active form of Cmk1p may be a dimer (71).

The physiological role(s) of Cmk1p and Cmk2p are not well understood. How-ever, like calcineurin, these enzymes seem to participate in a number of stressresponses.cmk1cmk2double mutants lose viability during prolonged incubationwith mating pheromone, and calcineurin and the calmodulin-dependent kinasesact additively to promote survival of cells under these conditions (93). The specificrole of calmodulin-stimulated kinases in maintaining viability during pheromonetreatment is not understood. However, the expression ofCMK2 is induced in aCa2+/calcineurin/CRZ1-dependent manner, suggesting that conditions that lead tocalcineurin activation also may stimulate calmodulin-dependent kinase activity(H. Yoshimoto & M. Cyert, unpublished observations).

Calmodulin-dependent kinases also influence two other stress responses: theability of yeast to grow in the presence of weak organic acids and the acquisitionof thermotolerance. At low pH, when exposed to weak organic acids such as sor-bate or benzoate, wild-type yeast show a period of growth inhibition followed byadaptation (116). Recovery of growth is mediated largely by increased expressionof PDR12, which encodes an ABC cassette-type transporter that catalyzes effluxof these anions (4, 116). In contrast,cmk1cells constitutively express resistanceto organic acids, andCMK1 seems to negatively regulate Pdr12p activity (48).The mechanism of this regulation is unclear, however, as neither the gene expres-sion, protein levels, nor phosphorylation state of Pdr12p are altered incmk11mutants (48). Finally, wild-type yeast cells are also able to tolerate exposure to

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high temperature, and prior treatment of cells with mild stress improves their sur-vival during subsequent heat shock.cmk11mutants show decreased levels of thisinduced thermotolerance, and therefore Cmk1p somehow positively regulates thisstress response (50).

Other Potential Targets

Iqg1p IQG1/CYK1 encodes a product with sequence similarity to mammalianIQGAP proteins that regulate the cytoskeleton and are thought to act as effec-tors for several small GTPases (30, 67, 108, 150). Iqg1p localizes to the bud neckduring anaphase and is required for formation and contraction of the actomyosinring during cytokinesis (30, 67, 108). Iqg1p contains IQ motifs, which are re-quired for Iqg1p localization to the bud neck. Iqg1p recruits actin to the bud neckvia an actin-binding domain and also interacts with the small GTP-binding pro-teins, Cdc42p and Tem1p, potentially linking these regulators to the cytoskeleton(108, 130).

Although mammalian IQGAPs bind calmodulin, it is unclear whether Iqg1p isa calmodulin target inS. cerevisiae. In vitro, Iqg1p binds to GST-Cmd1p in a Ca2+-dependent manner; however, the IQ motifs are neither necessary nor sufficient forthis interaction (130). In contrast, the IQ motifs are required for binding of Iqg1pto Mlc1p, a myosin light chain that also interacts with Myo1p and Myo2p (9, 136),and the Iqg1p-Mlc1p interaction mediates localization of Iqg1p to the actomyosinring (9, 131). Cmd1p localization is perturbed iniqg1mutant cells, suggesting thatthese two proteins may associate in vivo (108). However, calmodulin localizationmay be altered iniqg1 mutants owing to defects in actin organization rather thana disruption in calmodulin-Iqg1p binding.

VACUOLE FUSION Several observations, mostly from in vitro biochemical studies,suggest that calmodulin plays a role in vacuole fusion inS. cerevisiae. The fusionof yeast vacuolar vesicles to each other, or homotypic vacuolar fusion, has been re-constituted in a cell-free system and is similar in many respects to other membranefusion reactions (99). A priming stage is first required to activate SNAP/SNAREcomplexes in the two fusing membranes and is followed by a docking stage inwhich specific SNAP/SNARE complexes form between them. Finally, the lipid bi-layers mix, resulting in formation of a single membrane/compartment (78, 79, 149).Ca2+ and calmodulin are required during vacuole fusion in vitro for the final stageof bilayer mixing, and some calmodulin mutants, in particularcmd1-239, displayfragmented vacuoles in vivo at restrictive temperature (105, 115). Calmodulin alsobinds to vacuoles in a Ca2+-dependent manner and is found together with proteinphosphatase type I in a large protein complex that is required for bilayer mixing(113, 115). During vacuolar fusion, components of the vacuolar H+-ATPase thatform a proteolipid ring in each membrane interact with each other to form a dimer.Calmodulin is required for this dimerization, and Ca2+ is released from the vacuoleduring the fusion process. Thus, calmodulin is thought to act as a Ca2+ sensor to

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regulate the final stages of the fusion process (114). Although this biochemicalcharacterization strongly supports a role for calmodulin in vacuole fusion, the invitro findings are not completely consistent with in vivo observations. For exam-ple, the Ca2+-binding–deficient calmodulin, cmd1-3p, fails to support vacuolarfusion in vitro, but shows no defects in vacuolar morphology in vivo (39, 115).These differences may reflect redundancies in vivo that do not exist in the in vitrosystem. In any case, a direct target for calmodulin in this process has not beenestablished.

Gad1p GAD1 was identified by its ability when overexpressed to confer resis-tance to the oxidizing agents H2O2 and diamide, andgad1mutants are sensitive tothese oxidants (16). Gad1p shows high homology to glutamate decarboxylase, anenzyme that converts glutamate to GABA. A variety of genetic evidence suggeststhatGAD1encodes a glutamate decarboxylase, although the recombinant proteinpurified fromEscherichia colihad no activity in vitro (16). Gad1p binds calmod-ulin in vitro, suggesting that this protein may be modulated by Ca2+/calmodulin.However, there is currently no evidence that indicates a role for calmodulin in theoxidative stress response.

Dst1p/Ppr2p DST1/PPR2, which encodes theS. cerevisiaehomologue of the TFIIStranscription factor, was identified by direct screening of a library for calmodulin-binding proteins (137) and confirmed by two-hybrid studies (M. Stark, personalcommunication). However, the significance of these observations remains unclear,as no role for calmodulin in regulating TFIIS function has been demonstrated.

PERSPECTIVES

Calmodulin is one of the most highly conserved proteins and is present in alleukaryotes including animals, plants, and fungi. The amino acid sequences ofcalmodulins from all multicellular organisms are more than 90% identical (91).In contrast, calmodulin from the yeastS. cerevisiaeis the most divergent form ofcalmodulin that has been characterized and is 60% identical to vertebrate calmod-ulin. S. cerevisiaecalmodulin also differs significantly from vertebrate calmod-ulin in its structure and its biochemical properties. This calmodulin is the onlyone known that binds a maximum of three rather than four molecules of Ca2+

(72, 76, 77, 133). The Ca2+-bound form of calmodulin is also unique in exhibitinginteractions between its N- and C-terminal domains (63, 157). These differencesare reflected in the relatively poor ability of yeast calmodulin to activate mam-malian target enzymes in vitro and in the inability ofS. cerevisaecalmodulin tocomplement a calmodulin mutant ofS. pombe(72, 94, 107). Despite these dif-ferences, however, calmodulin functions are highly conserved in budding yeast.Calmodulin from vertebrates orS. pombecan complement the growth defectof a calmodulin mutant ofS. cerevisiae, and all known calmodulin targets in

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S. cerevisiaehave a functional homologue or orthologue in multicellular euka-ryotes (Table1).

One of the most surprising findings from the studies of yeast calmodulin is thedemonstration that calmodulin performs its essential functions in the budding yeastin its Ca2+-free form (39). Although this finding was initially quite controversial,subsequent analyses confirmed that calmodulin association with its two essen-tial targets, Nuf1p and Myo2p, is Ca2+ independent (11, 38, 137). Does Cmd1pmediate any Ca2+-dependent regulation of Nuf1p or Myo2p function? In neithercase can a role for Ca2+ be definitively ruled out. However, that Ca2+-binding–defective alleles ofCMD1 support normal growth and morphology indicates thatCa2+-dependent regulation of these targets by calmodulin, if it indeed exists, is notrequired for viability. Interestingly, inS. pombecalmodulin function at the SPBdoes require Ca2+ binding (92), and the ATPase activity of a mammalian class Vmyosin (p190) is regulated by Ca2+ in vitro (124). Thus, at least in some species,calmodulin’s role in associating with SPB proteins or myosins is to provide sometype of Ca2+ regulation. InS. cerevisiaethis regulation may not occur or may onlysubtly affect the function of these targets.

Calmodulin participates in Ca2+-dependent modulation of protein phosphory-lation in yeast through activation of the calcineurin phosphatase and the Cmk1pand Cmk2p kinases (21, 22, 62, 106, 111). These enzymes are components of sig-naling pathways that allow yeast cells to respond to a variety of environmentalstresses. Ca2+ signals are particularly well-suited for stress responses as cytosolicCa2+ levels can be rapidly and reversibly regulated. In yeast, the rate of entry ofCa2+ across the plasma membrane may serve as a direct indicator of the integrityof the cell surface. A full understanding of the role that Ca2+ signaling plays inyeast awaits identification of the substrates of calcineurin, Cmk1p and Cmk2p,and further characterization of the mechanisms that regulate cytosolic Ca2+ levels.

It is highly likely that additional calmodulin targets in yeast remain to be iden-tified. The roles of calmodulin in regulating endocytosis and vacuole fusion areonly partially understood, and there may be other functions of calmodulin thathave not yet been demonstrated. Ikura and colleagues have studied the interac-tion of calmodulin with its many different targets and have compiled a databaseof calmodulin-binding peptides (http://calcium.oci.utoronto.ca/). As methods forpredicting calmodulin-binding domains such as these improve, and proteomic andgenomic analyses evolve, the entire complement of calmodulin functions in eu-karyotic cells will continue to unfold.

ACKNOWLEDGMENTS

I thank Jeremy Thorner, Michael Stark, and Trisha Davis for helpful discussion, andMichael Stark and Tokichi Miyakawa for contributing unpublished information.I gratefully acknowledge Victoria Heath and James Withee for critical reading ofthe manuscript. The work described in this review was supported by NIH, grant# GM-48729.

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NOTE ADDED IN PROOF

Several new calmodulin binding proteins in yeast were recently discovered throughproteomic analysis (159).

Visit the Annual Reviews home page at www.AnnualReviews.org

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Documents

Genetic Analysis of Calmodulin and Its Targets in Saccharomyces cerevisiae