9
Abstract Fission yeast strains auxotrophic for leucine are unable to proliferate in normally supplemented minimal media adjusted to pH 6.4 or above. High-pH sensitivity can be suppressed by the loss of Pub1, an E3 ubiquitin ligase, or by the replacement of NH 4 + with a non-repressing source of nitrogen such as L-proline. In this report we show pub1 to be required for the rapid down-regulation of leucine up- take observed in response to the addition of NH 4 + to the growth media. Furthermore, we corroborate earlier results demonstrating the transport of leucine to be negatively influenced by high extracellular pH. pub1 is homologous to the budding yeast nitrogen permease inactivator, NPI1/RSP5, which mediates the ubiquitination and subse- quent destruction of NH 4 + -sensitive permeases. The high- pH sensitivity of cells auxotrophic for leucine thus seems to reflect an inability of NH 4 + -insensitive permeases to transport sufficient leucine under conditions where the pro- ton gradient driving nutrient transport is low, and NH 4 + - sensitive permeases have been destroyed. Intriguingly, the partial suppression of both high pH sensitivity, and the inactivating effect of NH 4 + on leucine transport, seen in pub1-1 point mutants, becomes as complete as seen in pub1backgrounds when cells have concomitantly lost the function of the spc1 stress-activated MAPK. Key words Fission yeast · Nitrogen catabolite inactivation · pub1 ubiquitin ligase · spc1 stress-activated MAPK · Leucine transport Introduction The primary electrochemical gradient across the yeast plasma membrane is provided by the extrusion of protons through the H + -translocating ATPase. Once established this gradient is utilized to drive various secondary trans- port processes including the uptake of both uracil and leu- cine through H + -symports (Horak 1986; Goffeau et al. 1989; Van der Rest et al. 1995). External pH conditions, as well as the presence of NH 4 + in the growth media, have been shown to affect the effi- ciency of these transport mechanisms. The effect of exter- nal pH is seen as a decrease in nutrient uptake as the pH rises. For example, the high-affinity uptake system for leu- cine has been shown to have a pH optimum of between 3.0 and 3.25. As the pH rises from this optimum, substrate affinity (K t ) remains unaffected whereas the capacity (J max ) for transport becomes significantly reduced (Sych- rova et al. 1989). In budding yeast NH 4 + has been shown to exert its ef- fect on nutrient transport through the negative regulation of a group of permeases involved in nutrient-specific up- take. Such NH 4 + -sensitive permeases include the Gap1p general amino-acid permease, the Put4p proline permease, the Gnp1p glutamine permease, and the Dal5p/Uep1p al- lantoin ureidosuccinate permease (Rai et al. 1988; Vanden- bol et al. 1989; Jauniaux and Grenson 1990; Zhu et al. 1996). Two distinct pathways regulating this process have been identified: the nitrogen catabolite repression (NCR) pathway which acts to suppress the synthesis of new NH 4 + - sensitive permeases, and the nitrogen catabolite inactiva- tion (NCI) pathway which acts to inhibit the function of pre-existing permeases (Grenson 1983; 1992). The NCI pathway requires the integrity of NPI1/RSP5 which has been shown to encode an E3 ubiquitin ligase (Hein et al. 1995). Ubiquitination, in addition to its well-defined role in mediating the rapid and selective proteolysis of targeted proteins via the 26 S proteasome, has also been shown to serve as a signal for the endocytosis of various proteins lo- Curr Genet (1999) 35: 593–601 © Springer-Verlag 1999 Received: 10 November 1998 / 15 March 1999 Jim Karagiannis · Reza Saleki · Paul G. Young The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH 4 + in fission yeast ORIGINAL PAPER J. Karagiannis · R. Saleki 1 · P. G. Young () Department of Biology, Queen’s University, Kingston, Ontario, K7L 3N6 Canada e-mail: [email protected] Tel.: +1-613-533 6148 Fax: +1-613-533 6617 Present address: 1 Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada Communicated by A. Goffeau

The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

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Page 1: The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

Abstract Fission yeast strains auxotrophic for leucine areunable to proliferate in normally supplemented minimalmedia adjusted to pH 6.4 or above. High-pH sensitivity canbe suppressed by the loss of Pub1, an E3 ubiquitin ligase,or by the replacement of NH4

+ with a non-repressing sourceof nitrogen such as L-proline. In this report we show pub1to be required for the rapid down-regulation of leucine up-take observed in response to the addition of NH4

+ to thegrowth media. Furthermore, we corroborate earlier resultsdemonstrating the transport of leucine to be negatively influenced by high extracellular pH. pub1 is homologousto the budding yeast nitrogen permease inactivator,NPI1/RSP5, which mediates the ubiquitination and subse-quent destruction of NH4

+-sensitive permeases. The high-pH sensitivity of cells auxotrophic for leucine thus seemsto reflect an inability of NH4

+-insensitive permeases totransport sufficient leucine under conditions where the pro-ton gradient driving nutrient transport is low, and NH4

+-sensitive permeases have been destroyed. Intriguingly, thepartial suppression of both high pH sensitivity, and theinactivating effect of NH4

+ on leucine transport, seen inpub1-1 point mutants, becomes as complete as seen inpub1∆ backgrounds when cells have concomitantly lost the function of the spc1 stress-activated MAPK.

Key words Fission yeast · Nitrogen catabolite inactivation · pub1 ubiquitin ligase · spc1 stress-activated MAPK · Leucine transport

Introduction

The primary electrochemical gradient across the yeastplasma membrane is provided by the extrusion of protonsthrough the H+-translocating ATPase. Once establishedthis gradient is utilized to drive various secondary trans-port processes including the uptake of both uracil and leu-cine through H+-symports (Horak 1986; Goffeau et al.1989; Van der Rest et al. 1995).

External pH conditions, as well as the presence of NH4+

in the growth media, have been shown to affect the effi-ciency of these transport mechanisms. The effect of exter-nal pH is seen as a decrease in nutrient uptake as the pHrises. For example, the high-affinity uptake system for leu-cine has been shown to have a pH optimum of between 3.0 and 3.25. As the pH rises from this optimum, substrateaffinity (Kt) remains unaffected whereas the capacity(Jmax) for transport becomes significantly reduced (Sych-rova et al. 1989).

In budding yeast NH4+ has been shown to exert its ef-

fect on nutrient transport through the negative regulationof a group of permeases involved in nutrient-specific up-take. Such NH4

+-sensitive permeases include the Gap1pgeneral amino-acid permease, the Put4p proline permease,the Gnp1p glutamine permease, and the Dal5p/Uep1p al-lantoin ureidosuccinate permease (Rai et al. 1988; Vanden-bol et al. 1989; Jauniaux and Grenson 1990; Zhu et al.1996). Two distinct pathways regulating this process havebeen identified: the nitrogen catabolite repression (NCR)pathway which acts to suppress the synthesis of new NH4

+-sensitive permeases, and the nitrogen catabolite inactiva-tion (NCI) pathway which acts to inhibit the function ofpre-existing permeases (Grenson 1983; 1992). The NCIpathway requires the integrity of NPI1/RSP5 which hasbeen shown to encode an E3 ubiquitin ligase (Hein et al.1995).

Ubiquitination, in addition to its well-defined role inmediating the rapid and selective proteolysis of targetedproteins via the 26 S proteasome, has also been shown toserve as a signal for the endocytosis of various proteins lo-

Curr Genet (1999) 35: 593–601 © Springer-Verlag 1999

Received: 10 November 1998 / 15 March 1999

Jim Karagiannis · Reza Saleki · Paul G. Young

The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4

+ in fission yeast

ORIGINAL PAPER

J. Karagiannis · R. Saleki1 · P. G. Young (½) Department of Biology, Queen’s University, Kingston, Ontario,K7L 3N6 Canadae-mail: [email protected].: +1-613-533 6148Fax: +1-613-533 6617

Present address:1 Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada

Communicated by A. Goffeau

Page 2: The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

calized to the plasma membrane. For example, endocyticdelivery to the vacuole of the Ste2p alpha-factor receptor,the Ste6p a-factor ABC transporter, the Pdr5p multidrugresistance transporter, the Mal1p maltose transporter, andthe Itr1p inositol permease has been shown to be precededby ubiquitination. Intriguingly, proteolytic degradation ofthese modified proteins is dependent not on the proteasomebut on functional vacuolar proteases (Kolling and Hollen-berg 1994; Egner et al. 1995; Lai et al. 1995; Riballo et al.1995; Egner and Kuchler 1996; Hicke and Riezman 1996).

A similar model has emerged with regards to the Npi1p-induced turnover of both the Fur4p uracil permease andthe Gap1p general amino-acid permease. Fur4p has beenshown to undergo an NPI1-dependent in vivo ubiquitina-tion. Its normal degradation is impaired in budding yeaststrains defective in either endocytic internalization or theproduction of functional vacuolar proteases. Mutations inthe regulatory or catalytic subunits of the proteasome, how-ever, do not affect the rate of uracil permease degradation(Galan et al. 1996).

Springael and Andre (1998) have also recently shownthat the addition of NH4

+ to proline grown budding yeastcells increases the conversion of Gap1p to ubiquitin-con-jugated forms. This conversion is followed by a rapid inter-nalization of the permease and subsequent degradation inthe vacuole. NPI1 mutants are defective in Gap1p ubiquit-ination and this defect results in the permease remainingstable on the plasma membrane, even in the presence ofNH4

+.In this report we demonstrate a role for the fission yeast

pub1 E3 ubiquitin ligase in modulating membrane trans-port processes. This role was uncovered as a result of stud-ies where it was found that fission yeast cells auxotrophicfor leucine were unable to grow in normally supplementedminimal media at high external pH (Saleki et al. 1997).Here we provide evidence suggesting that the underlying

mechanism behind this phenotype involves a pub1-medi-ated down-regulation of leucine uptake in response toNH4

+. Based on its homology to NPI1 we propose thisdown-regulation to be as a result of the ubiquitination andsubsequent vacuolar destruction of NH4

+-sensitive leucinepermeases.

Materials and methods

Strains and media. All Schizosaccharomyces pombe strains used inthis work (Table 1) were derived from wild-type strains 972 (h–S) or975 (h+N). Strains were grown in Edinburgh minimal medium (EMM),or in modified EMM in which NH4Cl was replaced with 10 mM ofL-proline. High pH EMM or modified EMM were adjusted to pH 6.4or 6.8 with NaOH and buffered with 10 mM of PIPES. Auxotrophicstrains were grown in media containing 0.1 mg/ml of adenine, uracil,or L-leucine unless otherwise stated. The preparation of EMM pH 3.5has been described elsewhere (Saleki et al. 1997).

Time-course experiments. Strains were grown to mid-log phase inEMM pH 5.5 [or modified EMM pH 5.5 for experiments where cellswere shifted from non-repressing (proline) to repressing (NH4

+)sources of nitrogen] at 30°C with constant shaking. Cells were thendiluted to approximately 1×105 cells/ml in 20 ml of the appropriatelypH-adjusted EMM or modified EMM and incubated at 30°C withconstant shaking. Samples were taken within 0.5 h of inoculation andat 6, 12, 24, 30, and 36 h thereafter. The number of cells/ml was de-termined with a Coulter Counter. Transition from non-repressing(proline) to repressing (NH4

+) sources of nitrogen was achieved bythe addition of 0.8 ml of a 2.5 M NH4Cl solution (final concentra-tion 100 mM of NH4Cl) after 6 h.

Leucine-uptake assays. Cultures were grown to 1–2×106 cells/ml at 30°C with constant shaking in modified EMM pH 5.5. The cul-tures were then split in two, one half being collected (3000 rpm for 5 min), washed, and re-suspended in modified EMM pH 5.5, andthe other half in modified EMM pH 6.8 (cells were re-suspended inthe same volume of media originally collected). Leucine uptake andA600 measurements were taken before the addition of NH4Cl to100 mM, as well as at the indicated time points later. Uptake was

594

Strain Genotype Source

Q250 972 h– Lab collectionQ360 ade6-216 leu1-32 ura3-D18 h+ Lab collectionQ868 leu1-32 h– Lab collectionQ1158 ade6-216 h– Lab collectionQ1163 ade6-216 leu1-32 h+ Lab collectionQ1164 ade6-216 ura4-D18 h– Lab collectionQ1166 leu1-32 ura4-D18 h– Lab collectionQ1411 ura4-D18 h– Lab collectionQ1477 pub1-1 mcs4-1 ade6-216 ura4-D18 leu1-32 h90 Saleki et al., 1997Q1485 pub1::ura4 ura4-D18 leu1-32 ade6-216 h– Nefsky and Beach, 1996Q1598 mcs4-1 ura4-D18 h+ This studyQ1599 mcs4-1 ura4-D18 h+/mcs4::ura4-D18 leu1-32 ade6-216 h– This studyQ1600 pub1-1 leu1-32 h+ This studyQ1601 pub1-1 spc1::ura4 ura4-D18 leu1-32 h– This studyQ1602 pub1::ura4 ura4-D18 leu1-32 h+ This studyQ1603 pub1-1 h– This studyQ1604 pub1-1 spc1::ura4 ura4-D18 h– This studyQ1605 spc1::ura4 ura4-D18 h+ This studyQ1606 pub1::ura4 ura4-D18 h+ This studyQ1607 pub1-1 wee1-50 ura4-D18 leu1-32 h– This studyQ1608 pub1::ura4 wee1-50 ura4-D18 leu1-32 h– This studyQ1609 mcs4::ura4-D18 leu1-32 ade6-216 h– (GP65) Cottarel, 1997Q1610 spc1::ura4 ura4-D18 leu1-32 h– (KS1366) Shiozaki and Russell, 1995

Table 1 Strain list

Page 3: The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

measured by adding 0.7 ml of culture to a microcentrifuge tube con-taining 1–5 µCi of L-[4,5-3H(N)]-leucine (50 Ci/mmol; NEN LifeScience Products) as well as unlabeled leucine (to bring the final leu-cine concentration to the indicated levels upon addition of the cul-ture). Tubes were vortexed and 0.2-ml samples were taken at 0.5,1.5, and 2.5 min (uptake was linear up to 3 min). Samples were fil-tered through 0.45-µm polycarbonate membranes which werewashed three times with 1 ml of 10 mM leucine before being trans-ferred to vials, and the radioactivity quantitated by liquid scintilla-tion counting.

Results

The high-pH sensitivity of auxotrophic cells is due to inefficient uptake of nutrients from the environment

During research investigating plasma membrane transport-ers and their role in pH homeostasis, it became apparentthat fission yeast cells carrying either the leu1-32 or ura4-D18 mutations were unable to proliferate when the pH ofthe growth media (normally pH 5.5) was adjusted to pH 6.4or above. In contrast, cells carrying the ade6-216 mutationshowed no sensitivity to high external pH (Fig. 1). Sincethe normal growth of cells harboring the leu1-32 or ura4-

D18 mutations is entirely dependent on efficient uptake ofleucine and uracil from the environment, we hypothesizedthat the inhibitory effect of high external pH was attribut-able to interference with the functioning of nutrient-spe-cific transport mechanisms.

As a simple test of this model, we increased the con-centration of uracil and leucine in the growth media tentimes (to 1 mg/ml). Under these conditions strains auxo-trophic for uracil or leucine were able to proliferate in me-dia adjusted to pH 6.4, while strains grown with more typ-ical concentrations of supplements (0.1 mg/ml) demon-strated a severe inhibition of growth. At pH 6.8, increasedsupplement concentrations also suppressed the growth in-hibition seen in uracil auxotrophs, while the growth of leu-cine auxotrophs remained inhibited (Fig. 2). The inability

595

Fig. 1 Fission yeast strains auxotrophic for uracil and/or leucine areunable to proliferate in normally supplemented minimal media athigh external pH. Strains as per legend were plated on EMM pH 5.5or EMM pH 6.8 and incubated for 72 h at 30°C. Plates were supple-mented with 0.1 mg/ml of adenine, uracil, and leucine

Fig. 2 Increasing the concentration of uracil or leucine supplementssuppresses the high-pH sensitivity of cells auxotrophic for uracil orleucine, respectively. Actively growing cells auxotrophic for leucine(squares) or uracil (circles) were inoculated into EMM pH 5.5, EMMpH 6.4, or EMM pH 6.8 with either 0.1 mg/ml supplements (closedsymbols) or 1 mg/ml supplements (open symbols)

Page 4: The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

of leucine auxotrophs to proliferate at pH 6.8 in the pres-ence of 1 mg/ml of leucine most likely reflects a more-se-vere inhibition of leucine transport by high external pHconditions. The fact that adenine auxotrophs showed nohigh-pH sensitivity supports the contention that discretenutrient transport systems can be affected differentially bya high pH.

The pub1-1 mutation and spc1∆ show synergy in suppressing the high-pH sensitivity of cells auxotrophic for leucine

The pub1-1 (protein ubiquitin ligase) and elp3-1 (elongat-ed at low pH) mutations were isolated as extragenic sup-pressors of high-pH sensitivity from a fission yeast straincarrying the ade6-216, ura4-D18 and leu1-32 auxotroph-ic markers. Neither mutation alone was sufficient to sup-press growth inhibition of the auxotrophic strain at pH 6.8.Interestingly, these mutations also showed a negative inter-action at low external pH (pH 3.5). pub1 was cloned andshown to encode an E3 ubiquitin ligase (Saleki et al. 1997).The molecular identity of elp3, however, remained un-known.

The identity of elp3 was first suggested by a similarityof phenotype in strains carrying the recessive elp3-1 andmcs4::ura4 (mcs4∆) mutations. When streaked on EMMplates adjusted to pH 3.5 both elp3-1 and mcs4∆ strainsshow a cell-elongation phenotype indicative of a delay in cell-cycle progression. Free-spore analysis, as well asan analysis of 18 tetrads from a genetic cross between anelp3-1 and an mcs4∆ strain, revealed no wild-type segre-gants. To confirm the allelism of elp3 and mcs4, elp3-1ura4-D18 /mcs4::ura4 ura4-D18 leu1-32 ade6-216 dip-loids were constructed and tested by streaking on EMMpH 3.5 plates. These diploids demonstrated a pH-sensitivecell-cycle delay. The absence of complementation there-fore indicated that the elp3 and mcs4 loci were allelic.

The mcs4 locus encodes a protein with homology to bac-terial two-component system response regulators and hasbeen shown to be an upstream regulator of the spc1 stress-activated MAPK pathway (Cottarel 1997; Shieh et al. 1997;Shiozaki et al. 1997). mcs4 loss-of-function mutations wereoriginally isolated as suppressors of the lethal prematureinitiation of mitosis associated with cdc2-3w wee1-50strains at their restrictive temperature (Molz et al. 1989).

To determine whether mcs4 was acting through the spc1pathway, or an alternate pathway, we constructed pub1-1spc1::ura4 (spc1∆) double mutants. As shown in Fig. 3spc1∆, like the mcs4-1 point mutation, was able to sup-press the growth inhibition caused by leucine auxotrophyat pH 6.8 when placed in a pub1-1 background. However,leu1-32 pub1-1 mutants were unable to proliferate underthese same conditions. Interestingly, leu1-32 pub1::ura4(pub1∆) strains were able to grow at pH 6.8 while leu1-32spc1∆ strains remained sensitive to high external pH. Infact growth of the leu1-32 spc1∆ strain was completely in-hibited even at a less-restrictive pH of 6.4, conditionswhere pub1-1 leu1-32 cells proliferated well.

Interestingly, the synergistic relationship betweenpub1-1 and the spc1∆ is also discernible at low extracel-lular pH (pH 3.5). Under these conditions pub1-1 cells areslow growing, and slightly elongated, whereas spc1∆ cellsare elongated, but capable of colony formation. Doublemutants are synthetically lethal and arrest as microcolo-nies of highly elongated cells (data not shown).

The high-pH sensitivity of auxotrophic cells is dependenton the presence of NH4

+ in the growth media

Since NH4+ is known to negatively regulate the activity of

several amino-acid and nucleoside permeases (Grenson1983; Horak 1986; Grenson 1992; Hein et al. 1995; Galan

596

Fig. 3 The pub1-1 mutation and spc1∆ show synergy in suppress-ing the high-pH sensitivity of cells auxotrophic for leucine. Active-ly growing cells were inoculated into EMM pH 5.5, EMM pH 6.4,or EMM pH 6.8. Wild-type (P), leu1-32 (L), pub1-1 leu1-32 (G),spc1::ura4 ura4-D18 leu1-32 (p), pub1::ura4 ura4-D18 leu1-32(H), pub1-1 spc1::ura4 ura4-D18 leu1-32 (m)

Page 5: The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

et al. 1996; Springael et al. 1998) we decided to study theperformance of auxotrophic cells in the absence of NH4

+.Interestingly, when L-proline was used as a nitrogensource, auxotrophic cells showed no sensitivity to high ex-ternal pH. Conversely, the addition of NH4

+ to proline-grown cells at high external pH was sufficient to bringabout a rapid and complete growth arrest to cells auxo-trophic for leucine. leu1-32 cells carrying either the pub1∆,or the pub1-1 mutation plus spc1∆, were entirely insensi-tive to the addition of NH4

+ and demonstrated no growtharrest. As expected, leu1-32 cells carrying pub1-1 or spc1∆alone were unable to suppress the growth inhibitory effectof NH4

+ at pH 6.8 (Fig. 4).Taken together, the above data suggest a model in which

there exists two leucine-uptake systems: an NH4+-insensi-

tive system which is negatively regulated by high externalpH (such a system must exist since leucine auxotrophs areable to grow in a normally supplemented NH4

+-containingmedium at pH 5.5) as well as an NH4

+-sensitive system.The data also suggest that both pub1 and spc1 are involvedin negatively regulating this NH4

+-sensitive system of leu-cine uptake. To test these models we chose to monitor theuptake of 3H-labeled leucine in the presence or absence ofNH4

+, at both pH 6.8 and pH 5.5, in each of our mutantbackgrounds.

597

Fig. 4 The high-pH sensitivityof cells auxotrophic for leucineis dependent on the presence of NH4

+ in the growth media.Actively growing cells from anovernight culture of the indicat-ed strains were inoculated intotwo separate proline-containingmodified minimal media pH 6.8liquid cultures. One of the twocultures was supplemented withNH4Cl to a final concentrationof 100 mM 6 h after inoculation(open symbols) while the otherculture remained untreated (closed symbols). The arrow in-dicates the time of addition ofNH4Cl

Leucine uptake is sensitive to both NH4+

and extracellular pH

We first performed experiments looking at the response ofa wild-type strain to the addition of NH4

+ at a leucine con-centration of 0.76 mM (the concentration of leucine usedin our growth assays). As seen in Fig. 5 the initial rate ofuptake drops quickly and dramatically (from the rate ob-served in proline pH 5.5 media) upon the addition of NH4

+

(reaching a minimum between 30–60 min). Furthermore,the severity of the decrease is 2–3-fold greater at pH 6.8than at pH 5.5. We thus establish that leucine uptake is sen-sitive both to the presence of NH4

+ as well as to extracel-lular pH conditions.

Since previous work has demonstrated the existence oftwo leucine-uptake systems in fission yeast (a high-affin-ity system with a Kt of 0.05 mM, as well as a low-affinitysystem with a Kt of 1.25 mM; Sychrova et al. 1989) we al-so examined the effect of both high and low leucine con-centration on the response of leucine uptake to NH4

+. Atconcentrations where one would expect only the high-af-finity system to be importing leucine at a significant ca-pacity (0.01 mM) uptake drops greater than 30 fold (to al-most zero) 1 h after the addition of NH4

+ (Fig. 6). Howev-er, at concentrations where one would expect both systems

Page 6: The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

We suggest that the destruction of these leucine-specificpermeases results in a decrease in total uptake capacity se-vere enough to inhibit the growth of cells auxotrophic forleucine, at high external pH, in the presence of standardconcentrations of leucine supplements.

Both growth and leucine-uptake data are supportive ofsuch a model. We clearly demonstrate an NH4

+ sensitivityto both the growth of cells auxotrophic for leucine, as wellas to leucine uptake. In addition we demonstrate an abil-ity of pub1 mutants to suppress both this growth inhibi-tion, and the inactivating effect of NH4

+ on uptake (Figs. 3,4, 7). The fact that increased leucine concentration can sup-press growth inhibition at high external pH (Fig. 2) cor-roborates the idea that proliferation by pub1 mutants underthese conditions can be explained in terms of the modula-tion of leucine transport.

We also supply additional support for the contentionthat the pH-sensitive nature of this growth inhibition re-flects the dependence of nutrient uptake on the strength of

to be importing leucine efficiently (2 mM) the drop in re-sponse to NH4

+ is only 2–3 fold. These data suggest thatthe high-affinity system is under a strong negative regula-tion by NH4

+ and that the lower affinity system is relative-ly insensitive to NH4

+.

pub1 is required for the rapid down-regulation of leucine uptake observed in response to NH4

+

Unlike wild-type strains, pub1∆ as well as pub1-1 spc1∆strains are able to maintain leucine uptake at a level com-parable to that seen in proline media after the addition ofNH4

+ (Fig. 7). The pub1-1 mutant showed an intermediatephenotype between that of a wild-type and a pub∆ straini.e., it was unable to completely suppress the loss of up-take 1 h after the addition of NH4

+, but did show a statisti-cally significant higher level of uptake compared to thewild-type. Surprisingly, spc1∆ strains showed no ability tosuppress the inactivating effect of NH4

+ on leucine uptake.In fact its only impact was seen in pub1-1 backgrounds, inwhich it had the effect of converting the observed partialsuppression to a complete suppression (Fig. 7).

Discussion

Based on its homology to Npi1p of budding yeast we pro-pose Pub1 to be involved in the ubiquitination and degra-dation of leucine-specific permeases in response to NH4

+.

598

Fig. 6 Leucine uptake is more severely affected by NH4+ at low leu-

cine concentrations. Wild-type cultures were grown to 1–2×106

cells/ml in modified EMM pH 5.5, collected, and then re-suspend-ed, at the same density, in either modified EMM pH 5.5, or modifiedEMM pH 6.8. Leucine uptake and A600 measurements were takenjust before the addition of NH4

+ to 100 mM as well as 1 h later. Up-take assays were performed at leucine concentrations of 0.01 mMand 2 mM. Bars indicate standard errors, n=3

Fig. 5 Leucine uptake is sensitive to both NH4+ and extracellular

pH. Wild-type cultures were grown to 1–2×106 cells/ml in modifiedEMM pH 5.5, collected, and then re-suspended, at the same den-sity, in either modified EMM pH 5.5, or modified EMM pH 6.8. Leu-cine uptake and A600 measurements were taken from the culturesmarked “Proline + NH4

+ pH 5.5” and “Proline + NH4+ pH 6.8” at the

indicated time points after the addition of NH4Cl to 100 mM (time0). A separate modified EMM pH 5.5 culture, treated with water in-stead of NH4

+ (marked “Proline pH 5.5”), was used as a control. Ex-periments were run in parallel in a staggered fashion. Uptake assayswere performed at a leucine concentration of 0.76 mM. Bars indi-cate standard errors, n=3

Page 7: The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

Perhaps the most surprising and enigmatic finding ofthis study was the discovery of the allelism between elp3and mcs4. The mcs4 locus was initially isolated in a screenfor mutations capable of suppressing the premature and le-thal initiation of mitosis associated with cdc2-3w wee1-50strains at their restrictive temperature (Molz et al. 1989).In addition to its ability to modulate the mitotic control offission yeast, mcs4 has also recently been shown to be in-volved in the response to extracellular stress through itsregulation of the spc1/sty1/phh1 stress-activated MAPKpathway. Through formal genetic and biochemical analy-sis Mcs4, which has significant homology to bacterial two-component response regulators, has been placed upstreamof Wak1/Wik1 MAPKKK, Wis1 MAPKK, and Spc1MAPK (Cottarel 1997; Shieh et al. 1997; Shiozaki et al.1997).

The spc1 pathway is best known for its ability to mod-ulate mitotic initiation under conditions of cellular stress(for a review describing this and other MAPK pathways inyeast see Banuett et al. 1998). Loss-of-function mutationsin this pathway result in a delay in mitotic initiation that isexacerbated under conditions of high osmolarity and nu-trient limitation. Integrity of the pathway is also requiredfor proper responses to a range of environmental insultsincluding heat shock, UV exposure, oxidative stress (Shio-zaki and Russell 1995; Degols et al. 1996; Wilkinson et al.1996; Samejima et al. 1997; Shieh et al. 1998), and lowextracellular pH (this study).

Interestingly, the spc1 MAPK pathway has also beenshown to regulate membrane transport processes in fissionyeast. For example, wis1, the MAPKK of the spc1 path-way, is required for the proper onset of gluconate transportunder conditions of glucose starvation (Caspari 1997). Inaddition, spc1 positively regulates both the Hba2 and Pmd1ABC transporters involved in multidrug resistance (Tooneet al. 1998).

In another of its many roles, spc1 is also required forproper vacuolar dynamics in response to both hypo- andhyper-osmolarity (Bone et al. 1998). Since a reduced abil-ity to endocytose and/or deliver nutrient-specific per-meases to the vacuole for degradation is one hypothesisexplaining how spc1 mutations might negatively regulatenutrient uptake, and since loss-of-function mutations inNpi1 cause endocytosis defects in budding yeast (Zoladeket al. 1997), we tested our pub1 and spc1 mutants for en-docytosis defects using the fluorescent dyes FM4-64 andlucifer yellow. No defects were observed (data not shown).

Unlike mutations in mcs4, mutations in wak1, wis1 orspc1 are unable to suppress the mitotic catastrophe pheno-type of cdc2-3w wee1-50 mutants. This indicates that mcs4, in addition to its modulation of mitotic control viathe spc1 pathway, also provides modulation via a secondindependent pathway. With respect to its ability to suppresshigh-pH sensitivity, we show mcs4 to be acting throughmodulation of the spc1 pathway, as spc1∆, just like themcs4-1 point mutation, is able to suppress the growth in-hibition of leucine auxotrophs at pH 6.8, when placed in a pub1-1 background. We go on to show that the partialsuppression of the inactivating effect of NH4

+ on leucine

599

Fig. 7 Mutations in pub1 suppress the inactivating effect of NH4+

on leucine uptake. Cultures of the indicated strains were grown to1–2×106 cells/ml in modified EMM pH 5.5, collected, and then re-suspended, at the same density, in either modified EMM pH 5.5, ormodified EMM pH 6.8. Leucine uptake and A600 measurements weretaken just before the addition of NH4

+ to 100 mM as well as 1 h lat-er. Uptake assays were performed at a leucine concentration of0.76 mM. Bars indicate standard errors, n=3

the electrochemical proton gradient driving secondarytransport processes. In agreement with previous results(Sychrova et al. 1989) we show leucine uptake to be neg-atively affected at high extracellular pH (Figs. 5, 6, 7).

Furthermore, we provide preliminary evidence sug-gesting the high-affinity leucine-uptake system of fissionyeast to be under a strong negative regulation by NH4

+, and the low-affinity system to be relatively insensitive to NH4

+. Data presented in Fig. 6 are simply explained ifsuch a model is assumed. These findings are consistent withearlier growth data which suggested the existence of bothNH4

+-sensitive and NH4+-insensitive systems of uptake.

Page 8: The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

uptake, seen in pub1-1 backgrounds, is converted to a com-plete suppression by a concomitant loss of the function ofspc1.

The similarity of phenotypes between pub1-1 spc1∆ andpub1∆ strains, the fact that the pub1-1 mutation partiallysuppresses both high-pH sensitivity and the inactivatingeffect of NH4

+ on leucine uptake (whereas pub1∆ is able tocompletely suppress), together with the fact that we havebeen unable to discern any effect of spc1 pathway muta-tions on either high-pH sensitivity, or leucine uptake, whennot in a pub1-1 background, suggests a model in whichspc1∆ completes a partial loss of function of the pub1-1allele.

The notion that pub1-1 encodes a partial loss of func-tion is also corroborated by two independent lines of rea-soning. First, pub1-1 strains are slow growing but able toform colonies when plated at pH 3.5. pub1∆ strains, on theother hand, are inviable under these same conditions (datanot shown). Second, pub1-1 wee1-50 double mutants areslow growing but viable at 36°C (data not shown), whereaspub1∆ wee1-50 mutants are inviable (inviability is the re-sult of mitotic catastrophe due to the inability of pub1∆strains to negatively regulate cdc25, an inducer of mitosis;Nefsky and Beach 1996). The less-severe phenotypes ofpub1-1 mutants, when compared to pub1∆ mutants, stronglysuggest that pub1-1 mutants retain some residual Pub1function.

It should be noted that the synergy between the pub1-1allele and spc1∆ is discernible not only at a high external pH,but also at a low extracellular pH where strains carrying bothmutations give rise to a synthetic cdc phenotype. The natureof this synergy, however, remains enigmatic. Interactionsbetween MAPK pathways and ubiquitination pathways havebeen uncovered in other systems, most notably in human Band T cells where phosphorylation of the bcl-6 proto-onco-gene by MAPK targets its destruction via the ubiquitin/pro-teasome pathway (Niu and Dalla-Favera 1998).

Acknowledgements This work was supported by grants from theNatural Sciences and Engineering Research Council of Canada toP. G. Y. David Beach is gratefully acknowledged for the provisionof the pub1∆ strain. Paul Russell is gratefully acknowledged for theprovision of the spc∆ strain. Guillaume Cottarel is gratefully ac-knowledged for the provision of the mcs4∆ strain. We wish to thankthe other members of the laboratory for discussion and Nancy Rus-sell for technical assistance.

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