Curr Genet (1999) 35: 593–601
© Springer-Verlag 1999
O R I G I N A L PA P E R
Jim Karagiannis · Reza Saleki · Paul G. Young
The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast
Received: 10 November 1998 / 15 March 1999
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 uptake observed in response to the addition of NH4+ 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 subsequent destruction of NH4+-sensitive permeases. The highpH sensitivity of cells auxotrophic for leucine thus seems to reflect an inability of NH4+-insensitive permeases to transport sufficient leucine under conditions where the proton gradient driving nutrient transport is low, and NH4+sensitive permeases have been destroyed. Intriguingly, the partial suppression of both high pH sensitivity, and the inactivating effect of NH4+ on leucine transport, seen in pub1-1 point mutants, becomes as complete as seen in pub1∆ 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
J. Karagiannis · R. Saleki1 · P. G. Young (½) 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: Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada
1
Communicated by A. Goffeau
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 transport processes including the uptake of both uracil and leucine 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 efficiency of these transport mechanisms. The effect of external pH is seen as a decrease in nutrient uptake as the pH rises. For example, the high-affinity uptake system for leucine has been shown to have a pH optimum of between 3.0 and 3.25. As the pH rises from this optimum, substrate affinity (Kt) remains unaffected whereas the capacity (Jmax) for transport becomes significantly reduced (Sychrova et al. 1989). In budding yeast NH4+ has been shown to exert its effect on nutrient transport through the negative regulation of a group of permeases involved in nutrient-specific uptake. Such NH4+-sensitive permeases include the Gap1p general amino-acid permease, the Put4p proline permease, the Gnp1p glutamine permease, and the Dal5p/Uep1p allantoin ureidosuccinate permease (Rai et al. 1988; Vandenbol 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 NH4+sensitive permeases, and the nitrogen catabolite inactivation (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-
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calized to the plasma membrane. For example, endocytic delivery to the vacuole of the Ste2p alpha-factor receptor, the Ste6p a-factor ABC transporter, the Pdr5p multidrug resistance transporter, the Mal1p maltose transporter, and the Itr1p inositol permease has been shown to be preceded by ubiquitination. Intriguingly, proteolytic degradation of these modified proteins is dependent not on the proteasome but on functional vacuolar proteases (Kolling and Hollenberg 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 Npi1pinduced turnover of both the Fur4p uracil permease and the Gap1p general amino-acid permease. Fur4p has been shown to undergo an NPI1-dependent in vivo ubiquitination. Its normal degradation is impaired in budding yeast strains defective in either endocytic internalization or the production of functional vacuolar proteases. Mutations in the regulatory or catalytic subunits of the proteasome, however, do not affect the rate of uracil permease degradation (Galan et al. 1996). Springael and Andre (1998) have also recently shown that the addition of NH4+ to proline grown budding yeast cells increases the conversion of Gap1p to ubiquitin-conjugated forms. This conversion is followed by a rapid internalization of the permease and subsequent degradation in the vacuole. NPI1 mutants are defective in Gap1p ubiquitination and this defect results in the permease remaining stable on the plasma membrane, even in the presence of NH4+. In this report we demonstrate a role for the fission yeast pub1 E3 ubiquitin ligase in modulating membrane transport processes. This role was uncovered as a result of studies where it was found that fission yeast cells auxotrophic for leucine were unable to grow in normally supplemented minimal media at high external pH (Saleki et al. 1997). Here we provide evidence suggesting that the underlying Table 1 Strain list
mechanism behind this phenotype involves a pub1-mediated down-regulation of leucine uptake in response to NH4+. Based on its homology to NPI1 we propose this down-regulation to be as a result of the ubiquitination and subsequent vacuolar destruction of NH4+-sensitive leucine permeases.
Materials and methods Strains and media. All Schizosaccharomyces pombe strains used in this work (Table 1) were derived from wild-type strains 972 (h–S) or 975 (h+N). Strains were grown in Edinburgh minimal medium (EMM), or in modified EMM in which NH4Cl was replaced with 10 mM of L-proline. High pH EMM or modified EMM were adjusted to pH 6.4 or 6.8 with NaOH and buffered with 10 mM of PIPES. Auxotrophic strains were grown in media containing 0.1 mg/ml of adenine, uracil, or L-leucine unless otherwise stated. The preparation of EMM pH 3.5 has been described elsewhere (Saleki et al. 1997). Time-course experiments. Strains were grown to mid-log phase in EMM pH 5.5 [or modified EMM pH 5.5 for experiments where cells were shifted from non-repressing (proline) to repressing (NH4+) sources of nitrogen] at 30°C with constant shaking. Cells were then diluted to approximately 1×105 cells/ml in 20 ml of the appropriately pH-adjusted EMM or modified EMM and incubated at 30°C with constant shaking. Samples were taken within 0.5 h of inoculation and at 6, 12, 24, 30, and 36 h thereafter. The number of cells/ml was determined with a Coulter Counter. Transition from non-repressing (proline) to repressing (NH4+) sources of nitrogen was achieved by the addition of 0.8 ml of a 2.5 M NH4Cl solution (final concentration 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 cultures were then split in two, one half being collected (3000 rpm for 5 min), washed, and re-suspended in modified EMM pH 5.5, and the other half in modified EMM pH 6.8 (cells were re-suspended in the same volume of media originally collected). Leucine uptake and A600 measurements were taken before the addition of NH4Cl to 100 mM, as well as at the indicated time points later. Uptake was
Strain
Genotype
Source
Q250 Q360 Q868 Q1158 Q1163 Q1164 Q1166 Q1411 Q1477 Q1485 Q1598 Q1599 Q1600 Q1601 Q1602 Q1603 Q1604 Q1605 Q1606 Q1607 Q1608 Q1609 Q1610
972 h– ade6-216 leu1-32 ura3-D18 h+ leu1-32 h– ade6-216 h– ade6-216 leu1-32 h+ ade6-216 ura4-D18 h– leu1-32 ura4-D18 h– ura4-D18 h– pub1-1 mcs4-1 ade6-216 ura4-D18 leu1-32 h90 pub1::ura4 ura4-D18 leu1-32 ade6-216 h– mcs4-1 ura4-D18 h+ mcs4-1 ura4-D18 h+/mcs4::ura4-D18 leu1-32 ade6-216 h– pub1-1 leu1-32 h+ pub1-1 spc1::ura4 ura4-D18 leu1-32 h– pub1::ura4 ura4-D18 leu1-32 h+ pub1-1 h– pub1-1 spc1::ura4 ura4-D18 h– spc1::ura4 ura4-D18 h+ pub1::ura4 ura4-D18 h+ pub1-1 wee1-50 ura4-D18 leu1-32 h– pub1::ura4 wee1-50 ura4-D18 leu1-32 h– mcs4::ura4-D18 leu1-32 ade6-216 h– (GP65) spc1::ura4 ura4-D18 leu1-32 h– (KS1366)
Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Saleki et al., 1997 Nefsky and Beach, 1996 This study This study This study This study This study This study This study This study This study This study This study Cottarel, 1997 Shiozaki and Russell, 1995
595 measured by adding 0.7 ml of culture to a microcentrifuge tube containing 1–5 µCi of L-[4,5-3H(N)]-leucine (50 Ci/mmol; NEN Life Science Products) as well as unlabeled leucine (to bring the final leucine concentration to the indicated levels upon addition of the culture). 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 filtered through 0.45-µm polycarbonate membranes which were washed three times with 1 ml of 10 mM leucine before being transferred to vials, and the radioactivity quantitated by liquid scintillation counting.
Results
The high-pH sensitivity of auxotrophic cells is due to inefficient uptake of nutrients from the environment
D18 mutations is entirely dependent on efficient uptake of leucine and uracil from the environment, we hypothesized that the inhibitory effect of high external pH was attributable to interference with the functioning of nutrient-specific transport mechanisms. As a simple test of this model, we increased the concentration of uracil and leucine in the growth media ten times (to 1 mg/ml). Under these conditions strains auxotrophic for uracil or leucine were able to proliferate in media adjusted to pH 6.4, while strains grown with more typical concentrations of supplements (0.1 mg/ml) demonstrated a severe inhibition of growth. At pH 6.8, increased supplement concentrations also suppressed the growth inhibition seen in uracil auxotrophs, while the growth of leucine auxotrophs remained inhibited (Fig. 2). The inability
During research investigating plasma membrane transporters and their role in pH homeostasis, it became apparent that fission yeast cells carrying either the leu1-32 or ura4D18 mutations were unable to proliferate when the pH of the growth media (normally pH 5.5) was adjusted to pH 6.4 or above. In contrast, cells carrying the ade6-216 mutation showed no sensitivity to high external pH (Fig. 1). Since the normal growth of cells harboring the leu1-32 or ura4-
Fig. 1 Fission yeast strains auxotrophic for uracil and/or leucine are unable to proliferate in normally supplemented minimal media at high external pH. Strains as per legend were plated on EMM pH 5.5 or EMM pH 6.8 and incubated for 72 h at 30°C. Plates were supplemented with 0.1 mg/ml of adenine, uracil, and leucine
Fig. 2 Increasing the concentration of uracil or leucine supplements suppresses the high-pH sensitivity of cells auxotrophic for uracil or leucine, respectively. Actively growing cells auxotrophic for leucine (squares) or uracil (circles) were inoculated into EMM pH 5.5, EMM pH 6.4, or EMM pH 6.8 with either 0.1 mg/ml supplements (closed symbols) or 1 mg/ml supplements (open symbols)
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of leucine auxotrophs to proliferate at pH 6.8 in the presence of 1 mg/ml of leucine most likely reflects a more-severe inhibition of leucine transport by high external pH conditions. The fact that adenine auxotrophs showed no high-pH sensitivity supports the contention that discrete nutrient transport systems can be affected differentially by a 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 (elongated at low pH) mutations were isolated as extragenic suppressors of high-pH sensitivity from a fission yeast strain carrying the ade6-216, ura4-D18 and leu1-32 auxotrophic markers. Neither mutation alone was sufficient to suppress growth inhibition of the auxotrophic strain at pH 6.8. Interestingly, these mutations also showed a negative interaction at low external pH (pH 3.5). pub1 was cloned and shown to encode an E3 ubiquitin ligase (Saleki et al. 1997). The molecular identity of elp3, however, remained unknown. The identity of elp3 was first suggested by a similarity of phenotype in strains carrying the recessive elp3-1 and mcs4::ura4 (mcs4∆) mutations. When streaked on EMM plates adjusted to pH 3.5 both elp3-1 and mcs4∆ strains show a cell-elongation phenotype indicative of a delay in cell-cycle progression. Free-spore analysis, as well as an analysis of 18 tetrads from a genetic cross between an elp3-1 and an mcs4∆ strain, revealed no wild-type segregants. To confirm the allelism of elp3 and mcs4, elp3-1 ura4-D18 /mcs4::ura4 ura4-D18 leu1-32 ade6-216 diploids were constructed and tested by streaking on EMM pH 3.5 plates. These diploids demonstrated a pH-sensitive cell-cycle delay. The absence of complementation therefore indicated that the elp3 and mcs4 loci were allelic. The mcs4 locus encodes a protein with homology to bacterial two-component system response regulators and has been shown to be an upstream regulator of the spc1 stressactivated MAPK pathway (Cottarel 1997; Shieh et al. 1997; Shiozaki et al. 1997). mcs4 loss-of-function mutations were originally isolated as suppressors of the lethal premature initiation of mitosis associated with cdc2-3w wee1-50 strains at their restrictive temperature (Molz et al. 1989). To determine whether mcs4 was acting through the spc1 pathway, or an alternate pathway, we constructed pub1-1 spc1::ura4 (spc1∆) double mutants. As shown in Fig. 3 spc1∆, like the mcs4-1 point mutation, was able to suppress the growth inhibition caused by leucine auxotrophy at pH 6.8 when placed in a pub1-1 background. However, leu1-32 pub1-1 mutants were unable to proliferate under these same conditions. Interestingly, leu1-32 pub1::ura4 (pub1∆) strains were able to grow at pH 6.8 while leu1-32 spc1∆ strains remained sensitive to high external pH. In fact growth of the leu1-32 spc1∆ strain was completely inhibited even at a less-restrictive pH of 6.4, conditions where pub1-1 leu1-32 cells proliferated well.
Fig. 3 The pub1-1 mutation and spc1∆ show synergy in suppressing the high-pH sensitivity of cells auxotrophic for leucine. Actively 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)
Interestingly, the synergistic relationship between pub1-1 and the spc1∆ is also discernible at low extracellular pH (pH 3.5). Under these conditions pub1-1 cells are slow growing, and slightly elongated, whereas spc1∆ cells are elongated, but capable of colony formation. Double mutants are synthetically lethal and arrest as microcolonies of highly elongated cells (data not shown). The high-pH sensitivity of auxotrophic cells is dependent on the presence of NH4+ in the growth media Since NH4+ is known to negatively regulate the activity of several amino-acid and nucleoside permeases (Grenson 1983; Horak 1986; Grenson 1992; Hein et al. 1995; Galan
597 Fig. 4 The high-pH sensitivity of cells auxotrophic for leucine is dependent on the presence of NH4+ in the growth media. Actively growing cells from an overnight culture of the indicated strains were inoculated into two separate proline-containing modified minimal media pH 6.8 liquid cultures. One of the two cultures was supplemented with NH4Cl to a final concentration of 100 mM 6 h after inoculation (open symbols) while the other culture remained untreated (closed symbols). The arrow indicates the time of addition of NH4Cl
et al. 1996; Springael et al. 1998) we decided to study the performance of auxotrophic cells in the absence of NH4+. Interestingly, when L-proline was used as a nitrogen source, auxotrophic cells showed no sensitivity to high external pH. Conversely, the addition of NH4+ to prolinegrown cells at high external pH was sufficient to bring about a rapid and complete growth arrest to cells auxotrophic for leucine. leu1-32 cells carrying either the pub1∆, or the pub1-1 mutation plus spc1∆, were entirely insensitive to the addition of NH4+ and demonstrated no growth arrest. As expected, leu1-32 cells carrying pub1-1 or spc1∆ alone were unable to suppress the growth inhibitory effect of 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+-insensitive system which is negatively regulated by high external pH (such a system must exist since leucine auxotrophs are able to grow in a normally supplemented NH4+-containing medium at pH 5.5) as well as an NH4+-sensitive system. The data also suggest that both pub1 and spc1 are involved in negatively regulating this NH4+-sensitive system of leucine uptake. To test these models we chose to monitor the uptake of 3H-labeled leucine in the presence or absence of NH4+, at both pH 6.8 and pH 5.5, in each of our mutant backgrounds.
Leucine uptake is sensitive to both NH4+ and extracellular pH We first performed experiments looking at the response of a wild-type strain to the addition of NH4+ at a leucine concentration of 0.76 mM (the concentration of leucine used in our growth assays). As seen in Fig. 5 the initial rate of uptake drops quickly and dramatically (from the rate observed 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 6.8 than at pH 5.5. We thus establish that leucine uptake is sensitive both to the presence of NH4+ as well as to extracellular pH conditions. Since previous work has demonstrated the existence of two leucine-uptake systems in fission yeast (a high-affinity system with a Kt of 0.05 mM, as well as a low-affinity system with a Kt of 1.25 mM; Sychrova et al. 1989) we also examined the effect of both high and low leucine concentration on the response of leucine uptake to NH4+. At concentrations where one would expect only the high-affinity system to be importing leucine at a significant capacity (0.01 mM) uptake drops greater than 30 fold (to almost zero) 1 h after the addition of NH 4+ (Fig. 6). However, at concentrations where one would expect both systems
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Fig. 5 Leucine uptake is sensitive to both NH4+ and extracellular pH. Wild-type cultures were grown to 1–2×106 cells/ml in modified EMM pH 5.5, collected, and then re-suspended, at the same density, in either modified EMM pH 5.5, or modified EMM pH 6.8. Leucine uptake and A600 measurements were taken from the cultures marked “Proline + NH4+ pH 5.5” and “Proline + NH4+ pH 6.8” at the indicated time points after the addition of NH4Cl to 100 mM (time 0). A separate modified EMM pH 5.5 culture, treated with water instead of NH4+ (marked “Proline pH 5.5”), was used as a control. Experiments were run in parallel in a staggered fashion. Uptake assays were performed at a leucine concentration of 0.76 mM. Bars indicate standard errors, n=3
to be importing leucine efficiently (2 mM) the drop in response to NH4+ is only 2–3 fold. These data suggest that the high-affinity system is under a strong negative regulation by NH4+ and that the lower affinity system is relatively 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 comparable to that seen in proline media after the addition of NH4+ (Fig. 7). The pub1-1 mutant showed an intermediate phenotype between that of a wild-type and a pub∆ strain i.e., it was unable to completely suppress the loss of uptake 1 h after the addition of NH4+, but did show a statistically significant higher level of uptake compared to the wild-type. Surprisingly, spc1∆ strains showed no ability to suppress the inactivating effect of NH4+ on leucine uptake. In fact its only impact was seen in pub1-1 backgrounds, in which it had the effect of converting the observed partial suppression to a complete suppression (Fig. 7).
Discussion
Based on its homology to Npi1p of budding yeast we propose Pub1 to be involved in the ubiquitination and degradation of leucine-specific permeases in response to NH4+.
Fig. 6 Leucine uptake is more severely affected by NH4+ at low leucine concentrations. Wild-type cultures were grown to 1–2×106 cells/ml in modified EMM pH 5.5, collected, and then re-suspended, at the same density, in either modified EMM pH 5.5, or modified EMM pH 6.8. Leucine uptake and A600 measurements were taken just before the addition of NH4+ to 100 mM as well as 1 h later. Uptake assays were performed at leucine concentrations of 0.01 mM and 2 mM. Bars indicate standard errors, n=3
We suggest that the destruction of these leucine-specific permeases results in a decrease in total uptake capacity severe enough to inhibit the growth of cells auxotrophic for leucine, at high external pH, in the presence of standard concentrations of leucine supplements. Both growth and leucine-uptake data are supportive of such a model. We clearly demonstrate an NH4+ sensitivity to both the growth of cells auxotrophic for leucine, as well as to leucine uptake. In addition we demonstrate an ability of pub1 mutants to suppress both this growth inhibition, and the inactivating effect of NH4+ on uptake (Figs. 3, 4, 7). The fact that increased leucine concentration can suppress growth inhibition at high external pH (Fig. 2) corroborates the idea that proliferation by pub1 mutants under these conditions can be explained in terms of the modulation of leucine transport. We also supply additional support for the contention that the pH-sensitive nature of this growth inhibition reflects the dependence of nutrient uptake on the strength of
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Fig. 7 Mutations in pub1 suppress the inactivating effect of NH4+ on leucine uptake. Cultures of the indicated strains were grown to 1–2×106 cells/ml in modified EMM pH 5.5, collected, and then resuspended, at the same density, in either modified EMM pH 5.5, or modified EMM pH 6.8. Leucine uptake and A600 measurements were taken just before the addition of NH4+ to 100 mM as well as 1 h later. Uptake assays were performed at a leucine concentration of 0.76 mM. Bars indicate standard errors, n=3
the electrochemical proton gradient driving secondary transport processes. In agreement with previous results (Sychrova et al. 1989) we show leucine uptake to be negatively affected at high extracellular pH (Figs. 5, 6, 7). Furthermore, we provide preliminary evidence suggesting the high-affinity leucine-uptake system of fission yeast 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 if such a model is assumed. These findings are consistent with earlier growth data which suggested the existence of both NH4+-sensitive and NH4+-insensitive systems of uptake.
Perhaps the most surprising and enigmatic finding of this study was the discovery of the allelism between elp3 and mcs4. The mcs4 locus was initially isolated in a screen for mutations capable of suppressing the premature and lethal initiation of mitosis associated with cdc2-3w wee1-50 strains at their restrictive temperature (Molz et al. 1989). In addition to its ability to modulate the mitotic control of fission yeast, mcs4 has also recently been shown to be involved in the response to extracellular stress through its regulation of the spc1/sty1/phh1 stress-activated MAPK pathway. Through formal genetic and biochemical analysis Mcs4, which has significant homology to bacterial twocomponent response regulators, has been placed upstream of Wak1/Wik1 MAPKKK, Wis1 MAPKK, and Spc1 MAPK (Cottarel 1997; Shieh et al. 1997; Shiozaki et al. 1997). The spc1 pathway is best known for its ability to modulate mitotic initiation under conditions of cellular stress (for a review describing this and other MAPK pathways in yeast see Banuett et al. 1998). Loss-of-function mutations in this pathway result in a delay in mitotic initiation that is exacerbated under conditions of high osmolarity and nutrient limitation. Integrity of the pathway is also required for proper responses to a range of environmental insults including heat shock, UV exposure, oxidative stress (Shiozaki and Russell 1995; Degols et al. 1996; Wilkinson et al. 1996; Samejima et al. 1997; Shieh et al. 1998), and low extracellular pH (this study). Interestingly, the spc1 MAPK pathway has also been shown to regulate membrane transport processes in fission yeast. For example, wis1, the MAPKK of the spc1 pathway, is required for the proper onset of gluconate transport under conditions of glucose starvation (Caspari 1997). In addition, spc1 positively regulates both the Hba2 and Pmd1 ABC transporters involved in multidrug resistance (Toone et al. 1998). In another of its many roles, spc1 is also required for proper vacuolar dynamics in response to both hypo- and hyper-osmolarity (Bone et al. 1998). Since a reduced ability to endocytose and/or deliver nutrient-specific permeases to the vacuole for degradation is one hypothesis explaining how spc1 mutations might negatively regulate nutrient uptake, and since loss-of-function mutations in Npi1 cause endocytosis defects in budding yeast (Zoladek et al. 1997), we tested our pub1 and spc1 mutants for endocytosis defects using the fluorescent dyes FM4-64 and lucifer yellow. No defects were observed (data not shown). Unlike mutations in mcs4, mutations in wak1, wis1 or spc1 are unable to suppress the mitotic catastrophe phenotype of cdc2-3w wee1-50 mutants. This indicates that mcs4, in addition to its modulation of mitotic control via the spc1 pathway, also provides modulation via a second independent pathway. With respect to its ability to suppress high-pH sensitivity, we show mcs4 to be acting through modulation of the spc1 pathway, as spc1∆, just like the mcs4-1 point mutation, is able to suppress the growth inhibition of leucine auxotrophs at pH 6.8, when placed in a pub1-1 background. We go on to show that the partial suppression of the inactivating effect of NH4+ on leucine
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uptake, seen in pub1-1 backgrounds, is converted to a complete suppression by a concomitant loss of the function of spc1. The similarity of phenotypes between pub1-1 spc1∆ and pub1∆ strains, the fact that the pub1-1 mutation partially suppresses both high-pH sensitivity and the inactivating effect of NH4+ on leucine uptake (whereas pub1∆ is able to completely suppress), together with the fact that we have been unable to discern any effect of spc1 pathway mutations on either high-pH sensitivity, or leucine uptake, when not in a pub1-1 background, suggests a model in which spc1∆ completes a partial loss of function of the pub1-1 allele. The notion that pub1-1 encodes a partial loss of function is also corroborated by two independent lines of reasoning. First, pub1-1 strains are slow growing but able to form colonies when plated at pH 3.5. pub1∆ strains, on the other hand, are inviable under these same conditions (data not shown). Second, pub1-1 wee1-50 double mutants are slow growing but viable at 36°C (data not shown), whereas pub1∆ wee1-50 mutants are inviable (inviability is the result 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 of pub1-1 mutants, when compared to pub1∆ mutants, strongly suggest that pub1-1 mutants retain some residual Pub1 function. It should be noted that the synergy between the pub1-1 allele and spc1∆ is discernible not only at a high external pH, but also at a low extracellular pH where strains carrying both mutations give rise to a synthetic cdc phenotype. The nature of this synergy, however, remains enigmatic. Interactions between MAPK pathways and ubiquitination pathways have been uncovered in other systems, most notably in human B and T cells where phosphorylation of the bcl-6 proto-oncogene by MAPK targets its destruction via the ubiquitin/proteasome pathway (Niu and Dalla-Favera 1998). Acknowledgements This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada to P. G. Y. David Beach is gratefully acknowledged for the provision of the pub1∆ strain. Paul Russell is gratefully acknowledged for the provision of the spc∆ strain. Guillaume Cottarel is gratefully acknowledged for the provision of the mcs4∆ strain. We wish to thank the other members of the laboratory for discussion and Nancy Russell for technical assistance.
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