Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on August 22, 2005
Human Molecular Genetics 2005 14(19):2851-2858; doi:10.1093/hmg/ddi317

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Pas1, a G₁ cyclin, regulates amino acid uptake and rescues a delay in G₁ arrest in Tsc1 and Tsc2 mutants in Schizosaccharomyces pombe

Marjon van Slegtenhorst, Aladdin Mustafa and Elizabeth Petri Henske^*

Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA

^* To whom correspondence should be addressed at: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, Tel: +1 2157282428; Fax: +1 2152141623; Email: elizabeth.henske{at}fccc.edu

Received June 16, 2005; Accepted August 11, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Tuberous sclerosis complex is a tumor suppressor syndrome caused by mutations in either the TSC1 or the TSC2 gene. Previous studies have shown that deletion of the TSC1 or TSC2 ortholog in Schizosaccharomyces pombe results in an amino acid uptake defect, with conditional lethality. We identified a G₁ cyclin, pas1⁺, as a high-copy suppressor of this defect in tsc1. Disruption of pas1⁺ causes defects in arginine and leucine uptake that are remarkably similar to tsc1 and tsc2, whereas pas1tsc1 and pas1tsc2 double mutants have more severe amino acid uptake defects. In a second screen, we identified a novel G63D/S165 N mutant of the small GTPase Rhb1, the target of the Tsc1/Tsc2 protein complex. The Rhb1 mutant suppresses amino acid uptake in tsc1 yeast, but not in pas1 yeast. Hence, Pas1 does not regulate amino acid uptake through Rhb1. To determine whether Pas1 links nutrient availability to cell cycle progression downstream of the Tsc1/Tsc2 complex, we examined the kinetics of G₁ arrest in single and double mutant strains. After nitrogen starvation, tsc1 and tsc2 yeast had a delay in G₁ arrest when compared with wild-type, which was rescued by deletion of pas1⁺. In summary, we identified the G₁ cyclin, Pas1, as a novel regulator of amino acid uptake. Our data support a model in which Pas1 inhibits G₁ arrest downstream of Tsc1 and Tsc2, linking nutrient uptake and cell cycle progression in yeast.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Tuberous sclerosis complex (TSC) is a tumor suppressor gene disorder characterized by benign tumors (hamartomas), lymphangiomyomatosis and severe neurological problems including seizures, mental retardation and autism. TSC is caused by mutations in either TSC1 (1) or TSC2 (2). The protein products of TSC1 and TSC2 interact (3,4) and function as a complex. Studies in Drosophila and mammalian systems have shown that the small GTPase Rheb (Ras homolog enriched in brain) is a direct downstream target of the TSC1–TSC2 complex (5–11). Rheb binds and activates target of rapamycin (TOR) (12–14), an evolutionarily conserved kinase that integrates signals from nutrients (amino acids and energy) and growth factors (insulin) to regulate cell growth, cell cycle progression and nutrient uptake (15–17). Although many studies have focused on the downstream effectors of TSC1/TSC2 and TOR, little is known about the role of the TSC1/TSC2 complex in amino acid uptake and cell cycle regulation.

Schizosaccharomyces pombe contains genes with significant homology to mammalian TSC1, TSC2, RHEB and TOR, which are referred to as tsc1⁺, tsc2⁺, rhb1⁺, tor1⁺ and tor2⁺, respectively. We and others have previously shown that strains lacking tsc1⁺ and tsc2⁺ (tsc1 and tsc2) have defects in the uptake of arginine and leucine. This amino acid uptake defect results in a conditional lethal phenotype where growth of tsc1 and tsc2 is dependent on the concentrations of amino acids in the yeast growth medium (18,19). The defect in arginine uptake in cells lacking tsc2⁺ is rescued by expression of a dominant-negative form of rhb1⁺ (19) suggesting that Rhb1 is downstream of Tsc2 in S. pombe, as has been shown in Drosophila and mammals. Interestingly, loss of rhb1⁺ in S. pombe results in increased sensitivity to a toxic analog of arginine (20) and to a G₁ growth arrest, similar to that caused by nitrogen starvation (21,22), suggesting a link between amino acid uptake and the cell cycle.

To identify downstream targets of the Tsc1 and Tsc2 proteins in S. pombe that mediate amino acid uptake, we performed a screen for genes whose overexpression rescues the lethal growth phenotype in tsc1. This screen identified a G₁ cyclin, pas1⁺, leading us to hypothesize that pas1⁺ signals downstream of Tsc1 and Tsc2 in S. pombe to link nutrient availability to cell cycle progression. We found that pas1 yeast have defects in amino acid uptake similar to tsc1 and tsc2. However, our data suggest that the primary role of Pas1 is to inhibit G₁ arrest downstream of the Tsc1/Tsc2 complex in S. pombe. Our study therefore provides new insight into the molecular mechanisms linking amino acid uptake and cell cycle progression.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Pas1 rescues the conditional lethal growth phenotype of tsc1
We and others previously found that deletion of tsc1⁺ in the CHP429 (h^–, leu1-32, ura4-D18, ade6-216, his7-366) strain (referred to as F15tsc1) results in a conditionally lethal phenotype on essential minimal medium (EMM) supplemented with 50 mg/l of uracil, adenine, leucine and histidine, due to defects in amino acid uptake (18,19). Growth can be partially restored when the amounts of supplements are increased. To identify downstream targets of the Tsc1/Tsc2 complex that mediate this phenotype, we screened for high-copy suppressors that restore growth on 50 mg/l of supplements. We transformed F15tsc1 with a fission yeast genomic library (pAL18:leu⁺) and identified four transformants that grow on EMM with 50 mg/l of uracil, adenine and histidine. Sequence analysis indicated that tsc1⁺ was among the rescuers, serving as a positive control. One of the other rescuers contained a complete open-reading frame of SPAC19E9.03, also referred to as pas1⁺. The pas1⁺ gene was of particular interest because it encodes a putative G₁ cyclin in S. pombe (23).

We first confirmed the rescue in F15tsc1 by transformation of the original genomic fragment from the library containing the full-length pas1⁺ gene. Although pas1⁺ was identified as a rescuer of growth for F15tsc1, it also restored growth to a similar degree in F15tsc2 (Fig. 1A).

View larger version (45K): [in this window] [in a new window]   Figure 1. Rescue of conditional lethal growth phenotype of tsc1 by Pas1. (A) Tsc1, Tsc2 and Pas1 expression constructs in pAL (leu1+) were transformed into F15tsc1 and F15tsc2 and plated onto EMM plates supplemented with 50 mg/l uracil, adenine and histidine. Colonies were allowed to grow for 5 days. (B) Northern blot analysis of total RNA from the indicated strains. Hybridizations were with the pas1+, tsc1+, tsc2+ and gpd3+ probes. Signals were quantified with NIH image software and normalized to the control probe, gpd3+.

 
We had previously observed in a microarray experiment that pas1⁺ mRNA was upregulated 1.8-fold in the tsc1 strain compared with wild-type yeast (unpublished data). This led us to ask whether pas1⁺ mRNA expression is also upregulated in tsc2. We found that the mRNA expression of pas1⁺ was increased in tsc1 and tsc2 at least 2-fold. In contrast, tsc1⁺ and tsc2⁺ expressions were not significantly changed in the pas1 strain (Fig. 1B).

pas1 has amino acid uptake defects similar to tsc1 and tsc2
Tsc1 and Tsc2 are known to regulate arginine and leucine uptake in S. pombe (18,19). The arginine uptake defect results in resistance to 60 mg/l canavanine, a toxic analog of arginine. We found that the pas1 strain was also resistant to 60 mg/l canavanine, similar to tsc1 and tsc2 (Fig. 2A). To determine whether Pas1 regulates amino acid uptake downstream of Tsc1/Tsc2, we crossed pas1 with tsc2 and with tsc1 to generate pas1tsc1 and pas1tsc2 double mutant strains. We predicted that if Pas1 and Tsc1/Tsc2 are in the same pathway, the pas1tsc1 and pas1tsc2 double mutant strains would have equally impaired amino acid uptake as the single mutants. We found that the pas1tsc1 and pas1tsc2 double mutants were more resistant to canavanine than the single mutants, as reflected by enhanced growth on plates containing 60 mg/l canavanine (Fig. 2A). These results suggest that Pas1 and the Tsc1/Tsc2 complex regulate arginine uptake through parallel pathways. To determine whether the canavanine resistance in the pas1 strain was due to decreased arginine uptake by the mutant strains, uptake of ³H-arginine was measured. Arginine uptake was significantly reduced in the pas1 strain (2.5-fold, P<0.05) compared with wild-type (Fig. 2B), which is equivalent to tsc1 and tsc2. The pas1tsc1 and pas1tsc2 double mutants were more severely affected (10-fold reduction of arginine uptake) than the pas1, tsc1 or tsc2 single mutants (P<0.05) (Fig. 2B). These results show that canavanine resistance in the pas1, tsc1 and tsc2 strains is correlated with arginine uptake defects.

View larger version (52K): [in this window] [in a new window]   Figure 2. Disruption of pas1+ results in amino acid uptake and permease expression defects. (A) 972 wild-type, tsc1, tsc2, pas1, tsc1pas1 and tsc2pas1 yeast were grown in EMM overnight to midlog phase. Cells were then diluted to A595=0.4, and 10-fold different dilutions (4000 to 400 to 40 cells) were spotted on EMM plates (left panel), with 60 mg/l canavanine (middle panel) or with 30 mg/l DL-ethionine (right panel). Plates were incubated at 30°C for 3 days and then photographed. (B) Yeast cells were grown till midlog phase (A595=0.5) in EMM and were resuspended in 100 µM of L-arginine with 1 µCi of L-3H-arginine (40–70 Ci/mmol). Incorporation of L-3H-arginine was measured after 10 min. Experiments were done in triplicate and similar results were seen with two independent clones. Asterisk indicates P<0.05 for single mutants compared with wild-type; double asterisks indicate P<0.05 for double mutant compared with single mutants (T-test) (C) Incorporation of L-3H-leucine was measured after 10 min, using the same protocol outlined in (B). (D) Expression of three permease genes, SPAP7G5.06+, SPAC869.10+ and isp5+, was determined in wild-type, tsc1, tsc2, pas1, tsc1pas1 and tsc2pas1 yeast by northern blot analysis after 16 h of growth in EMM. Signals were quantified with NIH image and normalized to the control probe, gpd3+. (E) Resistance to the toxic analog of arginine, canavanine, was measured on plates for tsc1, tsc2 and pas1, overexpressing vector control, tsc1+, tsc2+ or pas1+ and compared with wild-type. Growth on canavanine (lower panel) was compared with growth on regular EMM (upper panel). (F) Incorporation of L-3H-arginine was measured after 10 min as described before in (B) in the tsc1 strain following overexpression of vector control, tsc1+ or pas1+, and in the tsc2 strain following overexpression of vector control, tsc2+ or pas1+.

 
Next, we tested whether tsc1, tsc2 and pas1 yeast are resistant to DL-ethionine, a toxic analog of methionine in S. pombe. We found that the tsc1, tsc2 and pas1 strains are resistant to 30 mg/l DL-ethionine. The pas1tsc1 and pas1tsc2 double mutant strains are more resistant to DL-ethionine than the single mutants (Fig. 2A), similar to our findings with canavanine. These data suggest that the uptake of multiple amino acids is affected by inactivation of Pas1 or the Tsc1/Tsc2 complex.

Because it was shown before that Tsc1 and Tsc2 regulate leucine uptake in S. pombe (18), we compared ³H-leucine uptake in the pas1, tsc1 and tsc2 single mutants and the pas1tsc1 and pas1tsc2 double mutants. We found that leucine uptake was reduced 2.5-fold (P<0.05) in pas1 relative to wild-type, which is comparable to tsc1 and tsc2 (Fig. 2C). The pas1tsc2 double mutants were again more severely affected than pas1 (P<0.05), tsc1 (P<0.05) or tsc2 (P<0.05) single mutants (Fig. 2C). Taken together, these data demonstrate that Pas1 and the Tsc1/Tsc2 complex regulate amino acid uptake. The additive effect for each phenotype in the double mutants suggests that Pas1 and the Tsc1/Tsc2 complex have independent and synergistic effects on amino acid uptake.

pas1 has decreased expression of amino acid permeases
We previously found that the mRNA expression of three amino acid permeases (SPAC869.10⁺, SPAP7G5.06⁺ and isp5⁺) is reduced in the S. pombe tsc1 and tsc2 strains (19). These amino acid permeases show high sequence similarity to the general amino acid permease in Saccharomyces cerevisiae, Gap1p. To determine whether permease expression in S. pombe is affected by deletion of pas1⁺, we examined the mRNA expression levels of SPAC869.10⁺, SPAP7G5.06⁺ and isp5⁺ by northern blot analysis in tsc1, tsc2, pas1, pas1tsc1 and pas1tsc2 yeast. The mRNA expression of all three amino acid permeases was reduced by deletion of tsc1⁺ or tsc2⁺, consistent with our previous study, and also by deletion of pas1⁺ (Fig. 2D). The tsc1pas1 and tsc2pas1 double mutants had reduced mRNA levels of SPAC869.10⁺, SPAP7G5.06⁺ and isp5⁺ comparable to the pas1, tsc1 or tsc2 single mutants. Together, the amino acid uptake studies and permease expression defects indicate that both Tsc1/Tsc2 and Pas1 are required for efficient amino acid uptake in S. pombe.

Pas1 overexpression partially restores the arginine uptake defect in tsc1 and tsc2
We next investigated whether overexpression of pas1⁺ could rescue the canavanine resistance of tsc1, tsc2 and pas1 mutants. As expected, the canavanine sensitivity in the pas1 strain was restored by expression of pas1⁺. Furthermore, expression of pas1⁺ restored canavanine sensitivity in the tsc1 and tsc2 strains almost to the level detected with tsc1⁺ or tsc2⁺ overexpression (Fig. 2E). We next measured radiolabeled arginine uptake when pas1⁺ was overexpressed in the tsc1 or tsc2 strain. Using this assay, we found that arginine uptake in tsc1 or tsc2 with overexpression of pas1⁺ was 50% restored (Fig. 2F). Taken together, these results suggest that arginine uptake in S. pombe is regulated through Tsc1/Tsc2 and Pas1 via independent pathways.

Rhb^G63DS165N mutant can restore canavanine sensitivity in tsc1
To complement our overexpression screen, we performed a second screen for mutations in endogenous genes that rescue the growth defect in F15tsc1 on 50 mg/l of supplements. We identified three clones (F15tsc1A2, F15tsc1A3 and F15tsc1A4) that could grow on minimal medium with 50 mg/l supplements. The F15tsc1A2 strain was sensitive to canavanine, whereas F15tsc1A3 and F15tsc1A4 were resistant to canavanine (Fig. 3A), suggesting that the mutation in the F15tsc1A2 strain rescued arginine uptake. Because we expected that dominant-negative or partial loss-of-function mutations in Rhb1 would rescue defects in tsc1, we sequenced the rhb1⁺ gene in all three clones. We identified two mutations in the rhb1⁺gene in F15tsc1A2, G63D and S165 N. No rhb1⁺mutations were identified in F15tsc1A3 or F15tsc1A4. Human and S. pombe Rheb are 52% identical at the amino acid level and an additional 35% of the residues are conserved changes (Fig. 3B). The G63D substitution occurs at a residue that is identical in the S. pombe and human sequences, whereas the S165 N substitution is at a non-conserved residue within a conserved region.

View larger version (50K): [in this window] [in a new window]   Figure 3. Rhb1G63D/S165N mutant can rescue arginine uptake in tsc1. (A) F15wt, F15tsc1 and three mutagenesis clones (A2, A3 and A4) cells were spotted (4000 to 400 to 40 cells) onto EMM plates supplemented with 50 mg/l of adenine, leucine, histidine and uracil (left panel) and with canavanine (right panel). (B) Alignment of human RHEB (Hm-Rheb) protein sequence with S. pombe Rhb1 (Sp-Rhb1) protein sequence using ClustalW (Biology Workbench 3.2). Two mutations (G63D and S165 N) that were identified in clone A2 in Rhb1 after a mutagenesis screen are indicated in boxes. Identical amino acids are denoted by an asterisk, conserved amino acids are denoted by a colon, and semi-conserved amino acids by a period. (C) 972 wild-type, tsc1, pas1, rhbG63D/S165N, tsc1rhbG63D/S165N and pas1rhbG63D/S165N yeast were grown in EMM overnight to midlog phase. Cells were then diluted to A595=0.4, and 10-fold different dilutions (4000–400–40 cells) were spotted on EMM plates (left panel) and on EMM with 60 mg/l canavanine (right panel). Plates were incubated at 30°C for 3 days and then photographed.

 
To study the effect of the Rhb1^G63D/S165N mutant on arginine uptake in pas1 and tsc2, we crossed F15tsc1A2 with the wild-type, tsc2 and pas1 strains to generate Rhb1^G63D/S165N, tsc1Rhb1^G63D/S165N, tsc2Rhb1^G63D/S165N and pas1Rhb1^G63D/S165N strains. After crossing, the presence of the Rhb1^G63D/S165N mutations in all clones was verified by sequencing. We were not able to generate Rhb1^G63D/S165N in combination with tsc2, suggesting that this genotype is lethal. We tested the effect of the Rhb1^G63D/S165N mutant on canavanine sensitivity in yeast lacking either Tsc1 or Pas1. We found that Rhb1^G63D/S165N restored canavanine sensitivity in the tsc1 strain, suggesting that Rhb1^G63D/S165N is a novel partial loss-of-function mutant, and confirmed our previous finding that Rhb1 is the target of the Tsc1/Tsc2 complex in S. pombe. However, Rhb1^G63D/S165N did not restore canavanine sensitivity in the pas1 strain (Fig. 3C), suggesting that Pas1 does not signal through Rhb1 to regulate amino acid uptake in S. pombe.

tsc1 and tsc2 have a delay in G₁ arrest, which is rescued by deletion of pas1⁺
Because Pas1 was originally identified in a search for new factors controlling the cell cycle start in S. pombe (23), we asked whether tsc1 and tsc2 yeast have a defect in G₁ arrest in S. pombe. G₁ arrest in S. pombe is induced by nitrogen starvation and results in one-copy peak in the cell cycle profile. When compared with wild-type yeast, the tsc1 and tsc2 yeast had a delay in G₁ arrest after 4 and 8 h of nitrogen starvation (Fig. 4A). We next tested whether nitrogen-induced G₁ arrest was also affected in the pas1tsc1 and pas1tsc2 double mutants. Deletion of pas1⁺ in tsc1 or tsc2 restored the delay in nitrogen-induced G₁ arrest observed in the tsc1 or tsc2 single mutants (Fig. 4A). This was unexpected, because pas1⁺ was identified in a screen for genes that by overexpression could rescue growth in F15tsc1 yeast.

View larger version (22K): [in this window] [in a new window]   Figure 4. Delay in nitrogen starvation-induced G1 arrest in tsc1 and tsc2 is rescued by deletion of Pas1. (A) 972 wild-type, tsc1, tsc2, pas1, tsc1pas1 and tsc2pas1 yeast were grown in EMM overnight to midlog phase. Cells were grown for an additional 8 h in EMM without nitrogen. Samples were fixed at 0, 4 and 8 h, and 30 000 cells were analyzed for DNA content using flow cytometry. (B) 972 wild-type, tsc1 and tsc2 yeast expressing either vector control, tsc1+, tsc2+ or pas1+ were grown in EMM overnight to midlog phase, starved for nitrogen during an 8-h time course and analyzed for DNA content as in (A). (C) Our data are consistent with a model in which Pas1 functions downstream of Tsc1/Tsc2 to inhibit G1 arrest in S. pombe.

 
Next, we tested the effect of overexpression of pas1⁺, tsc1⁺ or tsc2⁺ on the delay in nitrogen-induced G₁ arrest in tsc1 or tsc2. Exogenous expression of tsc1⁺ or tsc2⁺ rescued the delay in G₁ arrest in tsc1 or tsc2, respectively, but exogenous expression of pas1⁺ did not (Fig. 4B). Furthermore, exogenous expression of tsc1⁺, tsc2⁺ or pas1⁺ induced a small G₁ one-copy peak without nitrogen starvation, suggesting that Pas1, Tsc1 and Tsc2 regulate G₁ arrest in S. pombe (Fig. 4B). The rescue of tsc1 and tsc2 by deletion of Pas1 strongly suggests that Pas1 functions downstream of Tsc1/Tsc2 to inhibit G₁ arrest (Fig. 4C).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
We previously found that Tsc1 and Tsc2 mutants in S. pombe have amino acid uptake defects, resulting in a conditional lethal phenotype where growth of tsc1 and tsc2 is dependent on the concentrations of amino acids in the yeast growth medium (19). To identify downstream effectors that might mediate this phenotype, we used a genetic screen approach.

We report here the identification of a G₁ cyclin, pas1⁺, as a suppressor of the growth defect in tsc1 on low supplements. Disruption of pas1⁺ results in leucine and arginine uptake defects that are similar to the defects in tsc1 and tsc2. Importantly, tsc1pas1 and tsc2pas1 double mutants are more severely affected than tsc1, tsc2 or pas1 single mutants, suggesting that Pas1 regulates amino acid uptake in a pathway that is parallel to Tsc1 and Tsc2. Consistent with these results, we found that overexpression of pas1⁺ in tsc1 or overexpression of pas1⁺ in tsc1 only partially rescued the resistance of tsc1 and tsc2 to canavanine, a toxic analog of arginine. The finding that pas1⁺ mRNA was upregulated in tsc1 and tsc2 yeast suggests that all three proteins might be linked through a common downstream amino acid sensing mechanism.

Pas1 was originally identified as a multicopy suppressor of the res1 null mutant (23). Res1 is part of the Res1–Cdc10 transcriptional regulator complex that together with the redundant Res2–Cdc10 complex activate genes that are essential for the G₁–S transition in S. pombe. Pas1 is strongly conserved in evolution, with predicted orthologs in mammals, insects, worms and yeast, but nothing is known about its function in mammalian cells. The Pas1 protein in S. pombe signals in a complex with Pef1, a kinase that shows highest homology to the human CDK2 protein. Therefore, the Pas1 and Pef1 mammalian orthologs are interesting candidates for further analysis.

Overexpression of either TSC1 or TSC2 in mammalian or insect cells has also been linked to the G₁–S transition, either by lengthening G₁ or by inhibiting cell proliferation (24–26). We found that tsc1 and tsc2 cells have a delay in G₁ arrest in S. pombe, which is not rescued by pas1⁺ overexpression. However, deletion of pas1⁺ in tsc1 or tsc2 yeast restored the G₁ arrest. This result was unexpected, because Pas1 was identified in an overexpression screen, but strongly suggests that Pas1 inhibits G₁ arrest downstream of the Tsc1/Tsc2 complex. In addition, overexpression of pas1⁺, tsc1⁺or tsc2⁺ induced a small G₁ peak, consistent with the idea that both Pas1 and Tsc1/Tsc2 regulate G₁ arrest in S. pombe. The delay in nitrogen starvation-induced G₁ arrest in tsc1 and tsc2 is in agreement with the cell cycle arrest reported for Rhb1 inactivation in S. pombe (21,22). The isolation of a G₁ cyclin as a partial rescuer of growth defects of tsc1 could provide insight into the mechanisms that coordinate amino acid uptake and cell cycle progression.

We identified a novel double mutant form of Rhb1 (Rhb1^G63D/S165N) that restored the canavanine sensitivity in the tsc1 strain. Because the Rhb1 knockout in S. pombe is lethal (21,22), the Rhb1^G63D/S165N mutant is likely to result in a partial loss-of-function of Rhb1. This could prove to be a valuable tool for studying Rhb1 function in S. pombe. Crossing the Rhb1^G63D/S165N mutations into the tsc2 strain resulted in lethality, suggesting that the Rhb1^G63D/S165N strain is sensitive to inactivation by Tsc2's GAP domain. The G63D substitution changes a conserved non-polar glycine into a negatively charged aspartic acid, which is predicted to result in a hyperactive form of Rhb1 (27). S165 is not conserved in the human Rheb protein, but the surrounding amino acids are, suggesting that this residue is in a conserved domain. This mutation could lead to a conformational change in Rhb1, which in turn could directly affect interactions with downstream effector molecules. One possibility is that it may affect the ability of GTP-bound Rhb1 to activate either S. pombe Tor1 or S. pombe Tor2. In mammalian cells, GTP is not required for binding between Rheb and the TOR1C complex; however, GTP-binding is necessary for Rheb-dependent activation of the TOR1C kinase activity (14).

In summary, our data demonstrate that a G₁ cyclin, Pas1, regulates arginine and leucine uptake in S. pombe. The similarities between the pas1 and the tsc1 and tsc2 strains suggest a possible link between amino acid uptake and cell cycle regulation in S. pombe. In addition, our finding that tsc1 can be rescued by the novel Rhb1^G63D/S165N mutant supports previous findings that Tsc1/Tsc2 is upstream of Rhb1 in S. pombe. Finally, we found for the first time that tsc1 and tsc2 yeast have a delay in nitrogen starvation-induced G₁ arrest, which is rescued by deletion of pas1⁺, indicating that Pas1 inhibits G₁ arrest downstream of the Tsc1/Tsc2 complex.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Yeast strains, media and growth conditions
The yeast strains used in this study are listed in Table 1. The Pas1 mutant strain, K193-A1, was a gift from Koichi Tanaka (University of Tokyo). Wild-type strain 972 (28) was a gift from J. Bähler (Sanger Institute) and PN42 was a gift from Janni Petersen (Rockefeller University). The S. pombe cells were grown in EMM (Qbiogene, Carlsbad, CA, USA) at 30°C unless otherwise stated.

View this table: [in this window] [in a new window]   Table 1. S. pombe strains used in this study

 
High-copy suppressor screen and plasmid isolation
The pAL18 ssp41 Cs2-21 yeast genomic library, with insert size averaging 2.3 kb, was a gift from Norbert Käufer (TU Braunschweig). Yeast transformations were performed with the Frozen-EZ Yeast Transformation II kit (Zymo Research, Orange, CA, USA). After 5 days of growth, plasmids were isolated from colonies that had formed on EMM supplemented with 50 mg/l of uracil, adenine and histidine. pAL18 plasmids containing full-length tsc1⁺, tsc2⁺ and pas1⁺ were used in all transformation studies.

Construction of tsc1pas1 and tsc2pas1 double mutant strains
Tsc1⁺ and tsc2⁺ deficient strains MVS1, MVS2, MVS3, MVS4, MVS5 (F15tsc1) and MVS6 (F15tsc2) were constructed as described before (19). MVS3 and MVS4 were mated on malt extract medium with K193-A1 to generate MVS1P (referred to as tsc1pas1) and MVS2P (referred to as tsc2pas1) double mutant strains. After 2 days of mating, asci with spores were treated with snail enzyme for 6 h and subjected to random spore analysis on plates with G418 to select for tsc1, followed by EMM with no uracil to select for pas1. The double mutants were verified by PCR and loss of expression was determined by northern blot analysis.

Northern blot analysis
RNA was isolated by phenol extraction and 10 µg of total RNA was run on a 1% formaldehyde gel at 100 V for 2 h and transferred to nylon membrane overnight in 20x SSC. Probes for pas1⁺, tsc2⁺, c869.10, 7G5.06, isp5 and gpd3 were PCR amplified from cDNA, cleaned over 0.8% agarose gel and labeled with [-³²P]dCTP (Perkin Elmer, Wellesley, MA, USA) using standard methods. Hybridizations were performed in rapid hybridization buffer (Amersham Biosciences, Piscataway, NJ, USA).

Amino acid analog sensitivity
Cells were grown overnight to midlog phase (OD₅₉₅ =0.4–0.6), and OD₅₉₅ was adjusted to 0.4 (10 000 cells/µl). About 4 µl of 10x, 100x and 1000x dilution was spotted onto EMM as a growth control, or EMM containing canavanine (60 µg/ml) or DL-ethionine (30 µg/ml) (Sigma, St Louis, MO, USA) and incubated for 3 days at 30°C.

Radiolabeled amino acid uptake assays
Arginine and leucine uptake assays were performed in triplicate as described by Urano et al. (29), with minor modifications. Cells were grown in EMM minimal medium with no supplements to midlog phase. One microcurie of L-³H-labeled amino acid (40–70 Ci/mmol) (Perkin Elmer) and 100 µM of non-radioactive amino acid (Sigma) were added to 25 000 cells in 600 µl of EMM. Aliquots of 200 µl were removed at 0 and 10 min, injected into 5 ml of deionized water and immediately subjected to vacuum manifold filtration. Cells were collected on Whatman glass microfiber filters, washed twice and dried. ³H-arginine and ³H-leucine were measured by scintillation counting.

Flow cytometry
Cells were grown for 16 h in EMM until midlog phase. Cells were washed twice with H₂O and resuspended in EMM without nitrogen (Qbiogene). Cells were then grown for 4 h at 25°C to induce G₁ arrest. Cells were collected and fixed with 70% ice-cold ethanol. For FACS analysis, cells were resuspended in 50 mM sodium citrate, washed in the same buffer and treated with RNase for 16 h. Before analysis, the yeast cells were briefly sonicated and stained with propidium iodide at a final concentration of 16 µg/ml. Thirty thousand cells were collected with a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and data were analyzed with WINMDI 2.8 software (www.facs.scripps.edu).

Mutagenesis screen
Cells were grown till midlog phase, washed in TM buffer (50 mM Tris-maleate, pH 6.0) and resuspended in TM at 3.5x10⁸ cells/ml. About 350 µl of cell suspension was mixed with 150 µl of nitrosoguanidine NG (1 mg/ml in TM) and left at 30°C for 8 min to induce 30–50% cells survival (determined by kill curve). Fifty microliters of mutagenized cells were washed three times with 1 ml TM and plated onto EMM supplemented with 50 mg/l of uracil, leucine, adenine and histidine.

	ACKNOWLEDGEMENTS

 
We are grateful to Hiroto Okayama for the Pas1 mutant strain and to Warren Kruger and Mark Nellist for critical reading of the manuscript. This work was supported by the Department of Defense and by a fellowship from the Polycystic Kidney Disease Foundation (to M.v.S.).

Conflict of Interest statement. None declared.

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