Mol Gen Genet (1992) 234:193-200 © Springer-Verlag •992

ore2, a mutation affecting proline biosynthesis in the yeast Saccharomyces cerevisiae, leads to a cdc phenotype Philippe Neuville* and Michel Aigle Laboratoire de g~n~tique, Universit~ de Bordeaux II, CNRS URA 542, Avenue des facult~s, 33405 Talence Cedex, France Received September 6, 1991 / Accepted March 26, 1992

Summary. We report here the isolation of temperaturesensitive mutants of the yeast Saecharomyces cerevisiae which exhibit cde phenotypes. The recessive mutations defined four complementation groups, named orel, ore2, ore3 and ore4. At the non-permissive temperature, strains bearing these mutations arrested in the G1 phase of the cell cycle. The wild-type allele of the gene altered in ore2 mutants was cloned. The nucleotide sequence of a fragment which can complement the mutation showed the presence of an open reading frame capable of encoding a protein with 286 amino acid residues. The deduced amino acid sequence showed 25% identity with that of the Eseherichia coli Al-pyrroline-5-carboxylate reductase, an enzyme of the pathway for the biosynthesis of proline. The ore2 mutants, correspondingly, were found to be capable of growing at the non-permissive temperature on a synthetic medium supplemented with proline. In addition, the chromosomal location of the gene and its restriction map were compatible with those previously reported for the P R O 3 gene which encodes the S. cerevisiae Al-pyrroline-5-carboxylate reductase. Key words: Saccharomyces cerevisiae - Cell cycle Proline - DNA sequencing

Introduction In eukaryotic cells, the regulation of cell division occurs within the G1 period of the cell cycle, between the completion of cytokinesis and the onset of DNA replication. Our area of interest concerns the nature of the G1 regulation in the budding yeast Saccharomyces cerevisiae, a useful model for these studies. Many mutants defective in some specific event in the cell cycle have been isolated * Present address: Wetlcome Laboratories for Experimental Parasitology, University of Glasgow, Bearsden Road, Glasgow G61 1QH, Scotland Correspondence to: M. Aigle. EMBL Data Library accessionnumber for the nucleotide sequence: X57338

(for a review see Wheals 1987). Some of these have been used to define a major regulatory step in G1, which has been referred to as "Start" (Hartwell et al. 1974). In addition to the case of cells arrested at Start either by mutation or by the presence of one of the yeast pheromones, another form of Start arrest is represented by cells in stationary phase: when yeast cells are deprived of an essential nutrient, they complete cell division and arrest in G1. In that state, yeast cells are more resistant to zymolyase (Deutch and Parry 1974), nystatin (Snow 1966) and to heat treatment (Walton et al. 1979; Plesset et al. 1987) than dividing cells. These particular properties led some authors to suggest that stationary phase cells are actually in a unique developmental stage referred to as GO (Pifion 1978; Pifion and Pratt 1980; Iida and Yahara 1984). The existence of such a unique developmental stage implies the existence of unique gene functions required for resumption of growth after stationary phase. Two groups have tried to isolate mutations affecting these putative functions. While Bedard et al. (1981) isolated no specific mutations and concluded that GO is only a part of the G1 phase, Drebot et al. (1987) isolated a double mutant defective only for reentry into the mitotic cycle from the stationary phase. In order to study the mechanisms involved in the establishment, maintenance or termination of stationary phase, we used a novel selection scheme to isolate conditional mutants presenting stationary phase characteristics when transferred to a rich medium after starvation. All the mutants we obtained presented edc phenotypes. One of these mutants was more extensively studied and the gene affected was characterised. This gene was identified as the P R O 3 gene, coding for an enzyme of the proline biosynthesis pathway.

Materials and methods Strains and media. Escherichia coli: The strain used for plasmid propagation was DH1; (F-, f e c a l , 9yrA96, endA1, thr~l, hsdR17 (rK-, ink-), supE44, ~-).

194

S. cerevisiae: All the strains used in this study were derived from X2180; (a/a, SUC2/SUC2, mal/mal, mel/ mel, 9al2/gal2, CUP1/CUP1). LG17-1D (a, ura3, leu2) was used for mutagenesis. Mutant strains used for the physiological studies were orel-A (URA3, LEU2, orel), ore2-A (URA3, LEU2, ore2), ore3-A (URA3, LEU2, ore3) and ore4-A (URA3, LEU2, ore4), Strain ore2-25B (ura3, trpl, ore2) was used for the ORE2 gene cloning. E. coli was grown in dYT (1.6% Bacto tryptone, 1% yeast extract, 0.5 % NaC1). This medium was supplemented with 0.005% ampicillin when used for the selection and growth of transformed bacteria. Yeast YPD or YPGE media were used as the complete medium. YPD contained 1% yeast extract, 1% Bacto tryptone and 2% dextrose. In YPGE, the dextrose was replaced by 1% glycerol and 1% ethanol. Yeast synthetic media YNB contained 0.67 % yeast nitrogen base without amino acids (Difco Laboratories), 0.012 % of each amino acid except methionine and cysteine, and 0.01% adenine and uracil. These media were buffered at pH 5.8 with 1% succinate and 0.6% NaOH. The carbon sources were either 2% glucose (YNBG), 0.1% glucose (G1), or 0.5% ethanol (YNBE). In all cases, 100 ml liquid cultures were grown in 500 ml flasks with rotary shaking. The solid media were obtained by addition of 2 % (w/v) agar (DifCO).

Mutagenesis. Ethyl methane sulfonate (EMS) mutagenesis was performed according to Fink (1970) and nitrous acid mutagenesis according to Meuris et al. (1967). Ultra-violet mutagenesis was performed by exposing yeast cells in plate culture to a UV source, under conditions which allowed 1% of the ceils to remain viable.

Isolation procedure. Enrichment: The cells were cultured at the permissive temperature (21 ° C) in YNBE medium and progressively transferred to the non-permissive temperature (37 ° C) during exponential growth. The cultures were then diluted 1/10 in fresh YNBE medium at 37 ° C, in order to dilute the thermosensitive mutants. Three days after the cultures had reached stationary phase, they were diluted 1:3 in YNBG medium at 37 ° C. When the OD6o o had doubled, the cells were treated with nystatin (Snow 1966). The nystatin was then removed by washing the cells twice with sterile water at 48 ° C. This treatment preferentially kills the dividing cells. The mutagenised cells were submitted to two rounds of this enrichment procedure. Screening: yeast cells were plated at 21 ° C on complete solid medium (YPGE) after the enrichment procedure. Colonies were then picked onto G1 plates which were incubated at 37 ° C for 5 days, in order to allow the cells to enter stationary phase. The colonies were then replicated on YPD and YPGE media and incubated at either 21 ° C or 37 ° C. The mutant clones able to form colonies at the permissive temperature, but not at the non-permissive temperature, were thus selected.

Measurement of cellular parameters. Yeast cell concentration was determined by measurement of the optical

density at 600 n m (OD6oo) and the percentage of budding cells measured after brief sonication by using a haematocytometer. The cellular viability was determined using methylene blue colouration according to Pierce (1977). Resistance to heat treatment was determined on cells cultured in YNBE medium. The cultures were shifted from 21 ° C to 37 ° C when the OD6o o was 0.1. After 24 h at 37 ° C, the cultures were incubated in a 48 ° C water bath. Cells were sampled at different times thereafter, and dilutions were plated on YPD medium. The number of yeast colonies was determined after 5 days incubation at 21 ° C.

Yeast genomic DNA library. The genomic library was constructed by M. Crouzet (Universit6 de Bordeaux II) by cloning partial Sau3A digests of genomic DNA from the strain SKQ2n (a/a, adel/ADE1, ade2/ADE2, hisl/ HIS1) from B. Cox's collection (Oxford University), into the yeast-E, coli shuttle vector pUKC200 (University of Kent, Canterbury). This vector is a yeast monocopy vector which contains the yeast TRP1 selection marker. DNA manipulations. Yeast transformation was performed according to the LiCI method described by Ito et al. (1983). Plasmid recovery from yeast cells was performed as described by Lorincz (1984). Standard molecular techniques and E. coli transformation were performed according to Maniatis et al. (1982). Deletion-derivative plasmids were obtained using a kit containing exonuclease III and mung bean nuclease according to the manufacturer's instructions (Stratagene). DNA sequencing was performed on double-stranded plasmids according to the dideoxy chain-termination method (Sanger et al. 1977) using the commercially available Sequenase Version 2.0 sequencing kit (US Biochemicals) and 1000Ci/mmol a[35S]dATP (Amersham). Computer analysis of the nucleotide and amino acids sequences were done using the CITI2 mainframe computer facilities (Centre Interuniversitaire de Traitement de l'Information, 45 rue des Saints-Pbres 75270 Paris, France).

Results

Isolation and genetic analysis of yeast mutants showing an altered resumption of growth after stationary phase The procedure employed in this study was designed to isolate mutants conditionally defective in the initiation of the cell cycle. Three different mutagenesis procedures were used in order to increase the number of potential mutants. The enrichment procedure was based on fundamental characteristics of cells from stationary phase cultures: viability in nutrient-depleted medium, resistance to nystatin and heat treatments. The three sets of mutagenized cells were independently submitted to two enrichment cycles as described in Fig. 1. The average survival rate of cells was 10 .4 per enrichment cycle. The clones were then screened for their ability to resume growth after stationary phase. From 2700 independent clones screened, 36 mutants were isolated. These mutants

195

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were able to f o r m colonies on a rich m e d i u m at 21 ° C after the stationary phase, but n o t at 37 ° C. E a c h m u t a t i o n was crossed into other genetic backg r o u n d s for further study. The diploid strains obtained, each heterozygous for one o f the mutations, showed that each m u t a t i o n was recessive to its wild-type allele. E a c h o f the recessive m u t a t i o n s segregated as a single nuclear gene. Analysis o f the different m u t a t i o n s d e m o n s t r a t e d that they belong to four c o m p l e m e n t a t i o n groups, which were n a m e d o r e l , ore2, ore3 and ore4.

ore phenotypes

To characterize the ore m u t a t i o n s m o r e extensively, g r o w t h o f all 36 m u t a n t s was examined in liquid media. W h e n strains bearing individual m u t a t i o n s were shifted to the nonpermissive temperature, g r o w t h stopped and the m u t a n t p o p u l a t i o n a c c u m u l a t e d as u n b u d d e d cells. These studies were m a d e in complete or synthetic media (YP or Y N B ) containing either a fermentable ( Y P D and Y N B G ) or a respiratory c a r b o n source ( Y P G E a n d Y N B E ) . Similar results were obtained with the different media. Figure 2 shows the g r o w t h curve o f one m u t a n t f r o m each c o m p l e m e n t a t i o n g r o u p in Y P D medium. In this m e d i u m m o r e t h a n 80% o f the m u t a n t cells accumulate u n b u d d e d after transfer to 37 ° C. The p r o p o r t i o n o f viable cells was greater t h a n 85% after 55 h at the non-permissive temperature. In addition, the rate o f viable cells after treatment at 48 ° C was greatly increased in the case o f the m u t a n t arrested cells c o m p a r e d to the wild-type growing cells (Table 1). These phenotypes cor-

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respond to the cdc definition and not to the phenotype of mutants affected only for the resumption of growth after stationary phase. Because of their characteristics, we concluded that these are temperature-sensitive mutations which cause cells to arrest in a particular step of the G1 phase, where they show some properties comparable to stationary phase cells.

Molecular cloning of the ORE2 9ene The ore2 mutation appeared, during the genetic analysis, to be linked to the ura3-1 marker and thus to be located on chromosome V. The recombination frequency between the two mutations was 0.34. We further localized the ore2 mutation on the right arm of chromosome V since it was not linked to the canl-lO0 mutation, which is linked to ura3-1 but located on the left arm of the chromosome. Because no CDC gene has been localised to this position, we interpreted this to mean that the ore2 phenotype had not been described previously. The ORE2 gene was cloned by functional complementation. A yeast genomic library prepared in the monocopy vector pUKC200 was used to transform the mutant strain ore2-25B. Transformed cells able to grow at the non-permissive temperature were selected. Plasmid DNA from each of these transformants was then extracted and propagated in E. coli. These plasmids were then tested for their ability to complement the ore2 mutation. This enabled us to characterise three plasmids, p7, p9 and p21, which were able to complement, or to suppress, the

ore2 mutation. The restriction maps of the inserts harboured by these plasmids are presented in Fig. 3. The KpnI restriction fragment common to the different inserts was purified, labelled, and used to hybridize to yeast chromosomes separated by pulsed field electrophoresis. The DNA fragment hybridized to chromosome V on which the ore2 mutation had previously been localized (data not shown). Thus, it appeared more likely that the cloned DNA contained the wild-typed allele of the mutated gene rather than a suppressor of the mutation.

Sequence determination Deletions of the p7 insert were obtained using exonuclease III and mung bean nuclease. The resulting plasraids were first used to determine the minimal DNA fragment able to complement the ore2 mutation, and secondly to establish the of the nucleotide sequence gene. The sequencing strategy is shown in Fig. 4. The deduced 2419 bp DNA sequence is presented in Fig. 5. This sequence reveals the presence of an 858 bp open reading frame (ORF) encoding a putative protein of 286 amino acids. Comparison of the putative amino acid sequence with the Swissprot data base revealed a 25 % homology between the ORE2 protein and the E. coli A 1-pyrroline-5carboxylate reductase (Deutch et al. 1982). Alignment of the two amino acid sequences is presented in Fig. 6. Al-pyrroline-5-carboxylate reductase is involved in the proline biosynthesis pathway. This enzyme is encoded by the proC gene of E. coli, and PRO3 gene of S. cerevisiae (Brandriss 1979).

Relationship between ORE2 and PRO3 genes The PRO3 gene has been cloned and its restriction map published (Tomenchok and Brandriss 1987). The restriction maps of the ORE2 and PRO3 genes, despite a few differences, are compatible. The PRO3 gene is located on the right arm of the chromosome V, as is the ORE2 gene (see above). The genetic distance between ura3 and pro3 mutations was determined to be 37 cM (Tomenchok and Brandriss 1987) while we observed a 0.34 recombination frequency between ura3 and ore2. In order to confirm that the two genes are identical, we investigated whether the ore3 mutant was conditionally proline deficient. The proline biosynthesis step in which the Al-pyrroline-5carboxylate reductase is involved is presented in Fig. 7. This enzyme is the only one that is common to the two proline biosynthesis pathways, from arginine or glutamate. The pro3 mutants are unable to grow on complete medium, even when the medium is supplemented with proline. They are only abled to grow on a synthetic medium containing 1 g/1 proline (Brandriss 1979). Examination of the ore2 mutant on such a medium showed that the thermosensitive phenotype was suppressed by the addition of proline. All these results indicate that the ore2 mutation is a thermosensitive pro3 allele.

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Discussion Conditional mutations are useful for the study of essential cellular functions such as those involved in cell division (Pringle 1978). We have used a novel protocol which leads to the isolation in S. cerevisiae of temperaturesensitive mutations in genes whose products affect completion of the Start event. All the mutations obtained are recessive alleles of single genes. They define four complementation groups.

Physiological studies suggest that cells bearing these mutations arrest in G1 after transfer to the nonpermissive temperature. In the m u t a n t strains isolated in this study, in contrast to m o s t of the previously described Gl-arrested, temperature-sensitive mutants (Hartwell et al. 1974; Reed 1980), no loss o f viability is observed when they are incubated at the non-permissive temperature. The mutations are thus considered to lead to an arrest of cell division in a physiological state comparable to stationary phase.

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We have cloned and sequenced the wild-type allele of the ORE2 gene, defined by one of the complementation groups. The nucleotide sequence of a 2419 bp DNA fragment revealed the presence of an ORF which encodes a putative protein of 286 amino acid residues. The amino acid sequence is 25 % identical to the sequence of the E. coli Al-pyrroline-5-carboxylate reductase. The chromosomal location of the gene and its restriction map, as well as the mutant phenotype, allowed us to identify the affected gene as the S. cerevisiae PRO3 gene. This gene encodes At-pyrroline-5-carboxylate reductase, which is the last enzyme of the proline biosynthesis pathway. These experimental results raise two main questions. The first is the nature of the stationary phase and the existence of a specific GO state. We attempted to isolate mutations which specifically affect the resumption of growth from stationary phase. Such mutations would have been useful for the study of the stationary phase by defining genetic events specifically associated with this particular physiological state. Bedard et al. (1981) tried to isolate such mutants using different selection procedures mainly based on known characteristics of Start. All the mutations they isolated led to ode phenotypes. However, Drebot et al. (1987) described a mutant strain conditionally defective only for re-entry into the mitotic cell cycle from stationary phase. The fact that the phenotype of this mutant is due to the interaction be-

Fig. 6. Alignment of the deduced amino acid sequences of the O R E 2 (upper sequence), and Eseherichia eoli proc + (lower sequence) putative gened products. Dots correspond to identical amino acid residues

tween mutations in two different genes makes this result difficult to interpret, In this study we used an enrichment procedure based on the fundamental properties of the stationary phase. The fact that we failed to isolate the mutants we were seeking does not allow us to conclude that there are no specific genetic events required for the resumption of growth after stationary phase. The specificity of our selection procedure, however, suggests that the latter is probably the case. The second problem concerns the relationships between the biosynthesis of proline and the regulation of the cell cycle. It is known that amino acid starvation, or undercharging of a corresponding tRNA, can lead to a G1 arrest (Unger and Hartwell 1976; Shilo et al. 1978; Niederberger et al. 1983). This suggests that protein synthesis is involved in Start regulation. The initiation of the cell cycle is, however, much more sensitive to amino acid starvation than is total protein synthesis. A possible explanation is that a common product requiring different amino acids could control the attainment of Start and that this product could be a labile protein (Shilo et al. 1978). Although the cyclins have mainly been described as being involved in hormone responses, they are also involved in the response to nitrogen starvation (Hadwiger et al. 1989). One can envisage that cyclin synthesis, which is necessary for passage through G1 (Wittenberg

199

glutamate ADP PRO1 Tglutamyl-phosphate NADPH~ PRO2 L-glutamate-y-semialdehyde~.~

L-A-pyrmline-5-carboxylate L-proline

ornithine

Acknowledgements. This work was supported by the CNRS, the Minist6re de la Recherche et de la Technologic, and the Universit6 de Bordeaux II. The authors wish to thank Marjorie Brandriss for her help in identifying the PRO3 gene and for helpful discussions, Jacqui McKeand and Stephen Phillips for correcting the manuscript.

arginine /

[{ cytoplasm

ters are activated after osmotic stress (Higgins et al. 1987). Although involvement of several amino acids in the realisation of Start has been described, the mechanisms by which amino acid starvation leads to a G1 arrest remain to be determined. It will be particularly interesting to determine whether this phenotype results only from a general limitation of protein synthesis or if other mechanisms involving individual amino acids, such as proline, are concerned.

L'A1-pyrr°line-icarb°xylate glutamate

mitochondrion

Fig. 7. Proline metabolism in Saccharomyces cerevisiae. Proline biosynthesis occurs in the cytoplasm and its degradation in the mitochondrion. The steps where the different PRO genes are implicated are indicated according to Brandriss (1979)

et al. 1990), is very sensitive to the efficiency of protein biosynthesis. It is therefore possible that amino acid starvation could stop the cells in G1 because of the absence of cyclin synthesis. Such a mechanism would allow the cells to a d a p t and survive efficiently in a wide range of starvation conditions. Our selection procedure led to the isolation o f a mutant defective in the biosynthesis of proline. This can be explained by the fact that proline auxotrophs are unable to grow on a rich medium. Proline is the only amino acid for which such a property has been described and this suggests that a nitrogen, or carbon, catabolite-sensitive uptake system could prevent the uptake of proline in a medium where multiple preferred carbon and nitrogen sources are available (Brandriss 1979). Other reports suggest that proline could have a more specific function. Free proline is not detected in dividing yeast cells but becomes the m o s t a b u n d a n t amino acid present after sporulation (Miller 1989). The accumulation of proline appears to be involved in the viability o f the spores and in their resistance to desiccation, as well as in spore dormancy, since this amino acid is rapidly excreted during germination (Miller 1989). The protective function of proline is well described in plants, where there is a direct relationship between proline concentration and resistance to osmotic stress (McCue and H a n s o n 1990). In E. coli, genes encoding proline and betaine transpor-

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