Calcium-Sensitive c1s4 Mutant of Saccharomyces cerevisiae with a ...

JOURNAL OF BACTERIOLOGY, Jan. 1986, p. 28-33

Vol. 165, No. 1

0021-9193/86//010028-06$02.00/0 Copyright © 1986, American Society for Microbiology

Calcium-Sensitive c1s4 Mutant of Saccharomyces cerevisiae with Defect in Bud Formation

a

YOSHIKAZU OHYA, SHIGEMI MIYAMOTO, YOSHINORI OHSUMI, AND YASUHIRO ANRAKU*

Department of Biology, Faculty of Science, University of Tokyo,

Hongo,

Bunkyo-ku, Tokyo 113, Japan

Received 25 July 1985/Accepted 10 October 1985

A calcium-sensitive cls4 mutant of Saccharomyces cerevisiae ceased dividing in the presence of 100 mM CaC12, producing large, round, unbudded cells. Since its DNA replication and nuclear division still continued after interruption of normal budding, the cls4 mutant had a defect in bud formation in Ca2+-rich medium. Its calcium content and calcium uptake activity were the same as those of the wild-type strain, suggesting that the primary defect of the mutation was not in a Ca2+ transport system. Genetic analysis showed that the cls4 mutation did not complement the cdc24-1 mutation, which is known to be a temperature-sensitive mutation affecting bud formation and localized cell surface growth at a restrictive temperature. Moreover, cls4 was tightly linked to cdc24, and a yeast 3.4-kilobase-pair DNA fragment carrying both the CLS4 and CDC24 genes was obtained. These results suggest that the cls4 mutation is allelic to the cdc24 mutation. Thus, Ca2+ ion seems to control bud formation and bud-localized cell surface growth.

Genetic studies of the cell division cycle in the yeast more than 50 genes are needed for progression of the multijunctional pathways of cell division (10-12, 24). Genes primarily responsible for the cell division cycle have been recognized by the phenotypes of conditional-lethal cdc mutations, such as temperature-sensitive mutations (12) and cold-sensitive mutations (17). At a nonpermissive temperature, cdc mutants arrest growth at particular stages of the cell cycle, indicating that the wild-type CDC gene is indispensable at these points in the cell cycle (12). From extensive studies of these cdc mutants, Pringle and Hartwell (24) concluded that progression through the cell division cycle occurs as a well-defined program of discrete, gene-controlled events. Various lines of evidence indicated that Ca2+ ion is important in progression of yeast cell cycle. The divalent cation ionophore A23187 blocks nuclear division in budding yeast (23) and fission yeast (4). Therefore, a pool of Ca2+ ion or Mg2+ ion may be essential for nuclear division. Moreover, in synchronous cultures of S. cerevisiae, Ca2+ uptake was found to increase when the cells started budding and then to return to the basal value, which was maintained until the end of the first cell cycle (26). This observation suggested some unknown but crucial role of Ca2+ in the early stage of the cell cycle. Currently, we are carrying out genetic studies on the regulatory roles of Ca2+ ions in the cell cycle of S. cerevisiae. We reported the properties of a Ca2'-dependent mutant (call) with a defect at a particular stage of the cell cycle (20). The Ca2'-dependent mutant (call-i) requires a high concentration of Ca2+ ion for growth at a restrictive temperature, and in Ca2+-poor medium shows growth arrest at a stage with a tiny bud before nuclear division. The discovery of the call mutant and its trifluoperazine-dependent suppressor mutants suggested that Ca2+ ion and a calmodulinlike protein play some important roles in bud enlargement and nuclear division (20). In parallel with this work, we systematically isolated a number of Ca2+-sensitive mutants (Y. Ohya, Y. Ohsumi, and Y. Anraku, J. Gen. Microbiol., in press). Among them, only the cls4 mutant arrested growth at

specific stage of the cell cycle. In this paper we report that the cls4 mutant has a defect in bud formation in Ca2+-rich medium, although its DNA synthesis and nuclear division rate are normal. Genetic and gene-cloning studies suggest that the cls4 mutation is allelic to a cdc24-1 mutation. Also the temperature-sensitive cdc24-1 mutant, which was isolated by Hartwell et al. (12), cannot form buds at the restrictive temperature; it produces large, round, multinucleate cells. Fluorescence microscopic observations on chitin (30) and newly synthesized cell wall polysaccharides (29) have demonstrated that the cdc24 mutant shows undirected cell wall growth. In other words, the cdc24 mutant has defects in the processes of localized cell surface growth that occur in the budding pQrtion of wild-type yeast cells. a

Saccharomyces cerevisiae have shown that

*

MATERIALS AND METHODS

Media. YPD medium contained 2% glucose, 2% polypeptone (Daigo Chemicals, Saitama), and 1% Bactoyeast extract (Difco Laboratories, Detroit, Mich.). For measurement of Ca2+ sensitivity, YPD was supplemented with 100 mM CaC12. For testing antibiotic G418 sensitivity of yeast strains, YPD supplemented with 0.5 mg of G418 (GIBCO Laboratories, Grand Island, N.Y.) per ml was used. Chemicals. The radioactive compounds [14C]leucine, [3H]adenine, and 45CaC12 were obtained from Amersham Japan, Tokyo. Restriction endonucleases were obtained from Takara Chemicals, Kyoto. Strains and plasmid. The S. cerevisiae strains used are listed in Table 1. The Ca2+-sensitive mutant cls4 was isolated from the haploid strain A5-8-1A (a leul). For this, cells of strain A5-8-1A were subjected to ethyl methanesulfonate mutagenesis as described by Lindegren et al. (15) and then inoculated onto a YPD plate. After incubation for 2 days, the colonies obtained were replica plated onto a YPD plate and a Ca2+-rich plate at 30°C, and Ca2+-sensitive colonies were isolated after incubation for 2 days. The cls4 mutant is one of the Ca2'-sensitive mutants that could not grow on Ca2+-rich medium, but could grow on YPD at 30°C. Escherichia coli DH1 (F- recAl endAl gyrA96 thi-l hsdR17 [hsdR+ hsdM-] supE44) (8), supplied by K. Komeda, University of Tokyo,

Corresponding author. 28

Ca2+-SENSITIVE CELL CYCLE MUTANT OF S. CEREVISIAE

VOL. 165, 1986

Strain

A5-8-1A A56-1-1A YOC38 YOC138-2Aa YOC138-1C YOC338 395

182-6-3 YOC124-3A YOC124-3B YOC224-2D

TABLE 1. S. cerevisiae strains Source or reference Relevant genotype MATa leul (20) MATa adel (20) MATa leul cls4 This study MATa leul cls4 This study MATa adel cls4 This study MA Ta/MATa cls4/cls4 This study leull+ +/adel MATa cdc19 adel ade2 (12) ural his7 lys2 tyrl MATa cdc24-1 ural (12) tyrl arg4 thr4 ade his trp MATa cdc24-1 leul This study This study MATa cdc24-1 adel MATa cdc24-1 leu2 This study

a The haploid strain YOC138-2A was one of segregants from the diploid strain constructed by crossing YOC38 and A56-1-1A.

was used for bacterial transformation. The plasmid YCpG11 was a generous gift from H. Kojima, University of Tokyo. Macromolecular synthesis. Protein synthesis was determined by measuring the amount of [14C]leucine incorporated into the trichloroacetic acid-insoluble fraction as described by Hartwell (9). DNA synthesis was determined by measuring incorporation of [3H]adenine into the alkali-resistant, cold trichloroacetic acid-insoluble fraction as described by Dumas et al. (5). Nuclear morphology was observed as described by Ohya et al. (20). Determinations of calcium content and Ca2+ uptake. For measurement of the calcium content, exponentially growing cells (10-ml culture) were collected, washed twice with an equal volume of distilled water, and suspended in distilled water at a final concentration of 5 x 107 cells per ml. The concentration of calcium in the suspension was determined with a Perkin-Elmer 370 atomic absorption spectrophotometer. The standard solution of calcium used for calibration was prepared from a stock solution of 100 ,uM CaCl2 (Wako

Chemicals, Tokyo). For measurement of Ca2+ uptake activity, exponentially growing cells (10-ml culture) were collected, washed twice with an equal volume of distilled water, and suspended in 20 mM 2 (N-morpholino)ethanesulfonic acid-Tris (pH 6.0) buffer at a final concentration of 2 x 108 cells per ml. The cell suspension (100 ,ul) was preincubated for 15 min with 10 mM glucose at 30°C, and then 20 p.M 45CaC12 (50 mCi/mmol) was added to start the uptake reaction. After 30 s, the reaction was stopped by diluting the mixture with 5 ml of ice-cold 5 mM CaCl2 solution. The mixture was quickly filtered on a membrane filter (TM-2; Toyo Roshi Co.) and washed twice with 5 ml of the same solution. Two washings with 5 ml of 5 mM CaCl2 were sufficient to remove the extracellular, cellwall-bound 45Ca2+, as described by Penia (22). The radioactivity taken up by the cells was determined in a liquid scintillation counter. Construction of a pool of yeast genomic DNA in the vector YCpGll. The plasmid YCpGll contains the replication origin on pBR322 and the genes for Apr, Tcr, and Kmr (Fig. 1). YCpGll also contains a yeast replicator, ARS1, yeast CEN4 fragment (7) that encompasses the centromere of chromosome IV, and a marker selectable in yeast, TRPJ. Moreover, the gene for Kmr in E. coli expresses G418r in yeast (32). Plasmid YCpG11 was used for construction of a pool of yeast DNA fragments for three reasons. First, its

29

stability in transformants is higher than that of an ARS- or 2,um-containing vector. Second, we did not know whether our favored gene would be lethal at a high gene dosage (14), and YCpG11 has a copy number of only 1. Third, this plasmid contains a gene for G418r (Km'), which is useful for transformation of any yeast nonauxotrophic strain. A pool of YCpG11 plasmids bearing yeast DNA fragments was constructed as follows. Purified yeast DNA (100 jig/ml) from A5-8-1A was cleaved with 7.5 U of Sau3AI per ml for 1 h to give fragments with an average size of approximately 10 kilobases (kb). The fragments were pooled and fractionated by centrifugation on a 10 to 40% sucrose density gradients as described by Maniatis et al. (16). Fragments of 5 to 15 kb were collected, purified, and then ligated to YCpG11 DNA that had been digested with BamHI. Insertion into the BamHI site of YCpG11 rendered the plasmid Tcs. The DNA concentrations in the ligation reaction were 100 ,ug/ml. After incubation for 1 h at 37°C, the ligation mixture was used to transform E. coli strain DH1 to Apr Kmr as described by Davis et al. (3). We used plates for selection of Tcs colonies (1) to obtain an Apr Kmr Tcs colony directly. All 200 transformants examined on the Bochner plate containing ampicillin and kanamycin showed Tcs. Four hundred transformed Tc5 colonies were pooled, and 12 sets (4,800 colonies) of yeast genomic pools were made. Yeast transformation and genetic methods. The method of DNA transformation of lithium acetate-treated yeast cells, described by Ito et al. (13) and modified by Rodriguez and Tait (25), was used. After mixing 0.3 ml of cell suspension with 10 ,ul of DNA and 0.7 ml of polyethylene glycol 4000, the mixture was incubated for 1 h at 30°C and then collected by centrifugation. The pellet was suspended in 1 ml of YPD and incubated for 16 h at 23°C. This incubation step was required for expression of the G418r phenotype. Samples of the cell suspension were plated on appropriate selection

plates. The conventional techniques of Sherman et al. (28) were used for genetic analyses. RESULTS Calcium-sensitive cls4 mutant with a defect in bud emergence. Wild-type S. cerevisiae strains (A5-8-1A and A56-1-1A) could grow in the presence of 400 mM CaCl2 but not in the presence of 1.0 M CaCl2. We thought that mutants having defects in regulation of calcium metabolism might have altered sensitivities to extracellular Ca2+ ions. We isolated 30 calcium-sensitive mutants (cls) that could not

FIG. 1. Restriction nuclease map of the plasmid YCpG11. The thick line represents yeast DNA sequences; the thin lines represent pBR322 or pAJ5O DNA sequences.

J. BACTERIOL.

OHYA ET AL.

30

grow in the presence of 100 mM CaCl2. Each had a single recessive chromosomal mutation. The mutants divided into 18 complementation groups. The isolation and genetic characterization of these cis mutants will be described elsewhere (Ohya et al., in press). All of the cls mutants but one in the 18 complementation groups arrested growth randomly at all stages of the cell cycle (data not shown). The cIs4 mutant showed arrest at a specific stage in the cell cycle in YPD medium containing 100 mM CaCI2 (Ca2'-rich medium). The mutant could grow in the presence of 100 mM MgCl2, or 3 mM each MnCl2 or ZnC12, similarly to the wild-type strain. After incubation in Ca2'-rich medium for 4 h at 30°C, the increase in the number of cIs4 cells stopped (Fig. 2a), and about 90% of the cells were at the unbudded stage. The cell volume of the cls4 mutant increased continuously, resulting in cells that were 10 times larger than those of the wild-type strain. Figures 2b and c show the protein synthesis and DNA synthesis respectively, of the cIs4 mutant in Ca2+-rich medium. Even after transfer to Ca2+-rich medium, protein and DNA syntheses of the cIs4 mutant continued as in YPD. This continuous protein synthesis was responsible for the increase in cell volume. To understand how and why DNA synthesis progressed, we examined the morphology of nuclei of the cls4 mutant. Figure 3 shows that these big unbudded cells contained several nuclei. The number of nuclei per cell varied from one to more than eight, but most cells contained two or four nuclei. The DNA content of the nuclei in the same haploid cell varied from 1 to 4 c (c represents the DNA content of the haploid Gl nucleus; see reference 20 for details), indicating that DNA replication and nuclear division in the cls4 mutant occurred asynchronously after interruption of normal budding. One possible explanation for this Ca2 sensitivity was a defect of Ca21 transport systems. We examined this possibility by measuring the calcium content and Ca2+ uptake activity of cls mutants. Most cls mutants had higher calcium contents or higher initial rates of Ca2+ uptake (or both) than the wild-type strain. But the cIs4 mutant had a normal

5rYPD4-C 5-4_

3

YPD mi

(cls4)wererownat3°

Ic*

*2F 4 icbtowacotne.A and

.24

YPD

6

wdCaC2 ()

Time (hr)

CaCI2

4

6

Time(hr)

(

2

Y

CaCl2

2

4 Time

6

(hr)

FIG. 2. Cell number and macromolecular syntheses of a cIs4

mutant in the presence of 100 mM CaCl2. Cells of strain Y0C38 (cls4) were grown at 30'C in YPD medium to approximately iO1 cells per ml. ['4C]leucine (b) or [3H]adenine (c) was added at zero time, and incubation was continued. After 1 h, CaCl2 (@) or MgCl2 (A) at

a final concentration of 100 mM was added. The other portion without added CaCI2 (0) was incubated in YPD medium as a

control. Samples (200 ,ul) were removed every 45 min, and the cell number (a), protein synthesis (b), and DNA synthesis (c) were determined as described in Materials and Methods.

&r* _

43

0 _ P .1 -P *esv p a

FIG. 3. Phase contrast and fluorescence photomicrographs of cells of the cIs4 mutant. Cells of strain YOC338 (cls4/cls4) were incubated in YPD (a) or in Ca2l-rich medium (b, c) at 30°C for 8 h. Cell morphology (a, b) was examined by phase-contrast photomicrography, and nuclear morphology (c) was examined by fluorescence photomicrography (c). Bars, 10 p.m.

calcium content and normal uptake activity. Cells of the wild-type strain grown in YPD contained 11.0 ± 0.9 nmol of calcium per mg of protein, whereas cells of the cls4 strain contained 10.8 ± 0.7 nmol of calcium per mg of protein. At a CaC12 concentration of 20 ,uM, the initial rate of Ca2+ uptake of the wild-type strain in the presence of 10 mM glucose was 4.4 nmol/min per mg of protein, whereas that of the cIs4 strain was 3.6 nmol/min per mg of protein. These results suggested that the cIs4 mutant has a defect other than in the Ca2+ transport system. Complementation test between cls4 and cdc24. It is known that the cdc24 mutant (ts mutant) cannot form buds at restrictive temperatures and recently additional mutants (cdc42 and cdc43) were isolated with similar phenotypes to that of cdc24 (12, 24). Therefore, it seemed possible that the cIs4 mutation is allelic to one of these cdc mutations. We performed a complementation test between the cls4 mutation and the cdc24 mutation (Table 2). Each mutation is recessive to its wild-type allele. The Ca2+-sensitive cls4 mutant could not grow in Ca2'-rich medium, and the temperature-sensitive cdc24 mutant could not grow at 37°C. Therefore, both mutants could not grow in Ca2'-rich medium at 37°C. A diploid constructed by crossing the cls4 strain with the cdc24-1 strain could not grow in Ca2+-rich medium at 37°C (Table 2), indicating that the cdc24-1 mutation did not complement the cls4 mutation. The diploid was unable to grow at 37°C with or without 100 mM CaC12, having the same phenotype as the cdc24 mutant. Judging from this observation, the cls4 allele is thought to be recessive to the cdc24-1 allele. Genetic linkage between cls4 and cdc24. The diploid strain constructed by crossing the cls4 with the cdc24-1 mutant was sporulated and subjected to tetrad analysis to check whether the cIs4 and cdc24-1 mutations are allelic. More than 100 asci were analyzed, and all of them were of the parental ditype (Table 3). This meant that these two genes are closely linked.

Ca2`-SENSIT1VE CELL CYCLE

VOL. 165, 1986

MUTANT OF S.

CEREVISIAE

31

TABLE 2. Complementation test between (cs4 and (dc24" Growth at:

300C

23°C

Strain (genotype)

37°C

YPD

+Ca

YPD

+Ca

YPD

+Ca

+ + +

+

+ + +

+

+ +

+

+ + +

+ + + ±

Haploids

A5-8-1A (+) YOC138-2A (cls4) YOC124-3A (cdc24)

± +

+

-

Diploids

A5-8-1A (+) x A56-1-1A (+) A5-8-1A (+) x YOC138-1C (cIs4) A5-8-1A (+) x YOC124-3B (cdc24) YOC138-2A (cls4) x YOC138-1C (cIs4) YOC138-2A (cIs4) x YOC124-3B (cdc24) YOC124A (cdc24) x YOC124-3B (cdc24)

+ + +

Cells wereIgrown on YPD plates (YPD) or YPD plates containing 100 mM scored as follows: +, good; +, intermediate; -, poor or negative.

Since a recent genetic map (18) showed that the cdc24 was located on the left arm of chromosome I, the linkages betw,een cls4 and markers on this chromosome were analyzed. Results showed that cls4 is linked with cdcl9 on left arm of the chromosome I (Table 3). Isolation of recombinant plasmids complementing both the cdc24 mutation and the cls4 mutation. If the cIs4 mutation is allelic to the cdc24 mutation, there should be a DNA fragment that complements both the cls4 and cdc24 mutations. To examine this possibility, we first cloned the yeast genomic DNA fragment capable of complementing the temperature-sensitive cdc24-1 mutation. This fragment was cloned from a S. cerevisiae DNA library carried in the yeast plasmid vector YCpG11. The library plasmids replicated autonomously in S. cerevisiae and expressed the gene for antibiotic G418r. The library was constructed like that described by Nasmyth and Reed (19). Complementing plasmids were cloned by selecting G418r Ts + transformants of S. cerevisiae YOC224-2D) (a Ieu2 cdc24-1). After incubation with polyethylene glycol 4000, cells were allowed to grow at 230C for 16 h in YPD medium. Ts' colonies were selected after incubation for 5 days at 37°C, and their G418 sensitivities were checked. Three independent colonies were isolated that were both Ts+ and G418r. Two of these simultaneously lost both phenotypic markers at an appropriate frequency for centromere vectors after growth in nonselective conditions (in YPD at 23°C). This instability suggested that the Ts' and G418. phenotypes are associated with the presence of a plasmid. The other colony showed instability for only G418r and was presumably a temperature-sensitive revertant. Total DNA was extracted from two Ts' G418r S. cerevi-

Interval'

cIs4 adel cIs4 cdc19 cls4 cdc24

TABLE 3. Mapping data of cIs4 No. of ascus type": PD

7 10 106

NPD

T

Map distance a isac (centinorgans)

2 0 0

19 4 0

>50 14