Correspondence to: Enrico Cabib, National Institutes of Health, 8 Center Drive MSC 0851, Bethesda, MD 20892., enricoc{at}bdg10.niddk.nih.gov (E-mail), (301) 496-1008 (phone), (301) 496-9431 (fax)
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of ß(13)glucan synthase. Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization. Cells of mutant rho1E45I in the G1 stage of the cell cycle did not bud at 37°C. In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature. Two mutants in adjacent amino acids, rho1V43T and rho1F44Y, showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature. This was also the case for rho1E45I when placed in a different genetic background. Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1E45I. Nuclear division did not occur in the mutant at 37°C, although replication of DNA proceeded slowly. The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone. The in vitro activity of ß(1
3)glucan synthase in rho1 E45I, although diminished at 37°C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1. Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and ß(1
3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds. Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.
Key Words: Saccharomyces cerevisiae, G proteins, cell polarity, actin, cell cycle
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
YEAST cells undergo polarization during the vegetative cycle, just before budding, and in the sexual cycle, before conjugation. This polarization is manifested in both cases by the reorganization of the actin cytoskeleton and by the localization of certain proteins at the budding site or at the mating projection (for reviews see 3)glucan synthase and is required for its activity (
Here we show that certain temperature-sensitive mutants of RHO1 are blocked at a cell cycle stage that precedes cell polarization. The defect does not appear to be related to ß(13)glucan synthase or Pkc1p activity. The mutants are also defective in cell polarization before conjugation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yeast Strains and Yeast Growth
The strains used in this study are listed in Table 1. Yeast cells were cultured either in minimal (2% glucose, 0.7% yeast nitrogen base without amino acids [Difco], plus requirements) or in YEPD medium (1% yeast extract [Difco], 2% peptone [Difco], and 2% glucose) to which adenine was added to a final concentration of 40 µg/ml. Solid media contained 2% agar.
|
Strain JDY4 was derived from OHNY1 (Table 1) by switching its mating type with HO recombinase as described by
Yeast transformation was carried out either by the lithium acetate method (
Plasmid and Strain Construction and Manipulation
Yeast genomic DNA was prepared as described in
To construct plasmids containing genomic mutations of RHO1, 720-bp fragments of rho1V43T, rho1F44Y, and rho1E45I were obtained by PCR from genomic DNA of strains HNY93, HNY95, and HNY97, respectively, with synthetic oligonucleotides (5'-ATGTCACAACAAGTTGGTAACA-3' for the 5' end [ATG in bold in this and subsequent oligonucleotides] and 5'-AAAGGCATACGTACATACAATGAGAAA-3' for the 3' end [SnaBI site underlined]). PCR fragments were digested with SnaBI, which cuts at a site located 118 bp downstream from the RHO1 ATG and at another site (included in the oligonucleotide), 86 bp downstream from the stop codon, and purified by agarose gel electrophoresis. Plasmid pRS316 (RHO1 [KpnI]) (kindly furnished by Y. Takai) was digested with SnaBI, dephosphorylated, and ligated with the fragments prepared above. The presence of the respective mutation was confirmed by sequencing the plasmid DNA with a synthetic oligonucleotide (5'-ATGTCACAACAAGTTGGTAACA-3') as a primer.
All of the above plasmids contained a KpnI site, inserted upstream of the open reading frame of RHO1 during construction of the original RHO1 plasmid (
Strain JDY6-7A(pRS316(RHO1[KpnI]) was a segregant dissected after sporulation of strain DHNY110 (Table 1). The resident plasmid was exchanged with any of those from the pRS314 series by shuffling. In turn, these were shuffled with the pRS316 plasmids (devoid of the KpnI site) when a different marker was required.
To reintroduce the mutated RHO1 into its chromosomal locus, the entire RHO1 coding sequence was deleted (in DHNY110, derived from W303-RHO1 [
The centromeric plasmid pGRT, containing the GAL1-RHO1 and the TRP1 gene, was constructed as follows: a synthetic oligonucleotide containing a HindIII restriction site (underlined) and the 5' end of the RHO1 reading frame, 5'-AAAATTAAGCTTGAAAGATGTCACAACAAG-3', was used as upstream primer and one bearing an EcoRI restriction site (underlined) and sequence 36 bp downstream of the RHO1 stop codon, 5'-TGCCACTAAGAATTCGACTGAGAGATC-3', as downstream primer in a PCR reaction with OHNY1 genomic DNA as template. The amplified product was digested with HindIII and EcoRI and ligated with pYES2.0, previously digested with the same restriction enzymes to yield plasmid pYES-RHO1. To transfer the GAL1-RHO1 fusion to a centromeric plasmid, a primer bearing a BamHI restriction site, 5'-CGGGATCCAGTACGGATTAGAAGCCG-3', and one with a KpnI site, 5'-GAGGTACCGGGCCGCAAATTAAAGCC-3', were used to amplify a 1,495-bp fragment of pYES-RHO1 containing the GAL1 promoter, the ORF of RHO1, and the transcription terminator. The PCR product was digested with BamHI and KpnI and ligated with pRS314 previously digested with the same enzymes to yield plasmid pGRT. This plasmid was found to support growth of rho1 mutants in galactose medium.
Strain DHY5D was transformed with pGRT and sporulated; tetrads were dissected on minimal medium containing 2% galactose and 0.2% sucrose in place of glucose. A Ura+ and Trp+ segregant, DHY1-5A(pGRT), was transformed with DNA fragments containing either RHO1, rho1V43T, rho1F44Y, or rho1E45I, obtained by digesting the respective pRS316 plasmids with SacI and XhoI. Ura- colonies were selected on glucose plates containing 5-fluoroorotic acid (
To place the rho1 mutation in a different genetic background, strain ECY44 was obtained by mating CRY1 and CRY2 (Table 1). A deletion of RHO1 was carried out by digesting pRS316(RHO1) with MluI and HpaI (as above), followed by blunting and treating with alkaline phosphatase; the excised fragment was replaced with HIS3, obtained by digestion of pJJ217 () was verified by PCR. ECY44
was transformed with pRS316(RHO1) or with pRS316(rho1E45I); the transformed strains were sporulated and segregants harboring the RHO1 disruption and the respective plasmid (His+ Ura+) were isolated after tetrad dissection.
The plasmid YCp50(PKC1R398P), carrying a constitutively active allele of PKC1, was provided by Y. Takai. To overcome marker limitations in some host strains, the mutated gene was excised from the plasmid with PstI and recloned into the PstI site of the centromeric vector pRS315.
DNA manipulations were performed according to standard protocols (
Human p21 (p01112) and yeast Rho1p (p06780) sequences were aligned with program PILEUP and LINEUP of the GCG package (Wisconsin Package Version 9.1; Genetics Computer Group) and the amino acids in yeast Rho1p corresponding to the p21 switch 1 domain were deduced from the alignment.
Isolation of G1 Cells and Determination of Budding
Yeast cells were grown in 160 ml of minimal medium to early log phase (0.3 g cells, wet weight/100 ml culture). The cells were harvested by centrifugation, suspended in 1 ml of 0.75 M methylmannoside, and sonicated briefly to break up clumps. Portions of the suspension (720 µl each) were applied on top of two 12-ml linear sucrose gradients (1540%) and centrifuged at 400 g for 10 min. The cells formed a wide band in the gradient. Three 0.5-ml fractions from the upper part of the band were collected with a J-shaped needle with the help of a peristaltic pump and checked microscopically. Those fractions that contained <5% of budding cells were pooled, washed with distilled water, and used to inoculate 5 ml of minimal medium. The G1 cells were cultivated at 26°C or 37°C and every 2 h cells were counted to determine percentage of budding. More than 300 cells were counted in each sample.
In the experiments with strain DL503 (pkc1ts), cells were fixed with 5% (final concentration) formaldehyde before counting, to prevent lysis. Small buds were counted with Nomarski optics under oil immersion. For photographic purposes, the tiny buds were best visualized in unfixed cells with phase-contrast under oil immersion.
Cell viability was estimated by staining with methylene blue. Cells were pelleted and suspended in 2 µg/ml methylene blue in 0.05 M KH2PO4. 5 min later, cells were observed in the microscope with Nomarski optics without removing the excess dye; the percentage of blue cells was determined. The Nomarski optics did not interfere with color observation and facilitated visualization of the unstained cells for counting.
Fluorescence Microscopy
For all experiments, cells were fixed with 5% formaldehyde at 4°C overnight as described by
Cdc42p was stained with affinity-purified rabbit anti-Cdc42p antibodies (kindly provided by D.I. Johnson) essentially as described by
Flow Cytometry
Cells were fixed with 70% ethanol and DNA was stained with propidium iodide as described in
Membrane Preparation and Measurement of ß(13)Glucan Synthase Activity
Membranes were prepared from logarithmic phase cells as previously described (
|
|
|
|
|
Inhibition of ß(13)Glucan Synthesis by L-733,560
An 80-ml culture of strain 1783 (wild-type) grown in minimal medium at 26°C and containing ~7 x 106 cells/ml was split in two. To one half L-733,560 (
Treatment of Cells with -Factor
To 5 ml of culture in minimal medium, containing ~107 cells/ml, two additions of 80 µg of -factor (Bachem Bioscience, Inc.) were made 1 h apart. After a total incubation time of 2 h at 26°C, most cells carrying wild-type RHO1 showed a mating projection. At this point, cells were photographed under phase-contrast or fixed for subsequent actin visualization.
Production of an Antibody against Rho1p and Western Blot Analysis
Polyclonal anti-Rho1p antiserum was raised in two rabbits (Alpha Diagnostic) against a purified maltose-binding protein (MBP)-Rho1p fusion obtained as follows. A 655-bp fragment containing the RHO1 coding sequence was amplified by PCR from genomic DNA with an upstream primer containing the initiation codon (5'-ATGTCACAACAAGTTGGTAACA-3') and a downstream primer containing an EcoRI restriction site (underlined) (5'-TGCCACTAAGAATTCGACTGAGAGATC-3'). The amplified fragment was digested with EcoRI and inserted into the pMAL-c2 expression vector (New England Biolabs), previously digested with XmnI and EcoRI, to yield the in-frame MBP-Rho1 fusion protein. The fusion protein was expressed in protease-deficient Escherichia coli BLR cells (Novagen), containing plasmid pDC952 (kindly provided by J.R. Walker) that encodes tRNAArgUCC to eliminate misincorporation of lysine in place of arginine (
For Western blot analysis, yeast cell lysates, obtained by glass bead disruption and clarified by centrifugation, were separated by SDS-polyacrylamide electrophoresis (40 µg of protein/lane) in a 14% gel (
Preimmune serum did not produce any signal. The antibody stained a band of the expected mobility (~26 kD) when wild-type cell lysates were used. The band was stronger when RHO1 was overexpressed on a high-copy plasmid and much weaker with extracts from strain HNY21 (rho1-104), which was found (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Certain Temperature-sensitive rho1 Mutants Are Blocked before Budding
Our initial interest in the RHO1 mutants rho1V43T, rho1F44Y, and rho1E45I (kindly provided by Y. Takai) stemmed from the report (3)glucan synthase, we thought that rho1V43T may be specifically impaired in glucan synthesis. However, we found that although glucan synthase was partially defective in rho1V43T after growth at 37°C, the same defect was shown by the other two mutants. In the course of those experiments we observed that at the nonpermissive temperature large unbudded cells predominated in the population, indicating a budding defect in all three strains. In contrast, under the same conditions, mutant rho1-104 cells mainly arrest with a very small bud (
Since uncertainty about ploidy would compromise the results, we constructed other strains harboring a disruption of RHO1 and a plasmid carrying either wild-type RHO1 or an appropriate mutation thereof (see Materials and Methods and Table 1). These strains remained haploid under usual laboratory conditions, although some aneuploids arose if the cells were left on plates for very long periods (Cabib, E., J. Drgonová, and T. Drgon, unpublished experiments).
Because Rho1p has different functions (see above) which may be executed at different stages of the cell cycle, it seemed desirable to study the presumed budding defect with a uniform cell population in the G1 stage of the cycle. Such a population can be obtained by gradient centrifugation (
|
Cells of rho1 Mutants Fail to Polarize
Cell polarization can be estimated by the distribution of actin (
|
In cdc42-1, a mutant with similar morphology to rho1E45I, DNA synthesis and nuclear division continue after the 37°C block. In contrast, DAPI staining showed a single nucleus per cell in all three rho1 mutants (Figure 2B, Figure D, Figure F, and Figure H). However, determination of DNA content per cell by fluorescence-activated cell sorting showed that DNA is slowly duplicated in the rho1E45I (Figure 4). This experiment also confirms that the initial cell population was in G1.
Another difference between cdc42 and rho1E45I is the randomized production of chitin at the cell surface as assessed by Calcofluor white staining. After incubation at 37°C, cdc42 cells stain very strongly with Calcofluor (
The rho1E45I Defect Results from Loss of a Previously Unknown Function
The defect in rho1E45I is recessive: neither strain ECY44(pRS316-RHO1) nor strain ECY44
(pRS316-rho1E45I) was temperature sensitive (result not shown). Both strains are diploids containing one chromosomal copy and one deletion of RHO1, plus a plasmid carrying either a wild-type or a mutated allele of the same gene, as indicated. However, segregants from sporulation of ECY44
(pRS316-rho1E45I), that harbored the RHO1 deletion and the plasmid with the mutation, were temperature sensitive, as expected (data not shown). These results show that the defect in rho1E45I is due to loss of function and not to interference with some other pathway caused by abnormal targeting of the mutated protein.
It was important to separate the function of Rho1p in cell polarization from those already known in ß(13)glucan synthesis and Pkc1p activation. Therefore, we studied the effect of temperature on glucan synthase activity in two different ways: in one of them the enzyme was measured at 30°C with membrane preparations obtained from cells grown at 26°C or from cells shifted to 37°C for 2 h; in the other, membranes from cells grown at 26°C were assayed both at 26°C and 37°C. The first condition assesses the irreversible inactivation of Rho1p in vivo at 37°C, whereas the second one takes into account the possibility that the inactivation at 37°C may be reversible upon cooling. Membranes from a wild-type strain and from mutant rho1-104 were included for comparison (Figure 5). Results for the latter two strains under the first condition were similar to those already reported (
As for Pkc1p,
These results indicate that the cell cycle block in mutant rho1E45I is not due to defects in glucan synthase or protein kinase C, although the latter may well be inactive in the mutant.
To confirm these findings and to obtain evidence on glucan synthesis independent of in vitro measurements of enzymatic activity, we used a different approach. It was reasoned that cells containing a wild-type allele of RHO1 but in which Pkc1 activity and synthesis of ß(13)glucan had been turned off should be able to form at least incipient buds, if the two latter functions were not required for cell polarization. To inactivate Pkc1, we used a temperature-sensitive pkc1 mutant. The terminal phenotype of such a mutant in asynchronous cultures at the nonpermissive temperature is that of a mother cell with a small bud lysing at the tip (
3)glucan synthesis, we employed the semisynthetic echinocandin L-733,560, that inhibits the formation of the polysaccharide in vivo and in vitro (
3)glucan synthesis, cells growing in the absence or in the presence of L-733,560 were labeled with 14C-glucose and the cell walls were digested with Zymolyase, an endo-ß(1
3)glucanase preparation. Incubation with this enzyme leads to solubilization of most of the cell wall components, resulting in a mixture of short ß(1
3)-linked glucose oligosaccharides and large mannoproteins attached to ß(1
6)glucan (
3)glucan synthesis. Since in the original wall the mannoprotein-ß(1
6)glucan complex is attached to ß(1
3)glucan (
3)glucan.
|
Having established the effectiveness of L-733,560, we proceeded to determine budding and viability (by methylene blue staining) of strain DL503 (pkc1) under conditions where either Pkc1p or ß(13)glucan synthase or both were not functional. At 26°C, in the presence of the inhibitor, cells gave rise to buds almost as efficiently as in its absence, but died rapidly (Figure 7 C) with a small bud (Figure 7 E). Many cells acquired an elongated shape, somewhat akin to that of the shmoos formed in the presence of
-factor (Figure 7 E, arrows). At 37°C, when only Pkc1p was inactivated, the cells also lost viability (Figure 7 D) and ended up with small buds (Figure 7 F), as previously found with unsynchronized cultures (
We conclude that a functional Rho1p is sufficient for cell polarization, even when Pkc1p and ß(13)glucan synthesis have been inactivated.
We previously found (
|
Polarization of Cells before Conjugation Is Defective in the rho1 Mutants
The polarization defect in the rho1 mutants is not limited to the budding cycle. Most cells in the mutant failed to produce a mating projection when incubated with the sexual pheromone -factor at the permissive temperature (26°C). After a 2-h incubation with the pheromone, the percentage of cells with a visible mating projection was 74 and 75 in two determinations with wild-type and 18 and 21 in the mutant. In the latter, most cells remained roundish with a large vacuole (Figure 9A and Figure B). Correspondingly, in the wild-type shmoos actin was recruited to the mating projection (Figure 9 C), whereas in the mutant cells there was some localization of actin only where a mating projection appeared (Figure 9 D). However, the block in mating projection was leaky enough to permit mating with the opposite mating type (results not shown). A similar defect in actin polarization was observed with the other two rho1 mutants (Figure 9E and Figure F). In an experiment in which wild-type yielded 55% cells with a mating projection, the values were 21 and 23% for rho1V43T and rho1F44Y, respectively. Thus the defect was somewhat decreased, relative to wild-type, in these two strains, in accordance with their leakiness in budding (Figure 1).
|
Variability in Penetration of the rho1E45I Mutation in Different Genetic Backgrounds
The observation that strain JDY6-7A[pRS316(RHO1)] grows on plates somewhat slowly at 37°C suggested the possibility that a temperature-sensitive mutation in another gene, either preexisting in the mother strain DHNY110 or introduced during our genetic manipulations, might contribute, together with rho1E45I, to generate the observed phenotype. Whether a mutation had been introduced artificially was investigated by using strain DHY1-5A(pGRT), which harbored a new deletion of the RHO1 gene (carried out in the diploid JDY7, isogenic with OHNY1) with a different marker (URA3) from that used previously (HIS3). Plasmid pGRT present in that strain was substituted by either pRS314(RHO1) or pRS314(rho1E45I). The resulting strains behaved undistinguishably from the previously used strains JDY6-7A[pRS316(RHO1)] and JDY6-7A[pRS316(rho1E45I)], i.e., at 37°C G1 cells carrying RHO1 budded and those carrying the mutation did not (results not shown). Since the probability of having introduced the same mutation with completely independent manipulations on different strains is vanishingly small, these results effectively eliminate that possibility.
It was still conceivable that both DHNY110 and OHNY1 originally harbored a temperature-sensitive mutation in another gene that was required for a stringent block of the cell cycle. To address this point, we used strain ECY44[pRS316(rho1E45I)] (Table 1), a diploid obtained by mating strains CRY1 and CRY2 to yield ECY44, followed by disruption of RHO1 and introduction of the plasmid carrying the rho1 mutation. Both CRY1 and CRY2, as well as ECY44 and ECY44
[pRS316(rho1E45I)], show robust growth at 37°C, clearly more vigorous than that of JDY6-7A[pRS316(RHO1)] or OHNY1 (results not shown). After sporulation of ECY44
[pRS316(rho1E45I)], six tetrads were analyzed. All scored 2:2 for temperature sensitivity (results not shown), as expected, since all contain rho1E45I, whereas half of them carry RHO1 and the other half rho1::HIS3. These results also confirm that the rho1 mutation is recessive. One of the tetrads was monitored for budding of G1 cells at 37°C (Figure 10 A), under the same conditions of the experiment of Figure 1. Although the temperature-sensitive segregants (A and D) budded much less than their temperature-resistant counterparts (B and C), there was still 1520% residual budding in A and D. Thus, there is some leakiness in this genetic background. To obtain a more precise estimate of the leakiness in terms of cells that had completed one or more cell cycles, we repeated the experiment with segregants C and D, but counted the total cell number, where a bud was counted as an independent cell (Figure 10 B). The increase in cell number in the wild-type segregant over a 6-h period was 5-fold with fairly synchronous growth, whereas in the rho1E45I segregant it was only 0.6-fold.
|
It remained to be ascertained whether the difference in penetration of the rho1E45I mutation between the JDY6-7A or OHNY1 backgrounds on one hand and the ECY44 background on the other is due to a single mutation or to a more general genetic variation. To elucidate this point, we mated ECY44
-1D (Figure 10A and Figure B) to JDY6-7A[pRS316(rho1E45I)] and sporulated the diploid. It was reasoned that, if the difference between the two strains was due to a single allele, the leakiness at 37°C should segregate 2:2, whereas if many genes were involved in the effect, a more randomized distribution would be found. All the progeny was temperature-sensitive (result not shown), as expected, since both mating partners carried a RHO1 deletion plus rho1E45I on a plasmid. Three tetrads were further analyzed by obtaining G1 cells from all segregants and incubating them at 26°C or 37°C. The number of cells was monitored as in the experiment of Figure 10 B. The increase in cell number in the different segregants over a 6-h period was variable, ranging between 0.2- and 2-fold, with one strain reaching 3.5-fold (Figure 10 C). There was no clear 2:2 segregation of the increase in cell number. Actin distribution was determined by fluorescence microscopy in all components of tetrad 6 of Figure 10 C, after incubation at 26°C or 37°C (Table 3). The results basically confirmed those observed by measuring cell number, since the great majority of cells did not show actin polarization at 37°C. In conclusion, these results support the notion that the penetration of mutation rho1E45I is determined by the genetic background rather than by a specific gene. The reasons for the variability will be discussed below.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The experiments described above show that mutant rho1E45I is defective in cell polarization: at 37°C cells do not bud; they enlarge and become round; actin is not reorganized and recruited to the presumptive bud site; certain proteins, such as Cdc42p and Spa2p, also usually found at the budding site, do not localize. The blocked cells show a single nucleus, although DNA duplication proceeds at 37°C, albeit at a greatly reduced rate. The mutant also shows a defective response to pheromone, both in the formation of a mating projection and in concentrating actin at the projection. Two rho1 mutants in adjacent amino acids, rho1V43T and rho1F44Y, also were defective in cell polarization at the nonpermissive temperature, but they showed some leakiness (Figure 1C and Figure D), consistent with a partial function of the mutated Rho1p.
The phenotype of the rho1 mutants analyzed in this study differs from that of mutant rho1-104, which arrests at the nonpermissive temperature with a preponderance of cells bearing a small bud (
The recessive character of the rho1E45I mutation indicates that the phenotype is a consequence of loss of function. That loss does not appear to be in one of the already known roles of Rho1p: the in vitro glucan synthase activity of the mutant, although reduced at 37°C, should be sufficient for normal in vivo ß(13)glucan synthesis, by comparison with that of mutant rho1-104 grown at 26°C (Figure 5). As for Pkc1p, transformation of our reconstructed strains with a constitutively active allele of the corresponding gene failed to suppress their temperature sensitivity at 37°C. In contrast,
Some uncertainty lingered because of this disagreement and of the difficulty in extrapolating from measurements of glucan synthase activity in vitro to its performance in vivo. Therefore, it was desirable to use a different approach to find out whether Pkc1p and synthase activity are required for cell polarization and budding. This was achieved by using a pkc1 temperature-sensitive mutant and an inhibitor that totally abolished ß(13)glucan synthesis, in cells containing a wild-type RHO1 allele. Turning off either Pkc1p activity or glucan synthesis or both did not abolish budding, an event subsequent to cell polarization, although at 37°C and in the presence of inhibitor the buds were extremely small. This reduction in size is not surprising, because each one of the two defects alone results in the production of small buds, followed by cell death. The rapid loss in cell viability stands in contrast with the very slow decrease in viable cells observed in rho1E45I at 37°C (Figure 1 B), another indication that the defect in the mutant does not reside in a lack of Pkc1p or glucan synthase function. These results provide a counterpart to those obtained with the rho1 mutants. Taken together, the two groups of findings indicate that Rho1p is endowed with a hitherto unknown function, necessary for polarization of the yeast cell.
It has not yet been established whether there is a relationship between specific regions of Rho1p and each of the functions performed by the protein. The mutations we examined are located in the "switch 1" domain of Ras-related proteins (Figure 11), one of the two regions that change their conformation in response to GTP/GDP exchange (3)glucan synthase. On the other hand, no conclusion can be made about protein kinase C activation. Constitutively active Pkc1p failed to suppress the temperature sensitivity of the mutants; therefore, an activation of the kinase by the mutated Rho1p would not have been detected in our experiments. In mammalian cells, however, a Ras1 mutation equivalent to rho1E45I resulted in impaired activation of the protein kinase Raf1, which, like Pkc1p, regulates a MAP kinase cascade (
|
As shown above, the penetration of the rho1E45I defect was influenced by the genetic background. This is not surprising, if one considers that Rho1p has several essential functions and that it may therefore be very difficult to obtain a mutant in which one of those functions has been completely abolished while maintaining enough of the others to survive at a permissive temperature. Thus, it seems probable that rho1E45I still maintains at 37°C some residual function for cell cycle progression that enables it to cross the block under favorable conditions. These may entail, for instance, certain levels of expression of other interacting proteins that may vary with the genetic background. This explanation also accounts for the finding that some segregants of cross JDY8 were more leaky than either of the two parents, depending on the gene mix they inherited. Anyway, none of the mutants was able to grow for more than one or a few generations, because they were all temperature-sensitive on plates.
What is the nature of the Rho1p function required for cell polarization? One aspect of the cell cycle block we examined is the lack of actin reorganization and it is certainly possible that Rho1p has a direct role in that process. As mentioned above, Rho has been shown to participate in actin organization in animal cells. Furthermore, Takai and associates have found interactions between Rho1p and Bni1p, a protein that binds to profilin, which in turn stimulates actin polymerization (
There are some indications that the Rho1p function discussed here involves more than actin organization: Cdc42p and Spa2p, two proteins that do not depend on actin for their localization, were not found in the presumptive budding area in rho1E45I. This result could be interpreted to mean that these proteins are unable to reach their destination or that they reach it but are unable to maintain localization in the presence of a defective Rho1p. However, this finding, together with the observation that the mutant does not undergo nuclear division and duplicates its DNA slowly, suggests that the execution point of Rho1p might precede that of Cdc42p. This is also in agreement with the lack of randomized deposition of chitin, which may require more than one round of DNA replication (
We sought information about the Rho1p effectors by looking for suppressors of the temperature sensitivity of rho1E45I. No effect was found by transformation with CDC42 on a high-copy plasmid (results not shown). This does not exclude the possibility that Cdc42p is a direct or indirect target of Rho1p, because mere increase in expression may be ineffective if an activation step is involved in the pathway and the rho1 mutant is unable to provide it. Sorbitol, at 1 M concentration, was able to suppress the temperature sensitivity (result not shown). This effect, however, may be due to protection of the mutated Rho1p protein by glycerol accumulated intracellularly in response to the external osmolyte, as we recently found for rho1-104 (Cabib, E., J. Drgonová, and D.-H. Roh, manuscript in preparation).
A genetic screen for high-copy suppressors yielded in seven cases RHO1 and in one the SSD1 gene (results not shown). The latter encodes a cytoplasmic RNA-binding protein (
In conclusion, although clarification of the mechanism by which Rho1p acts in G1 must await further experimentation, our results clearly show that this yeast protein, "at the interface between cell polarization and morphogenesis" (
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We are deeply indebted to Y. Takai for strains and plasmids. We thank D.I. Johnson for an anti-Cdc42p antibody and for very useful advice on its use. We also thank A. Bender, J. Pringle, J.R. Walker, M. Snyder, and B. Santos for plasmids, D. Levin for strains, and M. Kurtz for a sample of L-733,560. We are grateful to L. Crotti, J.C. Ribas, A. Robbins, M. Schmidt, and R. Wickner for a critical reading of the manuscript.
Submitted: July 29, 1998; Revised: June 11, 1999; Accepted: June 18, 1999.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, A.E.M., Johnson, D.I., Longnecker, R.M., Sloat, B.F., Pringle, J.R. (1990) Budding and establishment of cell polarity in the yeast Saccharomyces cerevisiae. J. Cell Biol. 111:131-142[Abstract].
Arellano, M., Duran, A., Perez, P. (1997) Localization of the Schizosaccharomyces pombe rho1p GTPase and its involvement in the organization of the actin cytoskeleton. J. Cell Sci. 110:2547-2555
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. (1997) Current Protocols in Molecular Biology. New York, John Wiley & Sons, Inc.
Ayscough, K.R., Stryker, J., Pokala, N., Sanders, M., Crews, P., Drubin, D.G. (1997) High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor Latrunculin-A. J. Cell Biol. 137:399-416
Cvrckova, F., Nasmyth, K. (1993) Yeast G1 cyclins CLN1 and CLN2 and a GAP-like protein have a role in bud formation. EMBO (Eur. Mol. Biol. Organ.) J. 12:5277-5286[Abstract].
Douglas, C.M., Marrinan, J.A., Li, W., Kurtz, M.B. (1994) A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-ß-D-glucan synthase. J. Bacteriol. 176:5686-5696[Abstract].
Drgonová, J., Drgon, T., Tanaka, K., Kollar, R., Chen, G.C., Ford, R.A., Chan, C.S., Takai, Y., Cabib, E. (1996) Rho1p, a yeast protein at the interface between cell polarization and morphogenesis. Science. 272:277-279[Abstract].
Drugan, J.K., Khosravi-Far, R., White, M.A., Der, C.J., Sung, Y.-J., Hwang, Y.-W., Campbell, S.L. (1996) Ras interaction with two distinct binding domains in Raf-1 may be required for Ras transformation. J. Biol. Chem. 271:233-237
Evangelista, M., Blundell, K., Longtine, M.S., Chow, C.J., Adams, N., Pringle, J.R., Peter, M., Boone, C. (1997) Bni1p, a yeast formin linking Cdc42p and the actin cytoskeleton during polarized morphogenesis. Science. 276:118-122
Gray, J.V., Ogas, J.P., Kamada, Y., Stone, M., Levin, D.E., Herskowitz, I. (1997) A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiae in bud emergence and identification of a putative upstream regulator. EMBO (Eur. Mol. Biol. Organ.) J. 16:4924-4937
Guthrie, C., Fink, G.R. (1991) Guide to Yeast Genetics and Molecular Biology. London, Academic Press.
Hall, A. (1998) Rho GTPases and the actin cytoskeleton. Science. 279:509-514
Hirai, A., Nakamura, S., Noguchi, Y., Yasuda, T., Kitagawa, M., Tatsuno, I., Oeda, T., Tahara, K., Terano, T., Narumiya, S. et al. (1997) Geranylgeranylated rho small GTPase(s) are essential for the degradation of p27Kip1 and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL-5 cells. J. Biol. Chem. 272:13-16
Hu, W., Bellone, C.J., Baldassare, J.J. (1999) RhoA stimulates p27Kip degradation through its regulation of cyclin E/CDK2 activity. J. Biol. Chem. 274:3396-3401
Imamura, H., Tanaka, K., Hihara, T., Umikawa, M., Kamei, T., Takahashi, K., Sasaki, T., Takai, Y. (1997) Bni1p and Bnr1p: downstream targets of the Rho family small G-proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae. EMBO (Eur. Mol. Biol. Organ.) J. 16:2745-2755
Kamada, Y., Qadota, H., Python, C.P., Anraku, Y., Ohya, Y., Levin, D.E. (1996) Activation of yeast protein kinase C by Rho1 GTPase. J. Biol. Chem. 271:9193-9196
Kapteyn, J.C., Montijn, R.C., Vink, E., De La Cruz, J., Llobell, A., Douwes, J.E., Shimoi, H., Lipke, P.N., Klis, F.M. (1996) Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked ß1,3-ß1,6-glucan heteropolymer. Glycobiology. 6:337-345[Abstract].
Kilmartin, J.V., Adams, A.E.M. (1984) Structural rearrangements of tubulin and actin during the cycle of the yeast Saccharomyces. J. Cell Biol. 98:922-933[Abstract].
Kohno, H., Tanaka, K., Mino, A., Umikawa, M., Imamura, H., Fujiwara, T., Fujita, Y., Hotta, K., Qadota, H., Watanabe, T., Ohya, Y., Takai, Y. (1996) Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae. EMBO (Eur. Mol. Biol. Organ.) J. 15:6060-6068[Abstract].
Kollár, R., Reinhold, B.B., Petraková, E., Yeh, H.J.C., Ashwell, G., Drgonová, J., Kapteyn, J.C., Klis, F.M., Cabib, E. (1997) Architecture of the yeast cell wall. ß(16)glucan interconnects mannoprotein, ß(1
3)glucan, and chitin. J. Biol. Chem. 272:17762-17775
Lee, K.S., Irie, K., Gotoh, Y., Watanabe, Y., Araki, H., Nishida, E., Matsumoto, K., Levin, D.E. (1993) A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C. Mol. Cell. Biol. 13:3067-3075[Abstract].
Levin, D.E., Bartlett-Heubusch, E. (1992) Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229[Abstract].
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275
Madaule, P., Axel, R., Myers, A.M. (1987) Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA. 84:779-783[Abstract].
Mitchison, J.M., Vincent, W.S. (1965) Preparation of synchronous cell cultures by sedimentation. Nature. 205:987-989.
Mol, P.C., Park, H.-M., Mullins, J.T., Cabib, E. (1994) A GTP-binding protein regulates the activity of (13)-ß-glucan synthase, an enzyme directly involved in yeast cell wall morphogenesis. J. Biol. Chem. 269:31267-31274
Nonaka, H., Tanaka, K., Hirano, H., Fujiwara, T., Kohno, H., Umikawa, M., Mino, A., Takai, Y. (1995) A downstream target of RHO1 a small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae. EMBO (Eur. Mol. Biol. Organ.) J. 14:5931-5938[Abstract].
Peppler, H.J., Rudert, F.J. (1953) Comparative evaluation of some methods for estimation of the quality of active dry yeast. Cereal. Chem. 30:146-152.
Peterson, J., Zheng, Y., Bender, L., Myers, A., Cerione, R., Bender, A. (1994) Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast. J. Cell Biol. 127:1395-1406[Abstract].
Qadota, H., Python, C.P., Inoue, S.B., Arisawa, M., Anraku, Y., Zheng, Y., Watanabe, T., Levin, D.E., Ohya, Y. (1996) Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-glucan synthase. Science. 272:279-281[Abstract].
Qiu, R.G., Chen, J., McCormick, F., Symons, M. (1995) A role for Rho in Ras transformation. Proc. Natl. Acad. Sci. USA. 92:11781-11785[Abstract].
Rose, M.D., Winston, F., Hieter, P. (1990) Methods in Yeast Genetics. A Laboratory Course Manual. NY, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Shaw, J.A., Mol, P.C., Bowers, B., Silverman, S.J., Valdivieso, M.H., Durán, A., Cabib, E. (1991) The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J. Cell Biol. 114:111-123[Abstract].
Sigal, I.S., Gibbs, J.B., D'Alonzo, J.S., Scolnick, E.M. (1986) Identification of effector residues and a neutralizing epitope of Ha-Ras-encoded p21. Proc. Natl. Acad. Sci. USA. 83:4725-4729[Abstract].
Sikorski, R.S., Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:19-27
Uesono, Y., Toh-e, A., Kikuchi, Y. (1997) Ssd1p of Saccharomyces cerevisiae associates with RNA. J. Biol. Chem. 272:16103-16109
Verma, R., Feldman, R.M.R., Deshaies, R.J. (1997a) SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34 and cyclin/CDK activities. Mol. Biol. Cell. 8:1427-1437[Abstract].
Verma, R., Anan, R.S., Huddleston, M.J., Carr, S.A., Reynard, G., Deshaies, R.J. (1997b) Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase. Science. 278:455-460
Yamochi, W., Tanaka, K., Nonaka, H., Maeda, A., Musha, T., Takai, Y. (1994) Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae. J. Cell Biol. 125:1077-1093[Abstract].
Ziman, M., Preuss, D., Mulholland, J., O'Brien, J.M., Botstein, D., Johnson, D.I. (1993) Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity. Mol. Biol. Cell. 4:1307-1316[Abstract].