Dynamic localization of protein phosphatase type 1 in the mitotic cell cycle of Saccharomyces cerevisiae.

Protein phosphatase type I (PP1), encoded by the single essential gene GLC7 in Saccharomyces cerevisiae, functions in diverse cellular processes. To identify in vivo subcellular location(s) where these processes take place, we used a functional green fluorescent protein (GFP)-Glc7p fusion protein. Time-lapse fluorescence microscopy revealed GFP-Glc7p localizes predominantly in the nucleus throughout the mitotic cell cycle, with the highest concentrations in the nucleolus. GFP-Glc7p was also observed in a ring at the bud neck, which was dependent upon functional septins. Supporting a role for Glc7p in bud site selection, a glc7-129 mutant displayed a random budding pattern. In alpha-factor treated cells, GFP-Glc7p was located at the base of mating projections, again in a septin-dependent manner. At the start of anaphase, GFP-Glc7p accumulated at the spindle pole bodies and remained there until cytokinesis. After anaphase, GFP-Glc7p became concentrated in a ring that colocalized with the actomyosin ring. A GFP-Glc7-129 fusion was defective in localizing to the bud neck and SPBs. Together, these results identify sites of Glc7p function and suggest Glc7p activity is regulated through dynamic changes in its location.

The JNM1 gene in the yeast Saccharomyces cerevisiae is required for nuclear migration and spindle orientation during the mitotic cell cycle.

JNM1, a novel gene on chromosome XIII in the yeast Saccharomyces cerevisiae, is required for proper nuclear migration. jnm1 null mutants have a temperature-dependent defect in nuclear migration and an accompanying alteration in astral microtubules. At 30 degrees C, a significant proportion of the mitotic spindles is not properly located at the neck between the mother cell and the bud. This defect is more severe at low temperature. At 11 degrees C, 60% of the cells accumulate with large buds, most of which have two DAPI staining regions in the mother cell. Although mitosis is delayed and nuclear migration is defective in jnm1 mutant, we rarely observe more than two nuclei in a cell, nor do we frequently observe anuclear cells. No loss of viability is observed at 11 degrees C and cells continue to grow exponentially with increased doubling time. At low temperature the large budded cells of jnm1 mutants exhibit extremely long astral microtubules that often wind around the periphery of the cell. jnm1 mutants are not defective in chromosome segregation during mitosis, as assayed by the rate of chromosome loss, or nuclear migration during conjugation, as assayed by the rate of mating and cytoduction. The phenotype of a jnm1 mutant is strikingly similar to that for mutants in the dynein heavy chain gene (Eshel, D., L. A. Urrestarazu, S. Vissers, J.-C. Jauniaux, J. C. van Vliet-Reedijk, R. J. Plants, and I. R. Gibbons. 1993. Proc. Natl. Acad. Sci. USA. 90:11172-11176; Li, Y. Y., E. Yeh, T. Hays, and K. Bloom. 1993. Proc. Natl. Acad. Sci. USA. 90:10096-10100). The JNM1 gene product is predicted to encode a 44-kD protein containing three coiled coil domains. A JNM1:lacZ gene fusion is able to complement the cold sensitivity and microtubule phenotype of a jnm1 deletion strain. This hybrid protein localizes to a single spot in the cell, most often near the spindle pole body in unbudded cells and in the bud in large budded cells. Together these results point to a specific role for Jnm1p in spindle migration, possibly as a subunit or accessory protein for yeast dynein.

Actin filaments in yeast are unstable in the absence of capping protein or fimbrin.

Many actin-binding proteins affect filament assembly in vitro and localize with actin in vivo, but how their molecular actions contribute to filament assembly in vivo is not understood well. We report here that capping protein (CP) and fimbrin are both important for actin filament assembly in vivo in Saccharomyces cerevisiae, based on finding decreased actin filament assembly in CP and fimbrin mutants. We have also identified mutations in actin that enhance the CP phenotype and find that those mutants also have decreased actin filament assembly in vivo. In vitro, actin purified from some of these mutants is defective in polymerization or binding fimbrin. These findings support the conclusion that CP acts to stabilize actin filaments in vivo. This conclusion is particularly remarkable because it is the opposite of the conclusion drawn from recent studies in Dictyostelium (Hug, C., P.Y. Jay, I. Reddy, J.G. McNally, P.C. Bridgman, E.L. Elson, and J.A. Cooper. 1995. Cell. 81:591-600). In addition, we find that the unpolymerized pool of actin in yeast is very small relative to that found in higher cells, which suggests that actin filament assembly is less dynamic in yeast than higher cells.

A Gip1p-Glc7p phosphatase complex regulates septin organization and spore wall formation.

Sporulation of Saccharomyces cerevisiae is a developmental process in which a single cell is converted into four haploid spores. GIP1, encoding a developmentally regulated protein phosphatase 1 interacting protein, is required for spore formation. Here we show that GIP1 and the protein phosphatase 1 encoded by GLC7 play essential roles in spore development. The gip1Delta mutant undergoes meiosis and prospore membrane formation normally, but is specifically defective in spore wall synthesis. We demonstrate that in wild-type cells, distinct layers of the spore wall are deposited in a specific temporal order, and that gip1Delta cells display a discrete arrest at the onset of spore wall deposition. Localization studies revealed that Gip1p and Glc7p colocalize with the septins in structures underlying the growing prospore membranes. Interestingly, in the gip1Delta mutant, not only is Glc7p localization altered, but septins are also delocalized. Similar phenotypes were observed in a glc7-136 mutant, which expresses a Glc7p defective in interacting with Gip1p. These results indicate that a Gip1p-Glc7p phosphatase complex is required for proper septin organization and initiation of spore wall formation during sporulation.

CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains two functional homologues of the ras oncogene family, RAS1 and RAS2. These genes are required for growth, and all evidence indicates that this essential function is the activation of adenylate cyclase. In contrast, ras in mammalian cells does not appear to influence adenylate cyclase activity. To clarify the relation between ras function in yeast and in higher eukaryotes, and the role played by yeast RAS in growth control, it is necessary to identify functions acting upstream of RAS in the adenylate cyclase pathway. The evidence presented here indicates that CDC25, identified by conditional cell cycle arrest mutations, encodes such an upstream function.

Mammalian and yeast ras gene products: biological function in their heterologous systems.

Activated versions of ras genes have been found in various types of malignant tumors. The normal versions of these genes are found in organisms as diverse as mammals and yeasts. Yeast cells that lack their functional ras genes, RASSC-1 and RASSC-2, are ordinarily nonviable. They have now been shown to remain viable if they carry a mammalian rasH gene. In addition, yeast-mammalian hybrid genes and a deletion mutant yeast RASSC-1 gene were shown to induce morphologic transformation of mouse NIH 3T3 cells when the genes had a point mutation analogous to one that increases the transforming activity of mammalian ras genes. The results establish the functional relevance of the yeast system to the genetics and biochemistry of cellular transformation induced by mammalian ras genes.

The role of RAP1 in the regulation of the MAT alpha locus.

The RAP1 gene of Saccharomyces cerevisiae encodes an abundant DNA-binding protein, also known as GRF1, TBA, or TUF, that binds to many sites in the yeast genome in vitro. These sites define a consensus sequence, [sequence: see text], and deletion analyses of genes that contain this sequence have implicated the involvement of RAP1 in numerous cellular processes, including gene activation and repression. The MAT alpha locus, required for determination of the alpha cell type in yeast cells, contains a RAP1 binding site; this site coincides with the MAT alpha upstream activating sequence (UAS) and is necessary for expression of the two genes encoded by the MAT alpha locus, MAT alpha 1 and MAT alpha 2. We show that the MAT alpha UAS is sufficient to activate transcription from a promoterless gene fusion of the yeast CYC1 upstream region and the lacZ gene. Constructs containing only the MAT alpha UAS generated elevated levels of beta-galactosidase activity which were indistinguishable from those of constructs containing the entire MAT alpha intergenic region. Further, the MAT alpha UAS has an intrinsic polarity of transcriptional activation; transcription of CYC1-lacZ was six- to sevenfold higher when the UAS was oriented in the direction normally associated with MAT alpha 2 transcription. Point mutations in the MAT alpha UAS that reduce MAT alpha expression three- to fivefold resulted in a bi-mating phenotype, while a mutation that reduced MAT alpha expression still further resulted in an a-mating phenotype. We isolated plasmids from a high-copy-number yeast library that suppressed the bi-mating defect of point mutations in the MAT alpha UAS, and the most effective dosage suppressor contained the gene encoding RAP1. A temperature-sensitive rap1 mutant bi-mates at the semipermissive temperature. Double mutants at rap1 and mat alpha mate exclusively as a cells, at all temperatures, and do not express detectable levels of MAT alpha RNA. These data provide evidence that the RAP1 gene product functions at the MAT alpha UAS in vivo.

Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase.

Mutations in the SRA1 or SRA3 gene eliminate the requirement for either RAS gene (RAS1 or RAS2) in Saccharomyces cerevisiae. We cloned SRA1 and SRA3 and determined their DNA sequences. SRA1 encodes the regulatory subunit of the cyclic AMP (cAMP)-dependent protein kinase and therefore is identical to REG1 and BCY1. This gene is not essential, but its deletion confers many traits: reduction of glycogen accumulation, temperature sensitivity, reduced growth rate on maltose and sucrose, inability to grow on galactose and nonfermentable carbon sources, and nitrogen starvation intolerance. SRA3 is homologous to protein kinases that phosphorylate serine and threonine and likely encodes the catalytic subunit of the cAMP-dependent protein kinase. The wild-type SRA3 gene either triplicated in the chromosome or on episomal, low-copy plasmids behaves like spontaneous dominant SRA3 mutations by suppressing ras2-530 (RAS2::LEU2 disruption), cdc25, and cdc35 mutations. These findings indicate that the yeast RAS genes are dispensable if there is constitutive cAMP-dependent protein kinase activity.

SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae.

sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity. We have cloned SRA5 by complementation. It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase. Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP. sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.

The REG2 gene of Saccharomyces cerevisiae encodes a type 1 protein phosphatase-binding protein that functions with Reg1p and the Snf1 protein kinase to regulate growth.

The GLC7 gene of Saccharomyces cerevisiae encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is essential for cell growth. We have isolated a previously uncharacterized gene, REG2, on the basis of its ability to interact with Glc7p in the two-hybrid system. Reg2p interacts with Glc7p in vivo, and epitope-tagged derivatives of Reg2p and Glc7p coimmunoprecipitate from cell extracts. The predicted protein product of the REG2 gene is similar to Reg1p, a protein believed to direct PP1 activity in the glucose repression pathway. Mutants with a deletion of reg1 display a mild slow-growth defect, while reg2 mutants exhibit a wild-type phenotype. However, mutants with deletions of both reg1 and reg2 exhibit a severe growth defect. Overexpression of REG2 complements the slow-growth defect of a reg1 mutant but does not complement defects in glycogen accumulation or glucose repression, two traits also associated with a reg1 deletion. These results indicate that REG1 has a unique role in the glucose repression pathway but acts together with REG2 to regulate some as yet uncharacterized function important for growth. The growth defect of a reg1 reg2 double mutant is alleviated by a loss-of-function mutation in the SNF1-encoded protein kinase. The snf1 mutation also suppresses the glucose repression defects of reg1. Together, our data are consistent with a model in which Reg1p and Reg2p control the activity of PP1 toward substrates that are phosphorylated by the Snf1p kinase.

Molecular cloning and characterization of the STE7 and STE11 genes of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, haploid cells occur in one of the two cell types, a or alpha. The allele present at the mating type (MAT) locus plays a prominent role in the control of cell type expression. An important consequence of the elaboration of cell type is the ability of cells of one mating type to conjugate with cells of the opposite mating type, resulting in yet a third cell type, an a/alpha diploid. Numerous genes that are involved in the expression of cell type and the conjugation process have been identified by standard genetic techniques. Molecular analysis has shown that expression of several of these genes is subject to control on the transcriptional level by the MAT locus. Two genes, STE7 and STE11, are required for mating in both haploid cell types; ste7 and ste11 mutants are sterile. We report here the molecular cloning of STE7 and STE11 genes and show that expression of these genes is not regulated transcriptionally by the MAT locus. We also have genetically mapped the STE11 gene to chromosome XII, 40 centimorgans from ura4.

The EGP1 gene may be a positive regulator of protein phosphatase type 1 in the growth control of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae GLC7 gene encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is required for cell growth. A cold-sensitive glc7 mutant (glc7Y170) arrests in G2/M but remains viable at the restrictive temperature. In an effort to identify additional gene products that function in concert with PP1 to regulate growth, we isolated a mutation (gpp1) that exacerbated the growth phenotype of the glc7Y170 mutation, resulting in rapid death of the double mutant at the nonpermissive temperature. We identified an additional gene, EGP1, as an extra-copy suppressor of the glc7Y170 gpp1-1 double mutant. The nucleotide sequence of EGP1 predicts a leucine-rich repeat protein that is similar to Sds22, a protein from the fission yeast Schizosaccharomyces pombe that positively modulates PP1. EGP1 is essential for cell growth but becomes dispensable upon overexpression of the GLC7 gene. Egp1 and PP1 directly interact, as assayed by coimmunoprecipitation. These results suggest that Egp1 functions as a positive modulator of PP1 in the growth control of S. cerevisiae.

The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit.

Loss-of-function gac1 mutants of Saccharomyces cerevisiae fail to accumulate normal levels of glycogen because of low glycogen synthase activity. Increased dosage of GAC1 results in increased activity of glycogen synthase and a corresponding hyperaccumulation of glycogen. The glycogen accumulation phenotype of gac1 is similar to that of glc7-1, a type 1 protein phosphatase mutant. We have partially characterized the GAC1 gene product (Gac1p) and show that levels of Gac1p increase during growth with the same kinetics as glycogen accumulation. Gac1p is phosphorylated in vivo and is hyperphosphorylated in a glc7-1 mutant. Gac1p and the type 1 protein phosphatase directly interact in vitro, as assayed by coimmunoprecipitation, and in vivo, as determined by the dihybrid assay described elsewhere (S. Fields and O.-k. Song, Nature [London] 340:245-246, 1989). The interaction between Gac1p and the glc7-1-encoded form of the type 1 protein phosphatase is defective, as assayed by either immunoprecipitation or the dihybrid assay. Increased dosage of GAC1 partially suppresses the glycogen defect of glc7-1. Collectively, our data support the hypotheses that GAC1 encodes a regulatory subunit of type 1 protein phosphatase and that the glycogen accumulation defect of glc7-1 is due at least in part to the inability of the mutant phosphatase to interact with its regulatory subunit.

Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.

The Saccharomyces cerevisiae SRK1 gene, a suppressor of bcy1 and ins1, may be involved in protein phosphatase function.

The Saccharomyces cerevisiae SRK1 gene, when expressed on a low-copy shuttle vector, partially suppresses the phenotype associated with elevated levels of cyclic AMP-dependent protein kinase activity and suppresses the temperature-sensitive cell cycle arrest of the ins1 mutant. SRK1 is located on chromosome IV, 3 centimorgans from gcn2. A mutant carrying a deletion mutation in srk1 is viable. SRK1 encodes a 140-kDa protein with homology to the dis3+ protein from Schizosaccharomyces pombe. The ability of SRK1 to alleviate partially the defects caused by high levels of cyclic AMP-dependent protein kinase and the similarity of its encoded protein to dis3+ suggest that SRK1 may have a role in protein phosphatase function.

Comparison of thermosensitive alleles of the CDC25 gene involved in the cAMP metabolism of Saccharomyces cerevisiae.

The CDC25 gene from Saccharomyces cerevisiae is an essential component of the RAS-adenylate cyclase pathway. Genetic and biochemical evidence has led to the proposal that the gene product may act upstream of RAS, possibly as a guanine nucleotide exchange factor. We report here the cloning, sequencing and characterization of four mutations in the CDC25 gene. All four are missense mutations which reside within the carboxy-terminal quarter of the single open reading frame found within the gene. Three of the four are missense mutations in the same amino acid codon. A search of protein data bases reveals that the carboxy terminus of the putative CDC25 gene product is similar to that of LTE1, a gene required for growth at low temperature and SCD25, a suppressor of cdc25. Taken together these data indicate that the carboxy terminus of CDC25 plays a critical role in the function of the CDC25 gene product and that other proteins, such as LTE1 or SCD25, may have related activities.

Deletion of the gene encoding the cyclin-dependent protein kinase Pho85 alters glycogen metabolism in Saccharomyces cerevisiae.

Pho85, a protein kinase with significant homology to the cyclin-dependent kinase, Cdc28, has been shown to function in repression of transcription of acid phosphatase (APase, encoded by PHO5) in high phosphate (Pi) medium, as well as in regulation of the cell cycle at G1/S. We described several unique phenotypes associated with the deletion of the PHO85 gene including growth defects on a variety of carbon sources and hyperaccumulation of glycogen in rich medium high in Pi. Hyperaccumulation of glycogen in the pho85 strains is independent of other APase regulatory molecules and is not signaled through Snfl kinase. However, constitutive activation of cAPK suppresses the hyperaccumulation of glycogen in a pho85 mutant. Mutation of the type-1 protein phosphatase encoded by GLC7 only partially suppresses the glycogen phenotype of the pho85 mutant. Additionally, strains containing a deletion of the PHO85 gene show an increase in expression of GSY2. This work provides evidence that Pho85 has functions in addition to transcriptional regulation of APase and cell-cycle progression including the regulation of glycogen levels in the cell and may provide a link between the nutritional state of the cell and these growth related responses.

Suppressors of the ras2 mutation of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two members of the ras gene family. Strains with disruptions of the RAS2 gene fail to grow efficiently on nonfermentable carbon sources. This growth defect can be suppressed by extragenic mutations called sra. We have isolated 79 independent suppressor mutations, 68 of which have been assigned to one of five loci. Eleven additional dominant mutations have not been assigned to a specific locus. Some sra1 and SRA4 and all SRA3 mutations were RAS independent, allowing growth of yeast cells that lack a functional RAS gene. Mutations in sra1, SRA3, SRA4 and sra6 are linked to his6, ino1, met3 and ade6, respectively. Some sra mutants have pleiotropic phenotypes that affect glycogen accumulation, sporulation, viability, respiratory capacity and suppression of two cell-division-cycle mutations, cdc25 and cdc35. The proposed functions of many of the suppressor genes are consistent with the model in which RAS activates adenylate cyclase.

Genetic interactions between GLC7, PPZ1 and PPZ2 in saccharomyces cerevisiae.

GLC7 encodes an essential serine/threonine protein type I phosphatase in Saccharomyces cerevisiae. Three other phosphatases (Ppz1p, Ppz2p, and Sal6p) share >59% identity in their catalytic region with Glc7p. ppz1 ppz2 null mutants have no apparent growth defect on rich media. However, null alleles of PPZ1 and PPZ2, in combination with mutant alleles of GLC7, confer a range of growth defects varying from slow growth to lethality. These results indicate that Glc7p, Ppz1p, and Ppz2p may have overlapping functions. To determine if this overlap extends to interaction with targeting subunits, Glc7p-binding proteins were tested for interaction in the two-hybrid system with the functional catalytic domain of Ppz1p. Ppz1p interacts strongly with a number of Glc7p regulatory subunits, including Glc8p, a protein that shares homology with mammalian PP1 inhibitor I2. Genetic data suggest that Glc8p positively affects both Glc7p and Ppz1p functions. Together our data suggest that Ppz1p and Ppz2p may have overlapping functions with Glc7p and that all three phosphatases may act through common regulatory proteins.