The protein kinase Pho85 is required for asymmetric accumulation of the Ash1 protein in Saccharomyces cerevisiae.

The Ash1 protein is a daughter cell-specific repressor of HO gene transcription in Saccharomyces cerevisiae. Both ASH1 mRNA and protein are localized to the incipient daughter cell at the end of mitosis; Ash1 then inhibits HO transcription in the daughter cell after cytokinesis. Mother cells, in contrast, contain little or no Ash1 and thus are able to transcribe HO. We show that deletion of PHO85, which encodes a cyclin-dependent protein kinase, causes reduced transcription of HO and that this reduction is dependent on ASH1. In pho85 mutants, Ash1 protein is no longer asymmetrically localized and is present, instead, in both mother and daughter cells. Initially, it appears to be localized properly but then persists as daughter cells mature into mother cells. In contrast, ASH1 mRNA is localized appropriately to daughter cells in pho85 mutants. We observe that Ash1 protein is phosphorylated by Pho85 in vitro and that Ash1 stability increases in a pho85 mutant. These data suggest that phosphorylation of Ash1 by Pho85 governs stability of Ash1 protein.

Induction of distinct [URE3] yeast prion strains.

[URE3] is a non-Mendelian genetic element in Saccharomyces cerevisiae, which is caused by a prion-like, autocatalytic conversion of the Ure2 protein (Ure2p) into an inactive form. The presence of [URE3] allows yeast cells to take up ureidosuccinic acid in the presence of ammonia. This phenotype can be used to select for the prion state. We have developed a novel reporter, in which the ADE2 gene is controlled by the DAL5 regulatory region, which allows monitoring of Ure2p function by a colony color phenotype. Using this reporter, we observed induction of different [URE3] prion variants ("strains") following overexpression of the N-terminal Ure2p prion domain (UPD) or full-length Ure2p. Full-length Ure2p induced two types of [URE3]: type A corresponds to conventional [URE3], whereas the novel type B variant is characterized by relatively high residual Ure2p activity and efficient curing by coexpression of low amounts of a UPD-green fluorescent protein fusion protein. Overexpression of UPD induced type B [URE3] but not type A. Both type A and B [URE3] strains, as well as weak and strong isolates of type A, were shown to stably maintain different prion strain characteristics. We suggest that these strain variants result from different modes of aggregation of similar Ure2p monomers. We also demonstrate a procedure to counterselect against the [URE3] state.

Post-transcriptional regulation through the HO 3'-UTR by Mpt5, a yeast homolog of Pumilio and FBF.

Drosophila Pumilio (Pum) and Caenorhabditis elegans FBF bind to the 3'-untranslated region (3'-UTR) of their target mRNAs and repress translation. Pum and FBF are members of a large and evolutionarily conserved protein family, the Puf family, found in Drosophila, C.elegans, humans, and yeasts. Budding yeast, Saccharomyces cerevisiae, has five proteins with conserved Puf motifs: Mpt5/Uth4, Ygl014w, Yll013c, Jsn1, and Ypr042c. Here we report that Mpt5 negatively regulates expression of the HO gene. Loss of MPT5 increased expression of reporter genes integrated into the ho locus, whereas overexpression of MPT5 decreased expression. Repression required the 3'-UTR of HO, which contains a tetranucleotide, UUGU, also found in the binding sites of Pum and FBF. Mutation of UUGU to UACU in the HO 3'-UTR abolished Mpt5-mediated repression. Studies using a three-hybrid assay for RNA binding indicate that Mpt5 binds to the 3'-UTR of HO mRNA containing a UUGU sequence but not a UACU sequence. These observations suggest that the yeast Puf homolog, Mpt5, negatively regulates HO expression post-transcriptionally.

Ash1p is a site-specific DNA-binding protein that actively represses transcription.

ASH1 encodes a protein that is localized specifically to the daughter cell nucleus, where it has been proposed to repress transcription of the HO gene. Using Ash1p purified from baculovirus-infected insect cells, we have shown that Ash1p binds specific DNA sequences in the HO promoter. DNase I protection analyses showed that Ash1p recognizes a consensus sequence, YTGAT. Mutation of this consensus abolishes Ash1p DNA binding in vitro. We have shown that Ash1p requires an intact zinc-binding domain in its C terminus for repression of HO in vivo and that this domain may be involved in DNA binding. A heterologous DNA-binding domain fused to an N-terminal segment of Ash1p functions as an active repressor of transcription. Our studies indicate that Ash1p is a DNA-binding protein of the GATA family with a separable transcriptional repression domain.

The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein.

The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high beta-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, beta-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p.

The ins and outs of cell-polarity decisions.

The guanine-nucleotide-exchange factor Cdc24 is a critical regulator of cell polarity. Far1 is a key player in Cdc24 regulation, controlling Cdc24 activity in budding yeast by regulating its subcellular localization in response to two very different signals - one external and one internal.

Genetic and biochemical characterization of the yeast spo12 protein.

We have performed a genetic and biochemical analysis of the SPO12 gene, which regulates meiotic nuclear divisions in budding yeast. When sporulated, spo12 mutants undergo a single meiotic nuclear division most closely resembling meiosis II. We observed that Spo12 protein is localized to the nucleus during both meiotic divisions and that Clb1-Cdc28, Clb3-Cdc28, Clb4-Cdc28, and Clb5-Cdc28 kinase activities during meiosis were not affected by a spo12 mutation. Using two-hybrid analysis, we identified several genes, three of which are meiotically induced, that may code for proteins that interact with Spo12p. We also observed that two genes, BNS1 (Bypasses Need for Spo12p), which has homology to SPO12, and SPO13, whose mutant phenotype is like that of spo12, can partially suppress the meiotic defect of spo12 mutants when overexpressed. We found that Spo12p is also localized to the nucleus in vegetative cells and that its level peaks during G2/M. We observed that a spo12 mutation is synthetically lethal in vegetative cells with a mutation in HCT1, a gene necessary for cells to exit mitosis, suggesting that Spo12p may have a role in exit from mitosis.

Localization of Bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site.

Yeast cells of different cell type exhibit distinct budding patterns that reflect the organization of the actin cytoskeleton. Bud1p (Rsr1p), a Ras-like GTPase, and Bud2p, a GTPase-activating protein for Bud1p, are essential for proper budding pattern. We show that Bud2p is localized at the presumptive bud site in G(1) cells in all cell types and that this localization is independent of Bud1p. Bud2p subsequently localizes to the mother-bud neck after bud emergence; this localization depends on the integrity of the septins. These observations indicate that Bud2p becomes positioned in G(1) cells by recognizing cell type-specific landmarks at the presumptive bud site.

O-Glycosylation of Axl2/Bud10p by Pmt4p is required for its stability, localization, and function in daughter cells.

Cells of the yeast Saccharomyces cerevisiae choose bud sites in a manner that is dependent upon cell type: a and alpha cells select axial sites; a/alpha cells utilize bipolar sites. Mutants specifically defective in axial budding were isolated from an alpha strain using pseudohyphal growth as an assay. We found that a and alpha mutants defective in the previously identified PMT4 gene exhibit unipolar, rather than axial budding: mother cells choose axial bud sites, but daughter cells do not. PMT4 encodes a protein mannosyl transferase (pmt) required for O-linked glycosylation of some secretory and cell surface proteins (Immervoll, T., M. Gentzsch, and W. Tanner. 1995. Yeast. 11:1345-1351). We demonstrate that Axl2/Bud10p, which is required for the axial budding pattern, is an O-linked glycoprotein and is incompletely glycosylated, unstable, and mislocalized in cells lacking PMT4. Overexpression of AXL2 can partially restore proper bud-site selection to pmt4 mutants. These data indicate that Axl2/Bud10p is glycosylated by Pmt4p and that O-linked glycosylation increases Axl2/ Bud10p activity in daughter cells, apparently by enhancing its stability and promoting its localization to the plasma membrane.

The role of Far1p in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating.

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) determine tissue and cell polarity in a variety of organisms. In yeast, cells orient polarized growth toward the mating partner along a pheromone gradient by a mechanism that requires Far1p and Cdc24p. Far1p bound Gbetagamma and interacted with polarity establishment proteins, which organize the actin cytoskeleton. Cells containing mutated Far1p unable to bind Gbetagamma or polarity establishment proteins were defective for orienting growth toward their mating partner. In response to pheromones, Far1p moves from the nucleus to the cytoplasm. Thus, Far1p functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling marked by Gbetagamma to polarize assembly of the cytoskeleton in a morphogenetic gradient.

The transcriptional program of sporulation in budding yeast.

Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis. DNA microarrays containing nearly every yeast gene were used to assay changes in gene expression during sporulation. At least seven distinct temporal patterns of induction were observed. The transcription factor Ndt80 appeared to be important for induction of a large group of genes at the end of meiotic prophase. Consensus sequences known or proposed to be responsible for temporal regulation could be identified solely from analysis of sequences of coordinately expressed genes. The temporal expression pattern provided clues to potential functions of hundreds of previously uncharacterized genes, some of which have vertebrate homologs that may function during gametogenesis.

Identification of Kel1p, a kelch domain-containing protein involved in cell fusion and morphology in Saccharomyces cerevisiae.

We showed previously that protein kinase C, which is required to maintain cell integrity, negatively regulates cell fusion (Philips, J., and I. Herskowitz. 1997. J. Cell Biol. 138:961-974). To identify additional genes involved in cell fusion, we looked for genes whose overexpression relieved the defect caused by activated alleles of Pkc1p. This strategy led to the identification of a novel gene, KEL1, which encodes a protein composed of two domains, one containing six kelch repeats, a motif initially described in the Drosophila protein Kelch (Xue, F., and L. Cooley. 1993. Cell. 72:681- 693), and another domain predicted to form coiled coils. Overexpression of KEL1 also suppressed the defect in cell fusion of spa2Delta and fps1Delta mutants. KEL2, which corresponds to ORF YGR238c, encodes a protein highly similar to Kel1p. Its overexpression also suppressed the mating defect associated with activated Pkc1p. Mutants lacking KEL1 exhibited a moderate defect in cell fusion that was exacerbated by activated alleles of Pkc1p or loss of FUS1, FUS2, or FPS1, but not by loss of SPA2. kel1Delta mutants form cells that are elongated and heterogeneous in shape, indicating that Kel1p is also required for proper morphology during vegetative growth. In contrast, kel2Delta mutants were not impaired in cell fusion or morphology. Both Kel1p and Kel2p localized to the site where cell fusion occurs during mating and to regions of polarized growth during vegetative growth. Coimmunoprecipitation and two-hybrid analyses indicated that Kel1p and Kel2p physically interact. We conclude that Kel1p has a role in cell morphogenesis and cell fusion and may antagonize the Pkc1p pathway.

The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae.

The MAPKKK Ste11p functions in three Saccharomyces cerevisiae MAPK cascades [the high osmolarity glycerol (HOG), pheromone response, and pseudohyphal/invasive growth pathways], but its activation in response to high osmolarity stimulates only the HOG pathway. To determine what restricts cross-activation of MAPK cascades (cross talk), we have studied mutants in which the pheromone response pathway is activated by high osmolarity (1 M sorbitol). We found that mutations in the HOG1 gene, encoding the p38-type MAPK of the HOG pathway, and in the PBS2 gene, encoding the activating kinase for Hog1p, allowed osmolarity-induced activation of the pheromone response pathway. This cross talk required the osmosensor Sho1p, as well as Ste20p, Ste50p, the pheromone response MAPK cascade (Ste11p, Ste7p, and Fus3p or Kss1p), and Ste12p but not Ste4p or the MAPK scaffold protein, Ste5p. The cross talk in hog1 mutants induced multiple responses of the pheromone response pathway: induction of a FUS1::lacZ reporter, morphological changes, and mating in ste4 and ste5 mutants. We suggest that Hog1p may prevent osmolarity-induced cross talk by inhibiting Sho1p, perhaps as part of a feedback control on the HOG pathway. We have also shown that Ste20p and Ste50p function in the Sho1p branch of the HOG pathway and that a second osmosensor in addition to Sho1p may activate Ste11p. Finally, we have found that pseudohyphal growth exhibited by wild-type (HOG1) strains depends on SHO1, suggesting that Sho1p may be a receptor that feeds into the pseudohyphal growth pathway.

Gametogenesis in yeast is regulated by a transcriptional cascade dependent on Ndt80.

Gametogenesis requires the successful coordination of two key processes, meiotic nuclear division and gamete morphogenesis. A central regulatory step in progression through gametogenesis occurs at the pachytene stage of meiotic prophase. We find that Ndt80 functions at pachytene of yeast gametogenesis (sporulation) to activate transcription of a set of genes required for both meiotic division (e.g., B-type cyclins) and gamete formation (e.g., SPS1). Ectopic synthesis of Ndt80 in vegetative cells induces transcription of these genes, and recombinant Ndt80 protein binds to a conserved sequence in their upstream region. Transcription of NDT80 itself is dependent on Ime1, which activates expression of early sporulation genes. Transcription of the Ndt80-regulated gene CLB1 is mediated by the checkpoint gene RAD17. Thus Ndt80 is a pivotal component of a transcriptional cascade programming yeast gametogenesis and may also be a target of meiotic checkpoint control.

Nuclear proteins Nut1p and Nut2p cooperate to negatively regulate a Swi4p-dependent lacZ reporter gene in Saccharomyces cerevisiae.

The URS2 region of the Saccharomyces cerevisiae HO upstream region contains 10 binding sites for the Swi4p/Swi6p transcription factor and confers Swi4p dependence for transcription. Using a hybrid promoter, UASGAL (upstream activation sequence of GAL1)-URS2R, in which the GAL1-10 regulatory region is fused to the proximal 360 bp of URS2, we isolated mutants in which Swi4p is no longer required for transcription. Mutations of SIN4, ROX3, SRB8, SRB9, SRB10, SRB11, and two novel genes, NUT1 and NUT2, relieve the requirement of Swi4p for expression of this reporter. We found that NUT1 (open reading frame [ORF] YGL151w) is a nonessential gene, that NUT2 (ORF YPR168w) is essential, and that both Nut1p and Nut2p encode nuclear proteins. Deletion of NUT1 causes a constitutive, Swi4p-independent phenotype only in combination with the nut2-1 allele or an allele of CCR4. In contrast, inactivation of a temperature-sensitive allele of NUT2, nut2-ts70, alone causes constitutivity. nut1Delta nut2-1 cells and sin4Delta cells exhibit Swi4p-independent expression of an ho-lacZ reporter but not of an intact ho gene. Likewise, a pPHO5-lacZ construct is constitutively expressed in nut1 nut2 mutants relative to their wild-type counterparts. These results suggest that Nut1p, Nut2p, Sin4p, and Ccr4p define a group of proteins that negatively regulate transcription in a subtle manner which is revealed by artificial reporter genes.