Biomedical subjects
I Herskowitz
Publications and source records attributed to I Herskowitz.
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.
Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast.
Cyclin-dependent kinase inhibitors (CKIs) play key roles in controlling the eukaryotic cell cycle by coordinating cell proliferation and differentiation. Understanding the roles of CKIs requires knowledge of how they are regulated both through the cell cycle and in response to extracellular signals. Here we show that the yeast CKI, Far1p, is controlled by ubiquitin-dependent proteolysis. Wild-type Far1p was stable only in the G1 phase of the cell cycle. Biochemical and genetic evidence indicate that its degradation required the components of the G1-S ubiquitination system, Cdc34p, Cdc4p, Cdc53p, and Skp1p. We isolated a mutant form of Far1p (Far1p-22) that was able to induce cell cycle arrest in the absence of alpha-factor. Cells that overexpress Far1-22p arrested in G1 as large unbudded cells with low Cdc28p-Clnp kinase activity. Wild-type Far1p, but not Far1-22p, was readily ubiquitinated in vitro in a CDC34- and CDC4-dependent manner. Far1-22p harbors a single amino acid change, from serine to proline at residue 87, which alters phosphorylation by Cdc28p-Cln2p in vitro. Our results show that Far1p is regulated by ubiquitin-mediated proteolysis and suggest that phosphorylation of Far1p by the Cdc28p-Clnp kinase is part of the recognition signal for ubiquitination.
Osmotic balance regulates cell fusion during mating in Saccharomyces cerevisiae.
Successful zygote formation during yeast mating requires cell fusion of the two haploid mating partners. To ensure that cells do not lyse as they remodel their cell wall, the fusion event is both temporally and spatially regulated: the cell wall is degraded only after cell-cell contact and only in the region of cell-cell contact. To understand how cell fusion is regulated, we identified mutants defective in cell fusion based upon their defect in mating to a fus1 fus2 strain (Chenevert, J., N. Valtz, and I. Herskowitz. 1994. Genetics 136:1287-1297). Two of these cell fusion mutants are defective in the FPS1 gene, which codes for a glycerol facilitator (Luyten, K., J. Albertyn, W.F. Skibbe, B.A. Prior, J. Ramos, J.M. Thevelein, and S. Hohmann. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:1360-1371). To determine whether inability to maintain osmotic balance accounts for the defect in cell fusion in these mutants, we analyzed the behavior of an fps1Delta mutant with reduced intracellular glycerol levels because of a defect in the glycerol-3-phosphate dehydrogenase (GPD1) gene (Albertyn, J., S. Hohmann, J.M. Thevelein, and B.A. Prior. 1994. Mol. Cell. Biol. 14:4135-4144): deletion of GPD1 partially suppressed the cell fusion defect of fps1 mutants. In contrast, overexpression of GPD1 exacerbated the defect. The fusion defect could also be partially suppressed by 1 M sorbitol. These observations indicate that the fusion defect of fps1 mutants results from inability to regulate osmotic balance and provide evidence that the osmotic state of the cell can regulate fusion. We have also observed that mutants expressing hyperactive protein kinase C exhibit a cell fusion defect similar to that of fps1 mutants. We propose that Pkc1p regulates cell fusion in response to osmotic disequilibrium. Unlike fps1 mutants, fus1 and fus2 mutants are not influenced by expression of GPD1 or by 1 M sorbitol. Their fusion defect is thus unlikely to result from altered osmotic balance.
Actin-dependent localization of an RNA encoding a cell-fate determinant in yeast.
The cytoplasmic localization of messenger RNA creates an asymmetric distribution of proteins that specify cell fate during development in multicellular eukaryotes. The protein Ash1 is a cell-fate determinant in budding yeast which localizes preferentially to the presumptive daughter nucleus, where it inhibits mating-type switching. Here we show that Ash1 mRNA is localized to the distal tip of daughter buds in post-anaphase cells. Three-dimensional imaging reveals that Ash1 mRNA is assembled into particles that associate with the cell cortex. To achieve this localization, Ash1 mRNA must have its 3' untranslated region and the actin cytoskeleton must be intact. Ash1 mRNA is not localized correctly in the absence of a myosin (Myo4) and is mislocalized to the mother-bud neck in the absence of a regulator of the actin cytoskeleton known as Bnil. We propose that Ash1 mRNA particles are transported into the daughter bud along actin filaments and are anchored at the distal tip. Thus, as in higher eukaryotes, Saccharomyces cerevisiae employs RNA localization to generate an asymmetric distribution of proteins and hence to determine cell fate.
A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiae in bud emergence and identification of a putative upstream regulator.
The protein kinase C of Saccharomyces cerevisiae, Pkc1, regulates a MAP kinase, Mpk1, whose activity is stimulated at the G1-S transition of the cell cycle and by perturbations to the cell surface, e.g. induced by heat shock. The activity of the Pkc1 pathway is partially dependent on Cdc28 activity. Swi4 activates transcription of many genes at the G1-S transition, including CLN1 and CLN2. We find that swi4 mutants are defective specifically in bud emergence. The growth and budding defects of swi4 mutants are suppressed by overexpression of PKC1. This suppression requires CLN1 and CLN2. Inhibition of the Pkc1 pathway exacerbates the growth and bud emergence defects of swi4 mutants. We find that another dose-dependent suppressor of swi4 mutants, the novel gene HCS77, encodes a putative integral membrane protein. Hcs77 may regulate the Pkc1 pathway; hcs77 mutants exhibit phenotypes like those of mpk1 mutants, are partially suppressed by overexpression of PKC1 and are defective in heat shock induction of Mpk1 activity. We propose that the Pkc1 pathway promotes bud emergence and organized surface growth and is activated by Cdc28-Cln1/Cln2 at the G1-S transition and by Hcs77 upon heat shock. Hcs77 may monitor the state of the cell surface.
Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarity.
Cells of budding yeast organize their cytoskeleton in a highly polarized manner during vegetative growth. Selection of a site for polarization requires a group of proteins including a Ras-like GTPase, Bud1, and its regulators. Another group of proteins, which includes a Rho-like GTPase (Cdc42), its guanine nucleotide exchange factor (Cdc24), and Bem1, is necessary for organization of the actin cytoskeleton and for cell polarization. We have proposed previously that the Bud1 protein, through its GTPase cycle, determines the localization of one or more of the cell polarity proteins to the bud site. Herein we demonstrate that Bud1 directly interacts with Cdc24 and Bem1: Bud1 in its GTP-bound form associates preferentially with Cdc24, whereas the GDP-bound form of Bud1 associates with Bem1. We also present subcellular fractionation data for Bud1 that is consistent with the idea that Bud1 can travel between the site for budding on the plasma membrane and the cytosol. We propose that Bud1 can exist in two active states for association with different partners and that the switch from Bud1-GTP to Bud1-GDP provides a regulatory device for ordered assembly of a macromolecular complex at the bud site.
Building organs and organisms: elements of morphogenesis exhibited by budding yeast.
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Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast.
STE20 encodes a protein kinase related to mammalian p65Pak which functions in several signal transduction pathways in yeast, including those involved in pseudohyphal and invasive growth, as well as mating. In addition, Ste20 plays an essential role in cells lacking Cla4, a kinase with significant homology to Ste20. It is not clear how the activity of Ste20 is regulated in response to these different signals in vivo, but it has been demonstrated recently that binding of the small GTP binding protein Cdc42 is able to activate Ste20 in vitro. Here we show that Ste20 functionally interacts with Cdc42 in a GTP-dependent manner in vivo: Ste20 mutants that can no longer bind Cdc42 were unable to restore growth of ste20 cla4 mutant cells. They were also defective for pseudohyphal growth and agar invasion, and displayed reduced mating efficiency when mated with themselves. Surprisingly, however, the kinase activity of such Ste20 mutants was normal when assayed in vitro. Furthermore, these alleles were able to fully activate the MAP kinase pathway triggered by mating pheromones in vivo, suggesting that binding of Cdc42 and Ste20 was not required to activate Ste20. Wild-type Ste20 protein was visualized as a crescent at emerging buds during vegetative growth and at shmoo tips in cells arrested with alpha-factor. In contrast, a Ste20 mutant protein unable to bind Cdc42 was found diffusely throughout the cytoplasm, suggesting that Cdc42 is required to localize Ste20 properly in vivo.
Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity.
The pheromone signal in the yeast Saccharomyces cerevisiae is transmitted by the beta and gamma subunits of the mating response G-protein. The STE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G beta mutation. The same genetic screen identified BEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designated MDG1 (multicopy suppressor of defective G-protein). The MDG1 gene was independently isolated in a search for multicopy suppressors of a bem1 mutation. The MDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins. A fusion of Mdg1p with the green fluorescent protein from Aequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein. Deletion of MDG1 causes sterility in cells in which the wild-type G beta has been replaced by partly defective G beta derivatives but does not cause any other obvious phenotypes. The mating defect of cells deleted for STE20 is partially suppressed by multiple copies of BEM1 and CDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity. Elevated levels of STE20 and BEM1 are capable of suppressing a temperature-sensitive mutation in CDC42. This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating.
Identification of asymmetrically localized determinant, Ash1p, required for lineage-specific transcription of the yeast HO gene.
S. cerevisiae cells exhibit asymmetric determination of cell fate. Cell division yields a mother cell, which is competent to transcribe the HO gene and switch mating type, and a daughter cell, which is not. We have isolated a mutant in which daughters transcribe HO and switch mating type. This mutation defines the ASH1 gene (asymmetric synthesis of HO). Deletion and overexpression of ASH1 cause reciprocal cell fate transformations: im ash1delta strains, daughters switch mating type as efficiently as mothers. Conversely, overexpression of ASH1 inhibits switching in mother cells. Ash1p has a zinc finger motif related to those of GATA transcriptional regulators. Ash1p is localized to the daughter nucleus in cells that have undergone nuclear division. Thus, Ash1p is a cell fate determinant that is asymmetrically localized to the daughter nucleus where it inhibits HO transcription.
The BUD4 protein of yeast, required for axial budding, is localized to the mother/BUD neck in a cell cycle-dependent manner.
A and alpha cells of the yeast Saccharomyces cerevisiae exhibit an axial budding pattern, whereas a/alpha diploid cells exhibit a bipolar pattern. Mutations in BUD3, BUD4, and AXL1 cause a and alpha cells to exhibit the bipolar pattern, indicating that these genes are necessary to specify the axial budding pattern (Chant, J., and I. Herskowitz. 1991. Cell. 65:1203-1212; Fujita, A., C. Oka, Y. Arikawa, T. Katagi, A. Tonouchi, S. Kuhara, and Y. Misumi. 1994. Nature (Lond.). 372:567-570). We cloned and sequenced BUD4, which codes for a large, novel protein (Bud4p) with a potential GTP-binding motif. Bud4p is expressed and localized to the mother/bud neck in all cell types. Most mitotic cells contain two apparent rings of Bud4 immunoreactive staining, as observed for Bud3p (Chant, J., M. Mischke, E. Mitchell, I. Herskowitz, and J.R. Pringle. 1995. J. Cell Biol. 129: 767-778). Early G1 cells contain a single ring of Bud4p immunoreactive staining, whereas cells at START and in S phase lack these rings. The level of Bud4p is also regulated in a cell cycle-dependent manner. Bud4p is inefficiently localized in bud3 mutants and after a temperature shift of a temperature-sensitive mutant, cdc12, defective in the neck filaments. These observations suggest that Bud4p and Bud3p cooperate to recognize a spatial landmark (the neck filaments) during mitosis and support the hypothesis that they subsequently become a landmark for establishing the axial budding pattern in G1.
Pea2 protein of yeast is localized to sites of polarized growth and is required for efficient mating and bipolar budding.
Saccharomyces cerevisiae exhibits polarized growth during two phases of its life cycle, budding and mating. The site for polarization during vegetative growth is determined genetically: a and alpha haploid cells exhibit an axial budding pattern, and a/alpha diploid cells exhibit a bipolar pattern. During mating, each cell polarizes towards its partner to ensure efficient mating. SPA2 is required for the bipolar budding pattern (Snyder. M 1989. J. Cell Biol. 108:1419-1429; Zahner, J.A., H.A. Harkins, and J.R. Pringle. 1996. Mol. Cell. Biol. 16:1857-1870) and polarization during mating (Snyder, M., S. Gehrung, and B.D. Page. 1991. J. Cell Biol. 114: 515-532). We previously identified mutants defective in PEA2 and SPA2 which alter cell polarization in the presence of mating pheromone in a similar manner (Chenevert, J., N. Valtz, and I. Herskowitz. 1994. Genetics, 136:1287-1297). Here we report the further characterization of these mutants. We have found that PEA2 is also required for the bipolar budding pattern and that it encodes a novel protein with a predicted coiled-coil domain. Pea2p is expressed in all cell types and is localized to sites of polarized growth in budding and mating cells in a pattern similar to Spa2p, Pea2p and Spa2p exhibit interdependent localization: Spa2p is produced in pea2 mutants but fails to localize properly; Pea2p is not stably produced in spa2 mutants. These results suggest that Pea2p and Spa2p function together as a complex to generate the bipolar budding pattern and to guarantee proper polarization during mating.
Discrete developmental stages during teliospore formation in the corn smut fungus, Ustilago maydis.
Ustilago maydis is a dimorphic fungus with a yeast-like non-pathogenic form and a filamentous (hyphal) pathogenic form that induces tumor formation in maize. Within mature tumors, hyphae give rise to teliospores, which are round, diploid cells surrounded by a specialized cell wall. Here we describe the time course of fungal development in the plant with a focus on the morphological changes in the hyphae and the pathway of teliospore formation. We confirm and extend earlier observations that U. maydis hyphae branch extensively on the leaf surface and intracellularly before induction of tumors. We observe that at later stages the filaments undergo a series of discrete morphogenetic changes leading to teliospore formation. In particular, we show that the hyphae become embedded in a mucilaginous matrix within the tumor cells and the hyphal tips become modified. The hyphae then undergo fragmentation to release individual cells that exhibit a variety of shapes on their way to becoming rounded. Finally, a specialized cell wall is deposited. Support for the existence of such a pathway comes from analysis of a mutant defective in the fuz1 gene: inactivation of fuz1 blocks production of the mucilaginous matrix and fragmentation of the hyphae, leading to a defect in teliospore formation. The different morphological changes that occur while in the plant but not in culture suggest that plant inputs play a key role in fungal development.
Amino acid substitutions in the structured domains of histones H3 and H4 partially relieve the requirement of the yeast SWI/SNF complex for transcription.
Transcription of many yeast genes requires the SWI/SNF regulatory complex. Prior studies show that reduced transcription of the HO gene in swi and snf mutants is partially relieved by mutations in the SIN1 and SIN2 genes. Here we show that SIN2 is identical to HHT1, one of the two genes coding for histone H3, and that mutations in either can result in a Sin- phenotype. These mutations are partially dominant to wild type and cause amino acid substitutions in three conserved positions in the structured domain of histone H3. We have also identified partially dominant sin mutations that affect two conserved positions in the histone-fold domain of histone H4. Three sin mutations affect surface residues proposed to interact with DNA and may reduce affinity of DNA for the histone octamer. Two sin mutations affect residues at or near interfaces between (H2A-H2B) dimer and (H3-H4)2 tetramer subunits of the histone octamer and may affect nucleosome stability or conformation. The ability of mutations affecting the structure of the histone octamer to relieve the need for SWI and SNF products supports the proposal that the SWI/SNF complex stimulates transcription by altering chromatin structure and can account for the apparent conservation of SWI and SNF proteins in eukaryotes other than yeast.
Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins.
Studies of the temperature-sensitive cdc37-1 mutant of Saccharomyces cerevisiae suggest that Cdc37 is required for passage through the G1 phase of the cell cycle, but its precise function is not known. We have investigated the role of Cdc37 in the regulation of the cyclin-dependent protein kinase Cdc28. We find that G1 arrest in the cdc37-1 mutant is accompanied by a decrease in the Cdc28 activity associated with the G1 cyclin Cln2. This defect appears to be caused by a decrease in the binding of Cdc28 and Cln2. cdc37-1 mutants also exhibit a defect in the binding and activation of Cdc28 by the mitotic cyclin Clb2. Thus Cdc37 may be a regulator that is required for the association of Cdc28 with multiple cyclins.
MAP kinase pathways in yeast: for mating and more.
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Role of Bud3p in producing the axial budding pattern of yeast.
Yeast cells can select bud sites in either of two distinct spatial patterns. a cells and alpha cells typically bud in an axial pattern, in which both mother and daughter cells form new buds adjacent to the preceding division site. In contrast, a/alpha cells typically bud in a bipolar pattern, in which new buds can form at either pole of the cell. The BUD3 gene is specifically required for the axial pattern of budding: mutations of BUD3 (including a deletion) affect the axial pattern but not the bipolar pattern. The sequence of BUD3 predicts a product (Bud3p) of 1635 amino acids with no strong or instructive similarities to previously known proteins. However, immunofluorescence localization of Bud3p has revealed that it assembles in an apparent double ring encircling the mother-bud neck shortly after the mitotic spindle forms. The Bud3p structure at the neck persists until cytokinesis, when it splits to yield a single ring of Bud3p marking the division site on each of the two progeny cells. These single rings remain for much of the ensuing unbudded phase and then disassemble. The Bud3p rings are indistinguishable from those of the neck filament-associated proteins (Cdc3p, Cdc10p, Cdc11p, and Cdc12p), except that the latter proteins assemble before bud emergence and remain in place for the duration of the cell cycle. Upon shift of a temperature-sensitive cdc12 mutant to restrictive temperature, localization of both Bud3p and the neck filament-associated proteins is rapidly lost. In addition, a haploid cdc11 mutant loses its axial-budding pattern upon shift to restrictive temperature. Taken together, the data suggest that Bud3p and the neck filaments are linked in a cycle in which each controls the position of the other's assembly: Bud3p assembles onto the neck filaments in one cell cycle to mark the site for axial budding (including assembly of the new ring of neck filaments) in the next cell cycle. As the expression and localization of Bud3p are similar in a, alpha, and a/alpha cells, additional regulation must exist such that Bud3p restricts the position of bud formation in a and alpha cells but not in a/alpha cells.