George F. Sprague

Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae.

We have devised a screen for genes from the yeast Saccharomyces cerevisiae whose expression is affected by cell type or by the mating pheromones. From this screen we identified a gene, FUS1, whose pattern of expression revealed interesting regulatory strategies and whose product was required for efficient cell fusion during mating. Transcription of FUS1 occurred only in a and alpha cells, not in a/alpha cells, where it was repressed by a1 X alpha 2, a regulatory activity present uniquely in a/alpha cells. Transcription of FUS1 showed an absolute requirement for the products of five STE genes, STE4, STE5, STE7, STE11, and STE12. Since the activators STE4, STE5, and STE12 are themselves repressed by a1 X alpha 2, the failure to express FUS1 in a/alpha cells is probably the result of a cascade of regulatory activities; repression of the activators by a1 X alpha 2 in turn precludes transcription of FUS1. In addition to regulation of FUS1 by cell type, transcription from the locus increased 10-fold or more when a or alpha cells were exposed to the opposing mating pheromone. To investigate the function of the Fus1 protein, we created fus1 null mutants. In fus1 X fus1 matings, the cells of a mating pair adhered tightly and appeared to form zygotes. However, the zygotes were abnormal. Within the conjugation bridge the contained a partition that prevented nuclear fusion and mixing of organelles. The predicted sequence of the Fus1 protein (deduced from the FUS1 DNA sequence) and subcellular fractionation studies with Fus1-beta-galactosidase hybrid proteins suggest that Fus1 is a membrane or secreted protein. Thus, Fus1 may be located at a position within the cell where it is poised to catalyze cell wall or plasma membrane fusion.

The Regulation of Filamentous Growth in Yeast

Filamentous growth is a nutrient-regulated growth response that occurs in many fungal species. In pathogens, filamentous growth is critical for host–cell attachment, invasion into tissues, and virulence. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth, which provides a genetically tractable system to study the molecular basis of the response. Filamentous growth is regulated by evolutionarily conserved signaling pathways. One of these pathways is a mitogen activated protein kinase (MAPK) pathway. A remarkable feature of the filamentous growth MAPK pathway is that it is composed of factors that also function in other pathways. An intriguing challenge therefore has been to understand how pathways that share components establish and maintain their identity. Other canonical signaling pathways—rat sarcoma/protein kinase A (RAS/PKA), sucrose nonfermentable (SNF), and target of rapamycin (TOR)—also regulate filamentous growth, which raises the question of how signals from multiple pathways become integrated into a coordinated response. Together, these pathways regulate cell differentiation to the filamentous type, which is characterized by changes in cell adhesion, cell polarity, and cell shape. How these changes are accomplished is also discussed. High-throughput genomics approaches have recently uncovered new connections to filamentous growth regulation. These connections suggest that filamentous growth is a more complex and globally regulated behavior than is currently appreciated, which may help to pave the way for future investigations into this eukaryotic cell differentiation behavior.

Control of yeast cell type by the mating type locus: I. Identification and control of expression of the a-specific gene BAR1

Abstract The MATα allele of the yeast mating type locus confers the α mating phenotype and contains two complementation groups, MATα1 and MATα2. The α1–α2 hypothesis proposes that MATα1 is a positive regulator of α-specific genes and that MATα2 is a negative regulator of a-specific genes. According to this hypothesis, matα2 mutants, which are defective in mating and in production of extracellular α-factor, express both a-specific functions (because they lack MATα2 product) and α-specific functions (because they contain MATα1 product). Failure to produce extracellular α-factor results from antagonism between these functions; in particular, because α-factor (an α-specific function) is degraded by an a-specific function. If this view is correct, matα2 mutants should acquire the ability to produce α-factor if they also carry a defect in the gene(s) responsible for α-factor degradation. We have isolated a derivative of a matα2 mutant that produces α-factor and have characterized the suppressor mutation in this strain. (1) This strain carries a mutation (bar1-1) tightly linked to HIS6 (on chromosome IX) that allows matα2 mutants to produce α-factor. (2) It does not allow matα1 mutants to produce α-factor. (3) Haploids of the a mating type bearing the bar1-1 mutation still mate, but are unable to act as a barrier to the diffusion of α-factor. MATa bar1-1 cells display increased sensitivity to α-factor. (4) A mutation (sst1−2) that causes increased sensitivity to α-factor is allelic to bar1-1 and also allows α-factor synthesis by matα2 mutants. The ability of matα2 bar1 double mutants to produce extracellular α-factor indicates that matα2 mutants do produce α-factor but that it is degraded by the Barrier function. These results suggest that BAR1 is normally expressed only in a cells, and is negatively regulated in α cells by the MATα2 product.

Pheromone response elements are necessary and sufficient for basal and pheromone-induced transcription of the FUS1 gene of Saccharomyces cerevisiae.

The FUS1 gene of Saccharomyces cerevisiae is transcribed in a and alpha cells, not in a/alpha diploids, and its transcription increases dramatically when haploid cells are exposed to the appropriate mating pheromone. In addition, FUS1 transcription is absolutely dependent on STE4, STE5, STE7, STE11, and STE12, genes thought to encode components of the pheromone response pathway. We now have determined that the pheromone response element (PRE), which occurs in four copies within the FUS1 upstream region, functions as the FUS1 upstream activation sequence (UAS) and is responsible for all known aspects of FUS1 regulation. In particular, deletion of 55 bp that includes the PREs abolished all transcription, and a 139-bp fragment that includes the PREs conferred FUS1-like expression to a CYC1-lacZ reporter gene. Moreover, three or four copies of a synthetic PRE closely mimicked the activity conferred by the 139-bp fragment, and even a single copy of PRE conferred a trace of activity that was haploid specific and pheromone inducible. In the FUS1 promoter context, four copies of the synthetic PRE inserted at the site of the 55-bp deletion restored full FUS1 transcription. Sequences upstream and downstream from the PRE cluster were important for maximal PRE-directed expression but, by themselves, did not have UAS activity. Other yeast genes with PREs, e.g., STE2 and BAR1, are more modestly inducible and have additional UAS elements contributing to the overall activity. In the FUS1 promoter, the PREs apparently act alone to confer activity that is highly stimulated by pheromone.

MATα1 protein, a yeast transcription activator, binds synergistically with a second protein to a set of cell-type-specific genes

We show by electrophoresis mobility shift and by DNAase I footprinting assays that the alpha 1 product of the yeast alpha mating-type locus binds to homologous sequences within the control regions of the three known alpha-specific genes. Binding requires both alpha 1 and a second yeast protein(s) (called PRTF) that is present in all three cell types (a, alpha, and a/alpha); neither protein binds alone. Binding and competition experiments using synthetic oligonucleotides indicate that PRTF binds to only part of the homology found at alpha-specific genes and imply that alpha 1 binds to the remainder. Our results suggest that alpha 1 renders gene expression alpha-specific by creating a binding site for PRTF. Similar experiments lead to the idea that PRTF also plays a role in transcription of a-specific genes. Perhaps a-specificity is achieved through the occlusion of the PRTF binding site by alpha 2, the negative regulator encoded by the alpha mating-type locus.

Yeast peptide pheromones, a-factor and α-factor, activate a common response mechanism in their target cells

We show that in yeast the cell type specificity of pheromone response is determined solely by the species of receptor that a cell synthesizes. The two receptor-pheromone interactions are functionally interchangeable and involve the creation of a common intracellular signal. In particular, we find that provision of a-factor receptor or alpha-factor receptor in mat alpha 1 mutants, which normally do not express either receptor or any other a- or alpha-specific products, allows response to the appropriate pheromone. Moreover, provision of a-factor receptor in a cells lacking alpha-factor receptor restores mating competence to those cells. Finally, an aspect of pheromone response that is normally unique to a-factor action on alpha cells--increased transcription from the alpha-specific STE3 gene--can also be observed following alpha-factor treatment of pseudo-a cells (mat alpha 2 ste3 ste13), special mutants that respond to alpha-factor and also have an active STE3 promoter.

Attachment of the Ubiquitin-Related Protein Urm1p to the Antioxidant Protein Ahp1p

ABSTRACT Urm1p is a ubiquitin-related protein that serves as a posttranslational modification of other proteins. Urm1p conjugation has been implicated in the budding process and in nutrient sensing. Here, we have identified the first in vivo target for the urmylation pathway as the antioxidant protein Ahp1p. The attachment of Urm1p to Ahp1p requires the E1 for the urmylation pathway, Uba4p. Loss of the urmylation pathway components results in sensitivity to a thiol-specific oxidant, as does loss of Ahp1p, implying that urmylation has a role in an oxidative-stress response. Moreover, treatment of cells with thiol-specific oxidants affects the abundance of Ahp1p-Urm1p conjugates. These results suggest that the conjugation of Urm1p to Ahp1p could regulate the function of Ahp1p in antioxidant stress response in Saccharomyces cerevisiae.

Far3 and Five Interacting Proteins Prevent Premature Recovery from Pheromone Arrest in the Budding Yeast Saccharomyces cerevisiae

ABSTRACT In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest. Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR1 has been extensively characterized. In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins. We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted. Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other. Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex. The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant. Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G1 arrest. These mutants initially arrest in response to pheromone but resume budding after 10 h. Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h. We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.

Yeast pheromone response pathway: characterization of a suppressor that restores mating to receptorless mutants.

Saccharomyces cerevisiae haploid cells, alpha and a, mate after being appropriately stimulated by the pheromone secreted by the opposite cell type (a-factor and alpha-factor, respectively). The binding of a pheromone to its receptor is a signal that initiates a series of intracellular changes that lead to the specific physiological alterations required for mating. To identify components of the signal transduction pathway, we sought pseudorevertants that restored mating competence to receptor mutants (MAT alpha ste3::LEU2). The suppressor srm1-1 was isolated as a recessive mutation that conferred temperature-sensitive growth to all strains and mating ability to MAT alpha ste3::LEU2 strains at the nonpermissive temperature. In addition, when srm1-1 mutants were shifted to the nonpermissive temperature, they exhibited two phenotypes characteristic of pheromone response, induction of FUS1 transcription and accumulation of cells in the G1 phase of the cell cycle. The srm1-1 mutation also suppressed a deletion of the alpha-factor-receptor gene in a cells. Together, these phenotypes suggest that the wild-type SRM1 product is a component of the pheromone response pathway. Deletion of STE4 or STE5, which are required in both haploid cell types for mating and response to pheromone, was not suppressed by srm1-1, suggesting that the SRM1 product may function before the STE4 and STE5 products. SRM1 is an essential gene and is expressed in both haploid cell types as well as in the product of their mating, a/alpha diploids. Homozygous srm1-1 a/alpha diploids were temperature sensitive although they did not arrest in G1. Thus, the SRM1 product may also have a role in the vegetative life cycle of cells.

Yeast MEK-dependent signal transduction: response thresholds and parameters affecting fidelity.

Ste7p and Mkk1p are MEK (MAPK/ERK kinase) family members that function in the mating and cell integrity signal transduction pathways in Saccharomyces cerevisiae. We selected STE7 and MKK1 mutations that stimulated their respective pathways in the absence of an inductive signal. Strikingly, serine-to-proline substitutions at analogous positions in Ste7p (position 368) and Mkk1p (position 386) were recovered by independent genetic screens. Such an outcome suggests that this substitution in other MEKs would exhibit similar properties. The Ste7p-P368 variant has higher basal enzymatic activity than Ste7p but still requires induction to reach full activation. The higher activity associated with Ste7p-P368 allows it to compensate for defects in the cell integrity pathway, but it does so only when it is overproduced or when Ste5p is missing. This behavior suggests that Ste5p, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7p specificity.