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G F Sprague

Publications and source records attributed to G F Sprague.

At least 19 recordsLinked to original sources
PubMed2003

Autocrine activation of the pheromone response pathway in matalpha2- cells is attenuated by SST2- and ASG7-dependent mechanisms.

Yeast mat alpha2 mutants express both mating pheromones and both mating pheromone receptors. They show modest signaling in the pheromone response pathway, as revealed by increased levels of FUS1 transcript, yet are resistant to pheromone treatment. Together, these phenotypes suggest that alpha2- cells undergo autocrine activation of the pheromone response pathway, which is subsequently attenuated. We constructed a regulatable version of the alpha2 gene (GALalpha2) and showed that, upon loss of alpha2 activity, cells exhibit an initial robust response to pheromone that is attenuated within 3 h. We reasoned that the viability of alpha2- cells might be due to attenuation, and therefore performed a genome-wide synthetic lethal screen to identify potential adaptation components. We identified two genes, SST2 and ASG7. Loss of either of these attenuation components results in activation of the pheromone pathway in alpha2- cells. Loss of both proteins causes a more severe phenotype. Sst2 functions as a GTPase activating protein (GAP) for the Galpha subunit of the trimeric G protein. Asg7 is an a -cell specific protein that acts in concert with the alpha-cell specific a -factor receptor, Ste3, to inhibit signaling by Gbetagamma. Hence, our results suggest that mat alpha2 mutants mimic the intracellular signaling events that occur in newly fused zygotes.

Base Sequence↗
PubMed2001

The phosphotyrosyl phosphatase activator, Ncs1p (Rrd1p), functions with Cla4p to regulate the G(2)/M transition in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae p21-activated kinases, Ste20p and Cla4p, have individual functions but appear to share an essential function(s) as well because a strain lacking both kinases is inviable. To learn more about the shared function, we sought new mutations that were lethal in the absence of CLA4. This approach led to the identification of at least 10 complementation groups designated NCS (need CLA4 to survive). As for ste20 cla4-75 mutants, most ncs cla4-75 double mutants were defective for septin localization during budding. One group, NCS1/RRD1 (YIL153w), did not confer this defect, however, and we investigated its function further. ncs1Delta cla4Delta cells arrested with elongated buds and short mitotic spindles. The morphological defects and lethality were suppressed by mutations that abrogate the cell cycle morphogenetic checkpoint, CDC28Y19F or swe1Delta. The connection to the cell cycle may be direct, as we detected a Cla4p-Cdc28p complex. NCS1 encodes a protein with significant similarity to a mammalian phosphotyrosyl phosphatase activator (PTPA) regulatory subunit for type 2A protein phosphatases (PP2As). Genetic and biochemical evidence suggested that the phosphatase Sit4p is a target for Ncs1p. First, CLA4 and SIT4 were synthetically lethal. Second, Ncs1p and its yeast paralog, Noh1p (Rrd2p), bound to the catalytic domain of Sit4p in vitro, and Ncs1p could be immunoprecipitated with Sit4p but not with another PP2A (Pph21p) from yeast cell extracts. Strains lacking both NCS1 and NOH1 were inviable and arrested as unbudded cells, implying that PTPA function is required for proper G(1) progression.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2000

Glucose depletion causes haploid invasive growth in yeast.

Haploid yeast invades solid agar in response to nutrient limitation. To decipher the cues that underlie invasion, we have developed a single cell invasive growth assay. Using this assay, as well as the traditional plate-washing assay, we show that invasive growth occurs in response to glucose depletion. In the absence of glucose (or other fermentable sugar), individual cells adopted a nonaxial budding pattern and elongated morphology within the first cell divisions, and invasion into the agar was observed in microcolonies containing as few as 10 cells. In support of this observation, we found that glucose suppressed the hyperinvasive growth morphology of STE11-4, pbs2, hsl7, and RAS2V19 mutations. In addition, removal of glucose from YPD medium caused constitutive invasion in wild-type cells. We tested glucose control proteins for a role in invasion and found that Snf1, a protein required for derepression of glucose-repressed genes, was required for invasive growth. The transcription factor Sip4, which interacts with Snf1 and is induced during the diauxic shift, had an inhibitory role on invasive growth, suggesting that multiple mechanisms are required for glucose depletion-dependent invasion.

Glucose↗
PubMed2000

Defects in protein glycosylation cause SHO1-dependent activation of a STE12 signaling pathway in yeast.

In haploid Saccharomyces cerevisiae, mating occurs by activation of the pheromone response pathway. A genetic selection for mutants that activate this pathway uncovered a class of mutants defective in cell wall integrity. Partial loss-of-function alleles of PGI1, PMI40, PSA1, DPM1, ALG1, MNN10, SPT14, and OCH1, genes required for mannose utilization and protein glycosylation, activated a pheromone-response-pathway-dependent reporter (FUS1) in cells lacking a basal signal (ste4). Pathway activation was suppressed by the addition of mannose to hexose isomerase mutants pgi1-101 and pmi40-101, which bypassed the requirement for mannose biosynthesis in these mutants. Pathway activation was also suppressed in dpm1-101 mutants by plasmids that contained RER2 or PSA1, which produce the substrates for Dpm1. Activation of FUS1 transcription in the mannose utilization/protein glycosylation mutants required some but not all proteins from three different signaling pathways: the pheromone response, invasive growth, and HOG pathways. We specifically suggest that a Sho1 --> Ste20/Ste50 --> Ste11 --> Ste7 --> Kss1 --> Ste12 pathway is responsible for activation of FUS1 transcription in these mutants. Because loss of pheromone response pathway components leads to a synthetic growth defect in mannose utilization/protein glycosylation mutants, we suggest that the Sho1 --> Ste12 pathway contributes to maintenance of cell wall integrity in vegetative cells.

Cell Wall↗
PubMed1997

The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.

The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins).

Acyltransferases↗
PubMed1997

Human CPR (cell cycle progression restoration) genes impart a Far- phenotype on yeast cells.

Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexpressed were capable of specifically overcoming G1 arrest signals from the cell cycle branch of the mating pheromone pathway, while still maintaining the integrity of the transcriptional induction branch. We have identified 13 human CPR (cell cycle progression restoration) genes and 11 yeast OPY (overproduction-induced pheromone-resistant yeast) genes that specifically block the G1 arrest by mating pheromone. The CPR genes represent a variety of biochemical functions including a new cyclin, a tumor suppressor binding protein, chaperones, transcription factors, translation factors, RNA-binding proteins, as well as novel proteins. Several CPR genes require individual CLNs to promote pheromone resistance and those that require CLN3 increase the basal levels of Cln3 protein. Moreover, several of the yeast OPY genes have overlapping functions with the human CPR genes, indicating a possible conservation of roles.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of FAR3, a gene required for pheromone-mediated G1 arrest in Saccharomyces cerevisiae.

In haploid Saccharomyces cerevisiae cells, mating pheromones activate a signal transduction pathway that leads to cell cycle arrest in the G1 phase and to transcription induction of genes that promote conjugation. To identify genes that link the signal transduction pathway and the cell cycle machinery, we developed a selection strategy to isolate yeast mutants specifically defective for G1 arrest. Several of these mutants identified previously known genes, including CLN3, FUS3, and FAR1. In addition, a new gene, FAR3, was identified and characterized. FAR3 encodes a novel protein of 204 amino acid residues that is dispensable for viability. Northern blot experiments indicated that FAR3 expression is constitutive with respect to cell type, pheromone treatment, and cell cycle position. As a first step toward elucidating the mechanism by which Far3 promotes pheromone-mediated G1 arrest, we performed genetic and molecular experiments to test the possibility that Far3 participates in one of the heretofore characterized mechanisms, namely Fus3/Far1-mediated inhibition of Cdc28-Cln kinase activity, G1 cyclin gene repression, and G1 cyclin protein turnover. Our data indicate that Far3 effects G1 arrest by a mechanism distinct from those previously known.

Alleles↗
PubMed1995

Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity-establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae.

We have selected yeast mutants that exhibit a constitutively active pheromone-response pathway in the absence of the beta subunit of the trimeric G protein. Genetic analysis of one such mutant revealed that it contained recessive mutations in two distinct genes, both of which contributed to the constitutive phenotype. One mutation identifies the RGA1 locus (Rho GTPase activating protein), which encodes a protein with homology to GAP domains and to LIM domains. Deletion of RGA1 is sufficient to activate the pathway in strains lacking the G beta subunit. Moreover, in wild-type strains, deletion of RGA1 increases signaling in the pheromone pathway, whereas over-expression of RGA1 dampens signaling, demonstrating that Rga1p functions as a negative regulator of the pheromone response pathway. The second mutation present in the original mutant proved to be an allele of a known gene, PBS2, which encodes a putative protein kinase that functions in the high osmolarity stress pathway. The pbs2 mutation enhanced the rga1 mutant phenotype, but by itself did not activate the pheromone pathway. Genetic and two-hybrid analyses indicate that an important target of Rga1p is Cdc42p, a p21 GTPase required for polarity establishment and bud emergence. This finding coupled with recent experiments with mammalian and yeast cells indicating that Cdc42p can interact with and activate Ste20p, a protein kinase that operates in the pheromone pathway, leads us to suggest that Rga1p controls the activity of Cdc42p, which in turn controls the magnitude of signaling in the pheromone pathway via Ste20p.

Amino Acid Sequence↗
PubMed1995

Cloning and characterization of the Saccharomyces cerevisiae LYS7 gene: evidence for function outside of lysine biosynthesis.

The Saccharomyces cerevisiae LYS7 gene has been cloned based on its genetic map position and complementation of a lys7 mutant. The 1453-bp sequence contains an open reading frame (ORF) that predicts a unique 249 amino acid (aa) protein. A Northern blot experiment demonstrated that LYS7 transcription was not regulated by lysine-specific or general aa control mechanisms. To investigate the effects of total loss of LYS7 function, we created a complete deletion of the gene and introduced this allele into wild-type yeast. The lys7 delta mutant requires lysine and simultaneously displays an array of phenotypes that include pH and temperature sensitivity. The pleiotropic phenotypes of the lys7 delta mutant and the constitutive transcription pattern are at odds with the hypothesis that Lys7p functions solely in the lysine biosynthesis pathway.

Amino Acid Sequence↗
PubMed1995

Signal transduction and growth control in yeast.

An understanding of how an extracellular stimulus causes changes in cell growth is emerging from the study of four signal transduction pathways in Saccharomyces cerevisiae: the pheromone-response, pseudohyphal differentiation, osmolarity-response, and protein kinase C activated pathways. Each of these pathways contains at its core a distinct mitogen-activated protein kinase cascade. Biochemical and molecular studies have determined the functional order of the kinases in the pheromone-response pathway and have suggested that they are organized into a complex by a protein scaffold. The cell surface sensor system for the osmolarity-response pathway has been identified. It shows striking similarity to bacterial two-component sensor-responder systems. Finally, components that integrate information from these pathways and communicate it to cell growth regulators have been revealed.

Glycerol↗
PubMed1995

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.

Alleles↗
PubMed1994

Genetic mapping of STE20 to the left arm of chromosome VIII.

STE20 is a newly-discovered element of the Saccharomyces cerevisiae pheromone response pathway. We have isolated a recessive ste20 mutation and have used it to map the gene to the left arm of chromosome VIII, establishing the gene order STE20-CEN8-GPA1-ARG4.

Chromosome Mapping↗
PubMed1994

Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade.

We have used the two-hybrid system of Fields and Song to identify protein-protein interactions that occur in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Pathway components Ste4p, Ste5p, Ste7p, Ste11p, Ste12p, Ste20p, Fus3p and Kss1p were tested in all pairwise combinations. All of the interactions we detected involved at least one member of the MAP kinase cascade that is a central element of the response pathway. Ste5p, a protein of unknown biochemical function, interacted with protein kinases that operate at each step of the MAP kinase cascade, specifically with Ste11p (an MEKK), Ste7p (an MEK), and Fus3p (a MAP kinase). This finding suggests that one role of Ste5p is to serve as a scaffold to facilitate interactions among members of the kinase cascade. In this role as facilitator, Ste5p may make both signal propagation and signal attenuation more efficient. Ste5p may also help minimize cross-talk with other MAP kinase cascades and thus ensure the integrity of the pheromone response pathway. We also found that both Ste11p and Ste7p interact with Fus3p and Kss1p. Finally, we detected an interaction between one of the MAP kinases, Kss1p, and a presumptive target, the transcription factor Ste12p. We failed to detect interactions of Ste4p or Ste20p with any other component of the response pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1994

MCM1 point mutants deficient in expression of alpha-specific genes: residues important for interaction with alpha 1.

Complexes formed between MCM1 and several coregulatory proteins--alpha 1, alpha 2, and STE12--serve to govern transcription of the a- and alpha-specific gene sets in the yeast Saccharomyces cerevisiae. The N-terminal third of MCM1, MCM1(1-98), which includes a segment homologous to mammalian serum response factor, is capable of performing all of the functions necessary for cell-type-specific gene regulation, including DNA binding and interaction with coregulatory proteins. To explore the mechanisms by which MCM1(1-98) functions, we isolated point mutants that are specifically deficient in alpha-specific gene expression in vivo, anticipating that many of the mutants would be impaired for interaction with alpha 1. Indeed, in vitro DNA binding assays revealed that a substantial number of the mutants were specifically defective in the ability to bind cooperatively with alpha 1. Two other mutant classes were also found. One class, exemplified most clearly by substitutions at residues 22 and 27, exhibited a general defect in DNA binding. The second class, exemplified by substitutions at residues 33 and 41, was proficient at DNA binding and interaction with alpha 1 in vitro, suggesting that these mutants may be defective in achieving an alpha 1-mediated conformational change required for transcription activation in vivo. Most of the mutants defective for interaction with alpha 1 had substitutions within residues 69 to 81, which correspond to a region of serum response factor important for interaction with its coregulatory proteins. A subset of the mutants with changes in this region were also defective in the ability to bind with STE12 to DNA from an a-specific gene, suggesting that a common region of MCM1(1-98) mediates interaction with both alpha 1 and STE12. This region of MCM1 does not seem to constitute an independent domain of the protein, however, because some substitutions within this region affected DNA binding. Only two of the MCM1(1-98) point mutants showed significant defects in the ability to form complexes with alpha 2, suggesting that the mechanism by which MCM1 interacts with alpha 2 is distinct from that by which it interacts with alpha 1 and STE12.

Amino Acid Sequence↗
PubMed1994

The yeast pheromone response pathway: new insights into signal transmission.

The yeast (Saccharomyces cerevisiae) pheromone response pathway is one of the best understood eukaryotic signal transduction pathways. Nonetheless, it is likely that components and regulators of the pathway remain to be identified. We have employed three approaches to learn about interactions among known pathway components and to identify new components. First, the two-hybrid system of Fields and Song revealed that STE5, a protein of unknown biochemical function, interacts with each member of the MAP kinase cascade. One interpretation of this finding is that STE5 facilitates interactions between members of the cascade and thereby makes signal transmission more efficient. Second, genetic studies have identified new gene functions that appear to be involved in pheromone response. One of these is homologous to RHO-GAP proteins, an observation that suggests that a RHO protein (members of the RAS super-family) is part of the response pathway. A second gene function, FAR3, appears to be required only for a specific facet of pheromone response, arrest of the mitotic cell division cycle in G1.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Mutations that alter the third cytoplasmic loop of the a-factor receptor lead to a constitutive and hypersensitive phenotype.

The STE3 gene of Saccharomyces cerevisiae encodes a G protein-coupled receptor that is specific for the mating pheromone a-factor. The ste3L194Q mutation, which leads to the substitution of glutamine for leucine-194 within the third cytoplasmic loop of the receptor, resulted in a 20-fold increase in pheromone sensitivity and also caused partial constitutive activation of the response pathway. Moreover, other amino acid substitutions at the 194 position and several deletion mutations that collectively remove most of the third cytoplasmic loop resulted in hyperactive receptors. Therefore, we suggest that one role of the third cytoplasmic loop is to function as a negative regulatory domain involved in the maintenance of a nonsignaling state of the receptor. The constitutive activity and the pheromone hypersensitivity of ste3L194Q cells were recessive, suggesting that the wild-type receptor can antagonize the signal associated with the activated receptor. The ste3 delta 306 mutation, which results in truncation of most of the C-terminal domain of the receptor, led to a 20-fold increase in pheromone sensitivity, indicating that this domain also mediates negative regulation of the receptor. The ste3L194Q and ste3 delta 306 mutations appear to affect receptor activity independently, because the double mutant was associated with a 400-fold increase in pheromone sensitivity.

Alleles↗