Localization and signaling of G(beta) subunit Ste4p are controlled by a-factor receptor and the a-specific protein Asg7p.

Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein. A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed in MATa cells. Receptor inhibition blocks signaling by inhibiting the activity of the G protein beta subunit, Ste4p. To investigate how Ste4p activity is inhibited, its subcellular location was examined. In wild-type cells, alpha-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections. In cells expressing the a-factor receptor, alpha-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment. An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor. The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function. ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses. The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates. ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization. These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein beta subunit and causes its relocalization within the cell.

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.

Receptor inhibition of pheromone signaling is mediated by the Ste4p Gbeta subunit.

The pheromone response pathway of the yeast Saccharomyces cerevisiae is initiated in MATa cells by binding of alpha-factor to the alpha-factor receptor. MATa cells in which the a-factor receptor is inappropriately expressed exhibit reduced pheromone signaling, a phenomenon termed receptor inhibition. In cells undergoing receptor inhibition, activation of the signaling pathway occurs normally at early time points but decreases after prolonged exposure to pheromone. Mutations that suppress the effects of receptor inhibition were obtained in the STE4 gene, which encodes the beta-subunit of the G protein that transmits the pheromone response signal. These mutations mapped to the N terminus and second WD repeat of Ste4p in regions that are not part of its Galpha binding surface. A STE4 allele containing several of these mutations, called STE4(SD13), reversed the signaling defect seen at late times in cells undergoing receptor inhibition but had no effect on the basal activity of the pathway. Moreover, the signaling properties of STE4(SD13) were indistinguishable from those of STE4 in wild-type MATa and MATalpha cells. These results demonstrate that the effect of the STE4(SD13) allele is specific to the receptor inhibition function of STE4. STE4(SD13) suppressed the signaling defect conferred by receptor inhibition in a MATa strain containing a deletion of GPA1, the G protein alpha-subunit gene; however, STE4(SD13) had no effect in a MATalpha strain containing a GPA1 deletion. Suppression of receptor inhibition by STE4(SD13) in a MATa strain containing a GPA1 deletion was unaffected by deletion of STE2, the alpha-factor receptor gene. The results presented here are consistent with a model in which an a-specific gene product other than Ste2p detects the presence of the a-factor receptor and blocks signaling by inhibiting the function of Ste4p.

A nucleolar protein that affects mating efficiency in Saccharomyces cerevisiae by altering the morphological response to pheromone.

SSF1 and SSF2 are redundant essential yeast genes that, when overexpressed, increase the mating efficiency of cells containing a defective Ste4p Gbeta subunit. To identify the precise function of these genes in mating, different responses to pheromone were assayed in cells that either lacked or overexpressed SSF gene products. Cells containing null alleles of both SSF1 and SSF2 displayed the normal transcriptional induction response to pheromone but were unable to form mating projections. Overexpression of SSF1 conferred the ability to form mating projections on cells containing a temperature-sensitive STE4 allele, but had only a small effect on transcriptional induction. SSF1 overexpression preferentially increased the mating efficiency of a strain containing a null allele of SPA2, a gene that functions specifically in cell morphology. To investigate whether Ssf1p plays a direct physical role in mating projection formation, its subcellular location was determined. An Ssf1p-GFP fusion was found to localize to the nucleolus, implying that the role of SSF gene products in projection formation is indirect. The region of Ssf1p-GFP localization in cells undergoing projection formation was larger and more diffuse, and was often present in a specific orientation with respect to the projection. Although the function of Ssf1p appears to originate in the nucleus, it is likely that it ultimately acts on one or more of the proteins that is directly involved in the morphological response to pheromone. Because many of the proteins required for projection formation during mating are also required for bud formation during vegetative growth, regulation of the activity or amount of one or more of these proteins by Ssf1p could explain its role in both mating and dividing cells.

GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway.

The yeast RAS1 and RAS2 genes appear to be involved in control of cell growth in response to nutrients. Here we show that this growth control also involves a signal mediated by the heterotrimeric G protein alpha subunit homolog encoded by GPA2. A GPA2 null allele conferred a severe growth defect on cells containing a null allele of RAS2, although either mutation alone had little effect on growth rate. A constitutive allele of GPA2 could stimulate growth of a strain lacking both RAS genes. Constitutive GPA2 conferred heat shock sensitivity on both wild-type cells and cells lacking RAS function, but had no effect in a strain containing a null allele of SCH9, which encodes a kinase related to protein kinase A. The GPR1 gene was isolated and was found to encode a protein with the characteristics of a G protein-coupled receptor. Double Deltagpr1 Deltaras2 mutants displayed a severe growth defect that was suppressed by expression of the constitutive allele of GPA2, confirming that GPR1 acts upstream of GPA2. Gpr1p is expressed on the cell surface and requires sequences in the membrane-proximal region of its third cytoplasmic loop for function, as expected for a G protein-coupled receptor. GPR1 RNA was induced when cells were starved for nitrogen and amino acids. These results are consistent with a model in which the GPR1/GPA2 pathway activates the Sch9p kinase to generate a response that acts in parallel with that generated by the Ras/cAMP pathway, resulting in the integration of nutrient signals.

Loss of sustained Fus3p kinase activity and the G1 arrest response in cells expressing an inappropriate pheromone receptor.

The yeast pheromone response pathway is mediated by two G protein-linked receptors, each of which is expressed only in its specific cell type. The STE3DAF mutation results in inappropriate expression of the a-factor receptor in MATa cells. Expression of this receptor in the inappropriate cell type confers resistance to pheromone-induced G1 arrest, a phenomenon that we have termed receptor inhibition. The ability of STE3DAF cells to cycle in the presence of pheromone was found to correlate with reduced phosphorylation of the cyclin-dependent kinase inhibitor Far1p. Measurement of Fus3p mitogen-activated protein (MAP) kinase activity in wild-type and STE3DAF cells showed that induction of Fus3p activity was the same in both strains at times of up to 1 h after pheromone treatment. However, after 2 or more hours, Fus3p activity declined in STE3DAF cells but remained high in wild-type cells. The level of inducible FUS1 RNA paralleled the changes seen in Fus3p activity. Short-term activation of the Fus3p MAP kinase is therefore sufficient for the early transcriptional induction response to pheromone, but sustained activation is required for cell cycle arrest. Escape from the cell cycle arrest response was not seen in wild-type cells treated with low doses of pheromone, indicating that receptor inhibition is not simply a result of weak signaling but rather acts selectively at late times during the response. STE3DAF was found to inhibit the pheromone response pathway at a step between the G beta subunit and Ste5p, the scaffolding protein that binds the components of the MAP kinase phosphorylation cascade. Overexpression of Ste20p, a kinase thought to act between the G protein and the MAP kinase cascade, suppressed the STE3DAF phenotype. These findings are consistent with a model in which receptor inhibition acts by blocking the signaling pathway downstream of G protein dissociation and upstream of MAP kinase cascade activation, at a step that could directly involve Ste20p.

An essential gene pair in Saccharomyces cerevisiae with a potential role in mating.

In the yeast Saccharomyces cerevisiae, the signal generated by extracellular pheromone is transmitted through the beta and gamma subunits of a trimeric G-protein to downstream signaling molecules that mediate the cellular responses associated with mating. To isolate potential downstream signaling components, a yeast genomic library on a multicopy plasmid was screened for genes that increased the mating efficiency of a strain containing a temperature-sensitive G beta subunit mutation. Overexpression of STE5, STE18 (which encodes the G gamma subunit), and a previously unidentified gene, termed SSF1, partially suppressed the mating defect of a G beta mutant. Hybridization of yeast genomic DNA with an SSF1 probe revealed a closely related homolog, termed SSF2, which was isolated and also found to test positively in the assay for suppression. Null mutations in either SSF1 or SSF2 had no obvious phenotype, but disruption of both genes was lethal. Depletion of SSF gene products from growing cultures caused both an arrest of cell division and a significant decrease in the ability of cells to mate. Because mating efficiency was increased by extra copies of the SSF genes and decreased by elimination of the gene products, it is likely that these genes play a role in mating as well as in an essential function.

The pheromone receptors inhibit the pheromone response pathway in Saccharomyces cerevisiae by a process that is independent of their associated G alpha protein.

Dominant mutations at the DAF2 locus confer resistance to the cell-cycle arrest that normally occurs in MATa cells exposed to alpha-factor. One of these alleles, DAF2-2, has also been shown to suppress the constitutive signaling phenotype of null alleles of the gene encoding the alpha subunit of the G protein involved in pheromone signaling. These observations indicate that DAF2-2 inhibits transmission of the pheromone response signal. The DAF2-2 mutation has two effects on the expression of a pheromone inducible gene, FUS1. In DAF2-2 cells, FUS1 RNA is present at an increased basal level but is no longer fully inducible by pheromone. Cloning of DAF2-2 revealed that it is an allele of STE3, the gene encoding the a-factor receptor. STE3 is normally an alpha-specific gene, but is inappropriately expressed in a cells carrying a STE3DAF2-2 allele. The two effects of STE3DAF2-2 alleles on the pheromone response pathway are the result of different functions of the receptor. The increased basal level of FUS1 RNA is probably due to stimulation of the pathway by an autocrine mechanism, because it required at least one of the genes encoding a-factor. Suppression of a null allele of the G alpha subunit gene, the phenotype associated with the inhibitory function of STE3, was independent of a-factor. This suppression was also observed when the wild-type STE3 gene was expressed in a cells under the control of an inducible promoter. Inappropriate expression of STE2 in alpha cells was able to suppress a point mutation, but not a null allele, of the G alpha subunit gene. The ability of the pheromone receptors to block the pheromone response signal in the absence of the G alpha subunit indicates that these receptors interact with another component of the signal transduction pathway.

The INO1 promoter of Saccharomyces cerevisiae includes an upstream repressor sequence (URS1) common to a diverse set of yeast genes.

The INO1 promoter of Saccharomyces cerevisiae includes a copy of an upstream repression sequence (URS1; 5'AGCCGCCGA 3') observed in the promoters of several unrelated yeast genes. Expression of INO1-lacZ and CYC1-lacI'Z, activated by the INO1 UASINO, is significantly decreased by the INO1 URS1.

Analysis of sequences in the INO1 promoter that are involved in its regulation by phospholipid precursors.

The promoter region of the highly regulated INO1 structural gene of yeast has been investigated. The major transcription initiation start site (+1) was mapped to a position located five nucleotides upstream of the previously identified initiation codon. The INO1 TATA is located at -116 to -111. The INO1 promoter region was used to construct fusions to the Escherichia coli lacZ gene. All INO1 fusion constructs that retained regulation in response to the phospholipid precursors inositol and choline, contained at least one copy of a nine bp repeated element (consensus, 5'-ATGTG-AAAT-3'). The smallest fragment of the INO1 promoter found to activate and regulate transcription of the fusion gene from a heterologous TATA element was 40 nucleotides in length. This fragment contained one copy of the nine bp repeat and spanned the INO1 promoter region from -259 to -219. However, when an oligonucleotide containing the nine bp repeated sequence was inserted 5' to the CYC1 TATA element, it failed to activate transcription.

Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway.

Several domains of guanine nucleotide-binding proteins are conserved and form the guanine nucleotide-binding pocket. Mutations in these domains in EF-Tu, ras, and Gas have been shown to result in informative phenotypes. We made several analogous changes in SCG1, which encodes the alpha subunit of the G protein involved in pheromone response in yeast. The scg1Lys388 and scg1Ala391 mutations resulted in severe growth and cell morphology defects; this phenotype is similar to the null phenotype and results from constitutive activation of the pheromone response pathway. On the basis of the model for the action of the yeast G protein, the effect of these mutations is consistent with the effect of analogous mutations in ras, which result in a transforming phenotype. The SCG1Ala322 mutation resulted in pheromone response and mating defects. This effect is similar to the effect of the analogous G alpha s mutation, which results in a defect in stimulation of adenylate cyclase. The scg1Val50 mutation, which is analogous to the transforming mutation rasVal12, resulted in multiple effects, including defects in growth, cell morphology, and mating. Some of our results and interpretations are different from previously published results of others for the same mutation in SCG1; specifically, our gene replacement of this mutation resulted in high basal activation of the pheromone response pathway, consistent with a GTPase defect, which was not seen previously with scg1Val50 on a low-copy plasmid. Implications of these phenotypes are discussed.

The carboxyl terminus of Scg1, the G alpha subunit involved in yeast mating, is implicated in interactions with the pheromone receptors.

The carboxyl termini of alpha subunits of mammalian G proteins have been implicated in receptor interactions. We have used a genetic analysis to test such a role for the carboxyl terminus of Scg1, the alpha subunit involved in the yeast pheromone response pathway. A 22-amino-acid truncation (scg1Amb451) resulted in defects in growth and cellular morphology. This phenotype is similar to the null phenotype and represents constitutive activation of the pheromone response pathway; it could result from various effects, including protein instability or constitutive guanine nucleotide exchange, as reported for some altered mammalian G alpha s constructs. A 5-amino-acid truncation (SCG1Och468) resulted in pheromone response and mating defects in both a and alpha cells, which is consistent with defects in interactions with the pheromone receptors. Lysine-to-proline mutations near the carboxyl terminus (SCG1Pro467 and SCG1Pro468) resulted in pheromone response and mating defects, the severity of which differed in a and alpha cells. This differential effect in the two mating types suggests that the specificity for the interactions with the two pheromone receptors may involve different residues of the Scg1 carboxyl terminus. Mutations leading to constitutive activation of the pathway were recessive, whereas mutations that result in decreased pheromone response and mating were partially dominant. These relationships are consistent with the model for the mechanism of action of the G protein subunits in the pheromone response pathway and indicate the importance of the stoichiometry of components of this system.

The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper.

In Saccharomyces cerevisiae, recessive mutations at the OPI1 locus result in constitutively derepressed expression of inositol 1-phosphate synthase, the product of the INO1 gene. Many of the other enzymes involved in phospholipid biosynthesis are also expressed at high derepressed levels in opi1 mutants. Thus, the OPI1 gene is believed to encode a negative regulator that is required to repress a whole subset of structural genes encoding for phospholipid biosynthetic enzymes. In this study, the OPI1 gene was mapped to chromosome VIII and cloned. When transformed into an opi1 mutant, the cloned DNA was capable of complementing the mutant phenotype and restoring correct regulation to the INO1 structural gene. Construction of two opi1 disruption alleles and subsequent genetic analysis of strains bearing these alleles confirmed that the cloned DNA was homologous to the genomic OPI1 locus. Furthermore, the OPI1 gene was found to be nonessential to the organism since mutants bearing the null allele were viable and exhibited a phenotype similar to that of previously isolated opi1 mutants. Similar to other opi1 mutants, the opi1 disruption mutants accumulated INO1 mRNA constitutively to a level 2-3-fold higher than that observed in wild-type cells. The cloned OPI1 gene was sequenced, and translation of the open reading frame predicted a protein composed of 404 amino acid residues with a molecular weight of 40,036. The predicted Opi1 protein contained a well defined heptad repeat of leucine residues that has been observed in other regulatory proteins. In addition, the predicted protein contained polyglutamine residue stretches which have also been reported in yeast genes having regulatory functions. Sequencing of opi1 mutant alleles, isolated after chemical mutagenesis, revealed that several were the result of a chain termination mutation located within the largest polyglutamine residue stretch.

RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals.

The large subunit of RNA polymerase II contains a highly conserved and essential heptapeptide repeat (Pro-Thr-Ser-Pro-Ser-Tyr-Ser) at its carboxy terminus. Saccharomyces cerevisiae cells are inviable if their RNA polymerase II large subunit genes encode fewer than 10 complete heptapeptide repeats; if they encode 10 to 12 complete repeats cells are temperature-sensitive and cold-sensitive, but 13 or more complete repeats will allow wild-type growth at all temperatures. Cells containing C-terminal domains (CTDs) of 10 to 12 complete repeats are also inositol auxotrophs. The phenotypes associated with these CTD mutations are not a consequence of an instability of the large subunit; rather, they seem to reflect a functional deficiency of the mutant enzyme. We show here that partial deletion mutations in RNA polymerase II CTD affect the ability of the enzyme to respond to signals from upstream activating sequences in a subset of promoters in yeast. The number of heptapeptide repeats required for maximal response to signals from these sequences differs from one upstream activating sequence to another. One of the upstream elements that is sensitive to truncations of the CTD is the 17-base-pair site bound by the GAL4 transactivating factor.

[Results of the surgical treatment of common bile duct calculi in aged patients]. / Résultats du traitement chirurgical de la lithiase de la voie biliaire principale chez le sujet âgé.

One hundred and two patients (median age: 76 years old; range: 70-91) underwent choledocotomy for biliary lithiasis. Seventy patients had at least one risk factor, 31 had at least 2 factors and 12 had 3 or 4 factors. The indication for surgical treatment was based on evolutive complications in 64 cases; an elective operation was performed in 38 cases. Common bile duct lithiasis was present in only 87 cases but associated lesions made a choledocotomy necessary in every case. A choledocoduodenostomy was performed in 40 cases and a choledocojejunostomy in 3 cases. No postoperative complications occurred in 86 patients. Of 16 patients with complications, 2 were fatal being due to hemiplegia and respiratory failure respectively. Two patients had residual lithiasis. Ninety-four patients were followed on a long-term basis (median follow-up time: 6 years): 34 died from underlying diseases, while 60 are still alive. Treatment failures were, in one case, recurrent lithiasis treated by choledocoduodenostomy and, in the other case, biliary anastomosis stenosis. These results show that the surgical treatment of choledocolithiasis does not necessarily increase the mortality rate in the elderly.

Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis.

The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate synthase, which catalyzes the first committed step in the synthesis of inositol-containing phospholipids. The expression of this gene was analyzed under conditions known to regulate phospholipid synthesis. RNA blot hybridization with a genomic clone for INO1 detected two RNA species of 1.8 and 0.6 kb. The abundance of the 1.8-kb RNA was greatly decreased when the cells were grown in the presence of the phospholipid precursor inositol, as was the enzyme activity of the synthase. Complementation analysis showed that this transcript encoded the INO1 gene product. The level of INO1 RNA was repressed 12-fold when the cells were grown in medium containing inositol, and it was repressed 33-fold when the cells were grown in the presence of inositol and choline together. The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy. The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of inositol-1-phosphate synthase and results in excretion of inositol. The expression of INO1 RNA was also examined in cells containing a mutation (cho2) affecting the synthesis of phosphatidylcholine. In contrast to what was observed in wild-type cells, growth of cho2 cells in medium containing inositol did not result in a significant decrease in INO1 RNA abundance. Inositol and choline together were required for repression of the INO1 transcript in these cells, providing evidence for a regulatory link between the synthesis of inositol- and choline-containing lipids. The level of the 0.6-kb RNA was affected, although to a lesser degree, by many of the same factors that influence INO1 expression.