Interaction among Saccharomyces cerevisiae pheromone receptors during endocytosis.

This study investigates endocytosis of Saccharomyces cerevisiae α-factor receptor and the role that receptor oligomerization plays in this process. α-factor receptor contains signal sequences in the cytoplasmic C-terminal domain that are essential for ligand-mediated endocytosis. In an endocytosis complementation assay, we found that oligomeric complexes of the receptor undergo ligand-mediated endocytosis when the α-factor binding site and the endocytosis signal sequences are located in different receptors.

Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor are functional units of endocytosis.

alpha-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. Receptors solubilized with the detergent n-dodecyl beta-D-maltoside were found to sediment as a single 8S species in glycerol density gradients. When the membranes from cells coexpressing two differentially tagged receptors were solubilized with detergent and subjected to immunoprecipitation, we found that the antibodies specific for either epitope tag resulted in precipitation of both tagged species.

The C terminus of the Saccharomyces cerevisiae alpha-factor receptor contributes to the formation of preactivation complexes with its cognate G protein.

Binding of the alpha-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Galpha subunits in an alpha-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Galpha subunit.

Dual lipid modification of the yeast ggamma subunit Ste18p determines membrane localization of Gbetagamma.

The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein. The Gbetagamma subunit (a complex of Ste4p and Ste18p) is associated with both internal and plasma membranes, and a portion is not stably associated with either membrane fraction. Like Ras, Ste18p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmitoylation.

Yeast mutants affecting possible quality control of plasma membrane proteins.

Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive alpha-factor receptor (mutation ste2-3) and arginine permease (mutation can1(ts)). These suppressors inhibited the elimination of misfolded receptors (synthesized at 34 degrees C) as well as damaged surface receptors (shifted from 22 to 34 degrees C). The stp22 mutation (allelic to vps23 [M.

Elimination of defective alpha-factor pheromone receptors.

This report compares trafficking routes of a plasma membrane protein that was misfolded either during its synthesis or after it had reached the cell surface. A temperature-sensitive mutant form of the yeast alpha-factor pheromone receptor (ste2-3) was found to provide a model substrate for quality control of plasma membrane proteins. We show for the first time that a misfolded membrane protein is recognized at the cell surface and rapidly removed.

The G beta gamma complex of the yeast pheromone response pathway. Subcellular fractionation and protein-protein interactions.

Genetic evidence suggests that the yeast STE4 and STE18 genes encode G beta and G gamma subunits, respectively, that the G betagamma complex plays a positive role in the pheromone response pathway, and that its activity is subject to negative regulation by the G alpha subunit (product of the GPA1 gene) and to positive regulation by cell-surface pheromone receptors. However, as yet there is no direct biochemical evidence for a G betagamma protein complex associated with the plasma membrane. We found that the products of the STE4 and STE18 genes are stably associated with plasma membrane as well as with internal membranes and that 30% of the protein pool is not tightly associated with either membrane fraction.

Agonist-specific conformational changes in the yeast alpha-factor pheromone receptor.

The yeast alpha-factor pheromone receptor is a member of the G-protein-coupled receptor family. Limited trypsin digestion of yeast membranes was used to investigate ligand-induced conformational changes in this receptor. The agonist, alpha-factor, accelerated cleavage in the third intracellular loop, whereas the antagonist, desTrp1,Ala3-alpha-factor, reduced the cleavage rate.

Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor.

When Saccharomyces cerevisiae a cells bind alpha-factor pheromone, the ligand is internalized and its binding sites are lost from the cell surface in a time-, energy-, and temperature-dependent manner. This report presents direct evidence for alpha-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the alpha-factor receptors were predominantly found in the plasma membrane peak before alpha-factor treatment and then appeared in membranes of lesser buoyant density after alpha-factor exposure.

Mutational activation of the STE5 gene product bypasses the requirement for G protein beta and gamma subunits in the yeast pheromone response pathway.

The STE5 gene encodes an essential element of the pheromone response pathway which is known to act either after the G subunit encoded by the STE4 gene or at the same step. Mutations in STE5, designated STE5Hyp, that partially activate the pathway in the absence of pheromone were isolated. One allele (STE5Hyp-2) was shown to cause a single amino acid substitution near the N terminus of the predicted STE5 protein.

Genetic fine-structural analysis of the Saccharomyces cerevisiae alpha-pheromone receptor.

The alpha-pheromone receptor encoded by the STE2 gene contains seven potential transmembrane domains. Its ability to transduce the pheromone signal is thought to require the action of a G protein. As an initial step toward defining the structural features of the receptor required for its activity, we examined the phenotypic consequences of linker insertion mutations (12 bp) at 10 different sites in the STE2 gene.

Regulation of postreceptor signaling in the pheromone response pathway of Saccharomyces cerevisiae.

alpha-Factor pheromone inhibits division of yeast a cells. After prolonged exposure to alpha-factor, the cells adapt to the stimulus and resume cell division. The sst2 mutation is known to inhibit adaptation.

Constitutive mutants in the yeast pheromone response: ordered function of the gene products.

The alpha factor pheromone inhibits the division of yeast a cells. A general method was developed for isolating mutants that exhibit constitutive activation of the pheromone response pathway. A dominant allele of the STE4 locus was recovered in addition to recessive mutations in the SCG1 gene.

The C-terminus of the S. cerevisiae alpha-pheromone receptor mediates an adaptive response to pheromone.

STE2 encodes a component of the S. cerevisiae alpha-pheromone receptor that is essential for induction of physiological changes associated with mating. Analysis of C-terminal truncation mutants of STE2 demonstrated that the essential sequences for ligand binding and signal transduction are included within a region containing seven putative transmembrane domains.

Saccharomyces cerevisiae mutants unresponsive to alpha-factor pheromone: alpha-factor binding and extragenic suppression.

Mutations in six genes that eliminate responsiveness of Saccharomyces cerevisiae a cells to alpha-factor were examined by assaying the binding of radioactively labeled alpha-factor to determine whether their lack of responsiveness was due to the absence of alpha-factor receptors. The ste2 mutants, known to be defective in the structural gene for the receptor, were found to lack receptors when grown at the restrictive temperature; these mutations probably affect the assembly of active receptors. Mutations in STE12 known to block STE2 mRNA accumulation also resulted in an absence of receptors.

Down regulation of the alpha-factor pheromone receptor in S. cerevisiae.

The peptide pheromone, alpha-factor, was found to elicit down regulation of receptor sites on yeast a cell targets. Cellular uptake of alpha-factor accompanied the loss of receptor sites. Receptor-deficient a cells bearing a deletion of the STE2 gene were unable to internalize alpha-factor.

Binding of alpha-factor pheromone to Saccharomyces cerevisiae a cells: dissociation constant and number of binding sites.

The number of alpha-factor binding sites on yeast MATa cells (8,000) and the equilibrium dissociation constant (6 X 10(-9) M) were determined from direct binding experiments. These values correct our previously reported estimates (D. D.

Binding of alpha-factor pheromone to yeast a cells: chemical and genetic evidence for an alpha-factor receptor.

The division cycle of yeast a cells is inhibited by alpha-factor. Haploid a cells were found to bind 35S-labeled alpha-factor, whereas haploid alpha cells and diploid a/alpha cells showed little binding. The association of alpha-factor with a cells was reversible upon dilution.

Genetic characterization of the folding domains of the catalytic chains in aspartate transcarbamoylase.

In Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation, the bulk of the carbamoyl phosphate of the cell is consumed for the biosynthesis of pyrimidines. As a consequence, there is little substrate available for arginine synthesis and the cell growth is impeded. Suppression of arginine auxotrophy by mutations which block aspartate transcarbamoylase activity provides a positive selection technique for mutant strains defective in this enzyme activity.

pryB mutations as suppressors of arginine auxotrophy in Salmonella typhimurium.

Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation were found to grow more slowly in minimal medium than pyrH+ controls. The addition of arginine or citrulline but not ornithine restored normal growth rates. This requirement for arginine was completely suppressed by pyrB mutations and partially suppressed by pyrC and pyrD mutations.

Interactions between 30s ribosomal proteins and 50s subunits of Escherichia coli.

The interaction between ribosomal proteins of the 30s subunit with intact 50s subunits was investigated. Experiments with mixtures of total 30s proteins indicated that several 30s proteins including protein s4 would form a stable complex with 50s subunits. Further work with pure s4 indicates that this protein binds stoichiometrically to the 50s subunits, probably through protein-nucleic acid interaction.