GSBS Student Publications

Title

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

UMMS Affiliation

Graduate School of Biomedical Sciences; Department of Molecular Genetics and Microbiology

Date

9-12-2000

Document Type

Article

Medical Subject Headings

Cell Fractionation; Cell Membrane; Endocytosis; Genotype; Peptides; Pheromones; Protein Subunits; Receptors, Mating Factor; Receptors, Peptide; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Transcription Factors

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

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. Coprecipitation was not a consequence of incomplete detergent extraction because the abundant plasma membrane protein Pma1 did not coprecipitate with the receptors. Moreover, the receptor complexes were present prior to detergent extraction because coimmunoprecipitation was not observed when cells expressing the single tagged species were mixed prior to membrane preparation. Treatment of cultures with alpha-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. We conclude that alpha-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. Furthermore, we show for the first time that unoccupied receptors participate in these endocytosis-competent complexes.

Rights and Permissions

Citation: Mol Biol Cell. 2000 Sep;11(9):2873-84.

Related Resources

Link to Article in PubMed

Journal Title

Molecular biology of the cell

PubMed ID

10982387