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

alpha-Factor receptors from Saccharomyces cervisiae are G-protein-coupled r eceptors containing seven transmembrane segments. Receptors solubilized wit h the detergent n-dodecyl beta-D-maltoside were found to sediment as a sing le 85 species in glycerol density gradients. When the membranes from cells coexpressing two differentially tagged receptors were solubilized with dete rgent and subjected to immunoprecipitation, we found that the antibodies sp ecific for either epitope tag resulted in precipitation of both tagged spec ies. Coprecipitation was not a consequence of incomplete detergent extracti on 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 ce lls expressing the single tagged species were mixed prior to membrane prepa ration. Treatment of cultures with alpha-factor had little effect on the ex tent of oligomerization as judged by the sedimentation behavior of the rece ptor 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 recepto rs that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis sign al (Ste2-T326) became competent for ligand-mediated endocytosis when they w ere expressed in cells containing wild-type receptors. Coimmunoprecipitatio n experiments indicated that the C-terminal cytoplasmic domain and intermol ecular disulfide bonds were unnecessary for oligomer formation. We conclude that alpha-factor receptors form homo-oligomers and that these complexes a re subject to ligand-mediated endocytosis. Furthermore, we show for the fir st time that unoccupied receptors participate in these endocytosis-competen t complexes.