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Vol. 11, Issue 9, 2873-2884, September 2000
-Factor Pheromone
Receptor Are Functional Units of Endocytosis
Department of Molecular Genetics and Microbiology, University of
Massachusetts Medical School, Worcester, Massachusetts 01655
-Factor receptors from Saccharomyces cerevisiae
are G-protein-coupled receptors containing seven transmembrane
segments. Receptors solubilized with the detergent
n-dodecyl 
-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 -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 -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 -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.
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