A. Yesilaltay et Dd. Jenness, Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor arefunctional units of endocytosis, MOL BIOL CE, 11(9), 2000, pp. 2873-2884
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.