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When Saccharomyces cerevisiae a cells bind a-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 a-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the a-factor receptors were predominantly found in the plasma membrane peak before a-factor treatment and then appeared in membranes of lesser buoyant density after a-factor exposure. Second, receptors were susceptible to cleavage by extracellular proteases before a-factor treatment and then became resistant to proteolysis after exposure to pheromone, consistent with the transit of receptors from the cell surface to an internal compartment. presumably by interacting with a common G protein (6, 29), they share no obvious sequence homology. Mutational studies of the a-factor receptor (17, 61) and other receptors with a similar structure (23, 32) implicate the third cytoplasmic loop in the coupling of these receptors to their respective G proteins. Other mutational analyses indicate that the carboxyterminal cytoplasmic domain of the pheromone receptor is important for receptor down regulation, pheromone internalization, adaptation to the pheromone-induced signal, and pheromone-induced morphological changes (33,34,47,49).A large body of experimental evidence suggests that pheromones are internalized by receptor-mediated endocytosis. Radioactive at-factor becomes associated irreversibly with a cells in an energy-and receptor-dependent manner (14,30). This association is accompanied by a disappearance of ligandbinding sites from the cell surface (30). Clathrin, which plays a major role in receptor-mediated endocytosis in mammalian cells, has recently been shown to facilitate the internalization of a-factor (58). Following internalization, the pheromone appears to be translocated to the vacuole via vesicular intermediates (54), which may represent early and late endosomes (55). Although there is no direct evidence that the ao-factor receptor leaves the cell surface upon binding pheromone, a-factor receptors have been shown to pass from the plasma membrane to the vacuole, and this process is accelerated when at cells are treated with a-factor (19). There is as yet no direct evidence for the internalization of the a-factor ligand. We sought physical evidence for the ligand-induced endocytosis of the a-factor receptor. Three independent criteria were used to establish the movement of a-factor receptors from the plasma membrane to internal compartments of the cell. First, Renografin density gradient centrifugation was used to fractionate the cellular membranes, allowing us to follow the ligand-induced exit of the receptor from the plasma membrane upon at-factor exposure. Second, protease susceptibility assays 7245

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Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive ␣-factor receptor (mutation ste2-3) and arginine permease (mutation can1 ts ). These suppressors inhibited the elimination of misfolded receptors (synthesized at 34°C) as well as damaged surface receptors (shifted from 22 to 34°C). The stp22 mutation (allelic to vps23 [M. Babst and S. Emr, personal communication] and the STP26 mutation also caused missorting of carboxypeptidase Y, and ste2-3 was suppressed by mutations vps1, vps8, vps10, and vps28 but not by mutation vps3. In the stp22 mutant, both the mutant and the wild-type receptors (tagged with green fluorescent protein [GFP]) accumulated within an endosome-like compartment and were excluded from the vacuole. GFP-tagged Stp22p also accumulated in this compartment. Upon reaching the vacuole, cytoplasmic domains of both mutant and wild-type receptors appeared within the vacuolar lumen. Stp22p and Gef1p are similar to tumor susceptibility protein TSG101 and voltage-gated chloride channel, respectively. These results identify potential elements of plasma membrane quality control and indicate that cytoplasmic domains of membrane proteins are translocated into the vacuolar lumen.Plasma membrane proteins link the interior of the cell with the extracellular environment. Removal of defective membrane proteins prevents the accumulation of damage that might otherwise compromise the ability of the cell to maintain electrochemical gradients, transport nutrients, and respond to sensory information. However, degradation of integral membrane proteins presents special problems for the cell because the internal and external sides of the protein are exposed to different biochemical environments. In eucaryotic cells, membrane proteins which have not folded or assembled properly are normally eliminated by the endoplasmic reticulum (ER) quality control process (25); however, examples of post-ER quality control are known (16,31). Degradation of defective membrane proteins in the yeast vacuole has been recognized; however, the molecular details of this process are unknown. This report describes a genetic approach toward elucidating the steps that comprise the quality control of integral plasma membrane proteins.Recent work with Saccharomyces cerevisiae has shown that temperature-sensitive forms of plasma membrane ATPase (Pma1p) (5) and ␣-factor receptors (19) are delivered directly to the vacuole, where they are degraded. In a previous report (19), we described a temperature-sensitive form of the yeast ␣-factor receptor (Ste2-3p) as a model for investigating the consequences of structural defects of integral plasma membrane proteins. Operationally, we define "misfolded receptors" as mutant receptors that are synthesized at the nonpermissive temperature, whereas "damaged cell surface receptors" are receptors that are exposed to the nonpermissive temperature only after they have been expressed at the cell surface. Misfolded receptors are delivered to the va...