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

Binding of the ␣-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant G␣ subunits in an ␣-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant G␣ subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the ␣-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for ␣-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the ␣-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor-G-protein preactivation complexes.In the yeast Saccharomyces cerevisiae, the ␣-factor pheromone activates a cell-surface receptor on MATa cells, leading to cell division arrest and expression of genes necessary for conjugation (1,16,38). The ␣-factor receptor (encoded by STE2) belongs to the large family of G-protein-coupled receptors (GPCRs), which includes receptors for hormones, neurotransmitters, and sensory stimuli (11, 57). GPCRs transduce their signal by activating a heterotrimeric guanine nucleotide binding protein (G protein) that results in the exchange of GDP for GTP in the G␣ subunit (6,20). In the case of the yeast pheromone pathway, the GTP-bound G␣ subunit releases the G␤␥ subunits, and the free G␤␥ complexes then mediate the subsequent events in the response pathway (1, 16, 38). Although the yeast pheromone receptors and other GPCRs respond to different extracellular signals and share no significant sequence homology, they possess a common structural topology composed of seven transmembrane domains connected by intracellular and extracellular loops. In addition, these receptors exhibit a similar organization of functional domains. For example, as in many GPCRs, the third intracellular loop of the ␣-factor receptor functions in G-protein coupling (10, 58).Moreover, the cytoplasmic C-terminal domain of both yeast and mammalian receptors mediates ligand-induced endocytosis (46, 49) and plays a role in desen...

TraC is one of the proteins encoded by the F transfer region of the F conjugative plasmid which is required for the assembly of F pilin into the mature F pilus structure. Overproduction of this protein from the plasmid pKAS2, which carries only traC, resulted in the formation of inclusion bodies from which soluble TraC was purified. When small amounts of TraC were produced from pKAS2, the protein was localized to the cytoplasm by using anti-TraC antibodies. Similar analysis of a set of TraC-alkaline phosphatase fusion proteins localized all of these fusion proteins to the cytoplasm. However, when TraC was expressed from the F plasmid, much of it appeared associated with the bacterial membrane fraction. Under these conditions, TraC does not appear to be part of the tip of the F pilus, as neither anti-TraC antibodies nor purified TraC had any effect on the infection of F-containing bacteria by the filamentous bacteriophage f1. These data suggest that TraC is normally associated with the membrane through interactions with other proteins specified by the tra region. This interaction may be via the carboxyl-terminal region of the TraC protein, as a mutant TraC protein containing an Arg-Cys substitution at amino acid 811 exhibits an interaction with the membrane weaker than that of the wild-type protein in the presence of the other Tra proteins.

When Saccharomyces cerevisiae a cells bind alpha-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 alpha-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the alpha-factor receptors were predominantly found in the plasma membrane peak before alpha-factor treatment and then appeared in membranes of lesser buoyant density after alpha-factor exposure. Second, receptors were susceptible to cleavage by extracellular proteases before alpha-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. The median transit time in both assays was approximately 8 min. The ultimate target of the internalized receptors was identified as the vacuole, since the membranes containing internalized receptors cofractionated with vacuolar membranes, since the turnover of receptors was stimulated by alpha-factor exposure, and since receptor degradation was blocked in a pep4 mutant that is deficient for vacuolar proteases. The carboxy-terminal domain of the receptor that is required for ligand internalization was also found to be essential for endocytosis of the receptor. A receptor mutant, ste2-L236H, which is defective for pheromone response but capable of ligand internalization, was found to be proficient for receptor endocytosis. Hence, separate structural features of the receptor appear to specify its signal transduction and internalization activities.