DIRECT EVIDENCE FOR LIGAND-INDUCED INTERNALIZATION OF THE YEAST ALPHA-FACTOR PHEROMONE RECEPTOR

When Saccharomyces cerevisiae a cells bind alpha-factor pheromone, the ligand is internalized and its binding sites are lost from the cell s urface in a time-, energy-, and temperature-dependent manner. This rep ort 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 pred ominantly found in the plasma membrane peak before alpha-factor treatm ent and then appeared in membranes of lesser buoyant density after alp ha-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 compartm ent. The median transit time in both assays was approximately 8 min. T he ultimate target of the internalized receptors was identified as the vacuole, since the membranes containing internalized receptors cofrac tionated with vacuolar membranes, since the turnover of receptors was stimulated by alpha-factor exposure, and since receptor degradation wa s blocked in a pep4 mutant that is deficient for vacuolar proteases. T he 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 phero mone response but capable of ligand internalization, was found to be p roficient for receptor endocytosis. Hence, separate structural feature s of the receptor appear to specify its signal transduction and intern alization activities.