UBIQUITINATION OF THE YEAST A-FACTOR RECEPTOR

The a-factor receptor (Ste3p) is one of two pheromone receptors in the yeast Saccharomyces cerevisiae that enable the cell-cell communicatio n of mating. In this report, we show that this receptor is subject to two distinct covalent modifications-phosphorylation and ubiquitination . Phosphorylation, evident on the unstimulated receptor, increases upo n challenge by the receptor's ligand, a-factor. We suggest that this p hosphorylation likely functions in the adaptive, negative regulation o f receptor activity. Removal of phosphorylation by phosphatase treatme nt uncovered two phosphatase-resistant modifications identified as ubi quitination using a myc-epitope-tagged ubiquitin construct. Ste3p unde rgoes rapid, ligand-independent turnover that depends on vacuolar prot eases and also on transport of the receptor from surface to vacuole (i .e., endocytosis) (Davis, N.G., J.L. Horecka, and G.F. Sprague, Jr., 1 993 J. Cell Biol. 122:53-65). An end4 mutation, isolated for its defec t in the endocytic uptake of cr-factor pheromone (Raths, S., J. Rohrer , F. Crausaz, and H. Riezman. 1993. J. Cell Biol. 120:55-65), blocks c onstitutive endocytosis of the a-factor receptor, yet fails to block u biquitination of the receptor. In fact, both phosphorylation and ubiqu itination of the surface-bound receptor were found to increase, sugges ting that these modifications may occur normally while the receptor is at the cell surface. In a mutant strain constructed to allow for depl etion of ubiquitin, the level of receptor ubiquitination was found to be substantially decreased. Correlated with this was an impairment of receptor degradative turnover-receptor half-life that is normally simi lar to 20 min at 30 degrees C was increased to similar to 2 h under th ese ubiquitin-depletion conditions. Furthermore, surface residency, no rmally of short duration in wildtype cells (terminated by endocytosis to the vacuole), was found to be prolonged; the majority of the recept or protein remained surface localized fully 2 h after biosynthesis. Th us, the rates of a-factor receptor endocytosis and consequent vacuolar turnover depend on the available level of ubiquitin in the cell. In c ells mutant for two E2 activities, i.e., ubc4 Delta ubc5 Delta cells, the receptor was found to be substantially less ubiquitinated, and in addition, receptor turnover was slowed, suggesting that Ubc4p and Ubc5 p may play a role in the recognition of the receptor protein as substr ate for the ubiquitin system. In addition to ligand-independent uptake , the a-factor receptor also undergoes a ligand-dependent form of endo cytosis (Davis, N.G., J.L. Horecka, and G.F. Sprague, Jr. 1993. J. Cel l. Biol. 122:53-65). Concurrent with ligand-dependent uptake, we now s how that the receptor undergoes ligand-induced ubiquitination, suggest ing that receptor ubiquitination may function in the ligand-dependent endocytosis of the a-factor receptor as well as in its constitutive en docytosis. To account for these findings, we propose a model wherein t he covalent attachment of ubiquitin to surface receptor triggers endoc ytic uptake.