THE G-BETA-GAMMA COMPLEX OF THE YEAST PHEROMONE RESPONSE PATHWAY - SUBCELLULAR FRACTIONATION AND PROTEIN-PROTEIN INTERACTIONS

Genetic evidence suggests that the yeast STE4 and STE18 genes encode G beta and G gamma subunits, respectively, that the G beta gamma comple x plays a positive role in the pheromone response pathway, and that it s activity is subject to negative regulation by the G alpha subunit (p roduct of the GPA1 gene) and to positive regulation by cell-surface ph eromone receptors, However, as yet there is no direct biochemical evid ence for a G beta gamma protein complex associated with the plasma mem brane, We found that the products of the STE4 and STE18 genes are stab ly associated with plasma membrane as well as with internal membranes and that 30% of the protein pool is not tightly associated with either membrane fraction, A slower-migrating, presumably phosphorylated, for m of Ste4p is enriched in the non-membrane fraction, The Ste4p and Ste 18p proteins that had been extracted from plasma membranes with deterg ent were found to cosediment as an 8 S particle under low salt conditi ons and as a 6 S particle in the presence of 0.25 M NaCl; the Ste18p i n these fractions was precipitated with anti-Ste4p antiserum, Under th e conditions of our assay, Gpa1p was not associated with either partic le. The levels of Ste4p and Ste18p accumulation in mutant cells provid ed additional evidence for a G beta gamma complex. Ste18p failed to ac cumulate in ste4 mutant cells, and Ste4p showed reduced levels of accu mulation and an increased rate of turnover in ste18 mutant cells. The gpa1 mutant blocked stable association of Ste4p with the plasma membra ne, and the ste18 mutant blocked stable association of Ste4p with both plasma membranes and internal membranes, The membrane distribution of Ste4p was unaffected by the ste2 mutation or by down-regulation of th e cell-surface receptors, These results indicate that at least 40% of Ste4p and Ste18p are part of a G beta gamma complex at the plasma memb rane and that stable association of this complex with the plasma membr ane requires the presence of G alpha.