Volume 272, Number 1, Issue of January 3, 1997 pp. 240-248
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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The G Complex of the Yeast Pheromone Response Pathway
SUBCELLULAR FRACTIONATION AND PROTEIN-PROTEIN INTERACTIONS

(Received for publication, July 8, 1996, and in revised form, October 7, 1996)

Jodi E. Hirschman , Gerard S. De Zutter , William F. Simonds and Duane D. Jenness

From the Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0122 and the  MDB/NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1752

Genetic evidence suggests that the yeast STE4 and STE18 genes encode G and G subunits, respectively, that the G complex plays a positive role in the pheromone response pathway, and that its activity is subject to negative regulation by the G subunit (product of the GPA1 gene) and to positive regulation by cell-surface pheromone receptors. However, as yet there is no direct biochemical evidence for a G protein complex associated with the plasma membrane. We found that the products of the STE4 and STE18 genes are stably 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, form of Ste4p is enriched in the non-membrane fraction. The Ste4p and Ste18p proteins that had been extracted from plasma membranes with detergent were found to cosediment as an 8 S particle under low salt conditions and as a 6 S particle in the presence of 0.25 M NaCl; the Ste18p in these fractions was precipitated with anti-Ste4p antiserum. Under the conditions of our assay, Gpa1p was not associated with either particle. The levels of Ste4p and Ste18p accumulation in mutant cells provided additional evidence for a G complex. Ste18p failed to accumulate in ste4 mutant cells, and Ste4p showed reduced levels of accumulation and an increased rate of turnover in ste18 mutant cells. The gpa1 mutant blocked stable association of Ste4p with the plasma membrane, 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 the cell-surface receptors. These results indicate that at least 40% of Ste4p and Ste18p are part of a G complex at the plasma membrane and that stable association of this complex with the plasma membrane requires the presence of G.

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