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(Received for publication, July 8, 1996, and in revised form, October 7, 1996)
From the Department of Molecular Genetics and Microbiology,
University of Massachusetts Medical School,
Worcester, Massachusetts 01655-0122 and the Genetic evidence suggests that the yeast
STE4 and STE18 genes encode G
Volume 272, Number 1,
Issue of January 3, 1997
pp. 240-248
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Complex of the Yeast Pheromone Response Pathway
SUBCELLULAR FRACTIONATION AND PROTEIN-PROTEIN
INTERACTIONS
and
MDB/NIDDK,
National Institutes of Health,
Bethesda, Maryland 20892-1752
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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