Molecular and Cellular Biology, November 1999, p. 7705-7711, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Subunit
Ste18p Determines Membrane Localization of G
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0122
Received 26 April 1999/Returned for modification 14 June 1999/Accepted 22 July 1999
The pheromone response in the yeast Saccharomyces
cerevisiae is mediated by a heterotrimeric G protein. The G
subunit (a complex of Ste4p and Ste18p) is associated with both
internal and plasma membranes, and a portion is not stably associated
with either membrane fraction. Like Ras, Ste18p contains a
farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and
a cysteine residue (Cys 106) that is a potential site for
palmitoylation. Mutant Ste18p containing serine at position 106 (mutation ste18-C106S) migrated more rapidly than wild-type
Ste18p during sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The electrophoretic mobility of wild-type Ste18p (but not
the mutant Ste18p) was sensitive to hydroxylamine treatment, consistent
with palmitoyl modification at Cys 106. Furthermore,
immunoprecipitation of the G
complex from cells cultured in the
presence of [3H]palmitic acid resulted in two radioactive
species on nonreducing SDS-PAGE gels, with molecular weights
corresponding to G
and G
. Substitution of serine for either
Cys 107 or Cys 106 resulted in the failure of G
to associate with
membranes. The Cys 107 substitution also resulted in reduced
steady-state accumulation of Ste18p, suggesting that the stability of
Ste18p requires modification at Cys 107. All of the mutant forms of
Ste18p formed complexes with Ste4p, as assessed by
coimmunoprecipitation. We conclude that tight membrane attachment of
the wild-type G
depends on palmitoylation at Cys 106 and
prenylation at Cys 107 of Ste18p.
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