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Vol. 11, Issue 3, 957-968, March 2000
and
G
Subunits Are Required for Full Activity of the
Pheromone Response Pathway in Saccharomyces cerevisiae
Department of Cell Biology and Physiology, Washington University
School of Medicine, St. Louis, Missouri 63110
To establish the biological function of thioacylation
(palmitoylation), we have studied the heterotrimeric guanine
nucleotide-binding protein (G protein) subunits of the
pheromone response pathway of Saccharomyces
cerevisiae. The yeast G protein 
subunit (Ste18p) is
unusual among G
subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect.
In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to
the plasma membrane even in the absence of prenylation or
thioacylation. However, G protein activation released prenylation- or
thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the G subunit are dispensable for G
protein activation by receptor, but they are required to maintain the plasma membrane association of G
after
receptor-stimulated release from G
. The G protein 
subunit (Gpa1p) is tandemly modified at its N terminus with amide- and
thioester-linked fatty acids. Here we show that Gpa1p was thioacylated
in vivo with a mixture of radioactive myristate and palmitate. Mutation
of the thioacylation site in Gpa1p resulted in yeast cells that
displayed partial activation of the pathway in the absence of
pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are
required for a fully functional pheromone response pathway.
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