Partial constitutive activation of pheromone responses by a palmitoylation-site mutant of a G protein alpha subunit in yeast

被引:51
作者
Song, JP
Dohlman, HG
机构
[1] YALE UNIV, SCH MED, DEPT PHARMACOL, NEW HAVEN, CT 06536 USA
[2] YALE UNIV, SCH MED, BOYER CTR MOL MED, MOL CARDIOBIOL PROGRAM, NEW HAVEN, CT 06536 USA
关键词
D O I
10.1021/bi961846b
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
G protein alpha subunits are often myristoylated and/or palmitoylated near their amino terminus. The G protein a subunit in the yeast Saccharomyces cerevisiae (GPA1 gene product, Gpa1p) is known to be myristoylated. and this modification is essential for G protein activity in vivo. Here we examined whether Gpa1p is palmitoylated and determined the functional consequences of this modification. [H-3]-Palmitic acid was incorporated into Gpa1p in cells expressing myc-tagged Gpa1p or Gpa1p-Gst. The label was released upon hydroxylamine treatment. Substitution of the conserved Cys 3 for Ser blocked incorporation of the label (Gpa1p(C3S)). Palmitoylation was also blocked by a mutation that prevents myristoylation (Gly2Ala), whereas the palmitoylation-site mutation had no effect on myristoylation of Gpa1p. Gpa1p(C3S) complemented the gpal Delta mutation in vivo and formed a complex with G(beta gamma) that was able to undergo nucleotide exchange in vitro. However, basal and pheromone-induced FUS1-lacZ transcription were 2-5-fold higher in the C3S mutant. Pheromone-induced growth arrest was also enhanced by the mutation, but recovery from arrest was not affected. Like wild-type Gpa1p. the C3S mutant was predominantly membrane-associated. Upon Triton X-114 partitioning or high pH treatment, no difference in the membrane-binding properties of the wild-type Gpa1p and the C3S mutant was detected. By sucrose density gradient centrifugation of membranes, however, most of the mutant protein was mislocalized to a non-plasma membrane compartment, whereas G(beta gamma) localization was unaltered. Taken together, our data suggest that Gpa1p is palmitoylated via a thioester bond at Cys 3 and that palmitoylation plays a role in modulating Gpa1p signaling and membrane localization.
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页码:14806 / 14817
页数:12
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