Regulation of membrane and subunit interactions by N-myristoylation of a G protein alpha subunit in yeast.
Initiation of the mating process in yeast Saccharomyces cerevisiae requires the action of secreted pheromones and G protein-coupled receptors. As in other eukaryotes, the yeast G protein alpha subunit undergoes N-myristoylation (GPA1 gene product, Gpa1p). This modification appears to be essential for function, since a myristoylation site mutation exhibits the null phenotype in vivo (gpa1(G2A)). Here we examine how myristoylation affects Gpa1p activity in vitro. We show that the G2A mutant of Gpa1p, when fused with glutathione S-transferase, can still form a complex with the G protein betagamma subunits. The complex is stabilized by GDP and is dissociated upon treatment with guanosine 5'-O-(thiotriphosphate). In addition, there is no apparent difference in the relative binding affinity of Gbetagamma for mutant and wild-type Gpa1p. Using sucrose density gradient fractionation of cell membranes, Gpa1p associates normally with the plasma membrane whereas Gpa1pG2A is mislocalized to a microsomal membrane fraction. A portion of Gbetagamma is also mislocalized in these cells, as it is in a gpa1Delta strain. In contrast, wild-type Gpa1p reaches the plasma membrane in cells that do not express Gbetagamma or cell surface receptors. These findings indicate that mislocalization of Gpa1pG2A is not caused by a redistribution of Gbetagamma, nor is it the result of any difference in Gbetagamma binding affinity. These data suggest that myristoylation is required for specific targeting of Gpa1p to the plasma membrane, where it is needed to interact with the receptor and to regulate the release of Gbetagamma.
Song
J
J
Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812, USA.
Hirschman
J
J
Gunn
K
K
Dohlman
H G
HG
D001483:Base Sequence; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D044387:GTP-Binding Protein beta Subunits; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins; D008969:Molecular Sequence Data; D009226:Myristates / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction
D017931:DNA Primers; D005656:Fungal Proteins; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D009226:Myristates; D029701:Saccharomyces cerevisiae Proteins; C067576:Ste4 protein, S cerevisiae; D019204:GTP-Binding Proteins; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins