Abstract
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.
Highlights
Transferase, can still form a complex with the G protein ␥ subunits
In order to respond to extracellular signals, G proteins and receptors must associate with the plasma membrane of the cell
The absence of GPA1 would normally lead to constitutive signaling and growth arrest; the gpa1⌬ strain is viable at 34 °C due to a temperature sensitive mutation that blocks the signal downstream of the G protein
Summary
Transferase, can still form a complex with the G protein ␥ subunits. The complex is stabilized by GDP and is dissociated upon treatment with guanosine 5-O-(thiotriphosphate). All of our initial experiments were carried out at high magnesium, in subsequent experiments we found no difference in the extent of G␥ binding or dissociation at 3 mM versus 50 mM magnesium.) The resin was washed three times with 20 mM sodium Pi, pH 7.3, 350 mM NaCl. The bound proteins were eluted by boiling in 100 l of SDS-PAGE sample buffer for 10 min.
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