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

Biochemistry. 1996 Nov 26;35(47):14806-17. doi: 10.1021/bi961846b.

Abstract

G protein alpha subunits are often myristoylated and/or palmitoylated near their amino terminus. The G protein alpha 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. [3H]-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 (Gpa1pC3S). Palmitoylation was also blocked by a mutation that prevents myristoylation (Gly2Ala), whereas the palmitoylation-site mutation had no effect on myristoylation of Gpa1p. Gpa1pC3S complemented the gpa1 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 FUSl-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane / metabolism
  • Cysteine / metabolism
  • Esters / metabolism
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • GTP-Binding Protein alpha Subunits*
  • GTP-Binding Protein alpha Subunits, Gq-G11
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism*
  • Guanosine Diphosphate / metabolism
  • Heterotrimeric GTP-Binding Proteins*
  • Membrane Proteins
  • Mutagenesis
  • Palmitic Acid / metabolism*
  • Pheromones / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*
  • Signal Transduction
  • Sulfhydryl Compounds / metabolism

Substances

  • Esters
  • FUS1 protein, S cerevisiae
  • Fungal Proteins
  • GTP-Binding Protein alpha Subunits
  • Membrane Proteins
  • Pheromones
  • Saccharomyces cerevisiae Proteins
  • Sulfhydryl Compounds
  • Guanosine Diphosphate
  • Palmitic Acid
  • GTP-Binding Proteins
  • GPA1 protein, S cerevisiae
  • GTP-Binding Protein alpha Subunits, Gq-G11
  • Heterotrimeric GTP-Binding Proteins
  • Cysteine