The G Protein αs Subunit Incorporates [3H]Palmitic Acid and Mutation of Cysteine-3 Prevents This Modification

Michael Y. Degtyarev, Allen M. Spiegel, Teresa L.Z. Jones

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Abstract

We investigated whether αs could be acylated by palmitate by transfecting COS cells with the cDNA for the wild-type, long form of αs and metabolically labeling with [3H]palmitate or [35S]methionine. Cells were separated into particulate and soluble fractions and immunoprecipitated with a specific peptide antibody. [3H]Palmitate was incorporated into both endogenous and transfected αs. Inhibition of protein synthesis with cycloheximide did not block the radiolabeling of αs with [3H]palmitate. Hydroxylamine treatment caused a release of the tritium radiolabel, demonstrating that the incorporation was through a thioester bond. The tritium radiolabel was base-labile and comigrated with [3H]palmitate on thin-layer chromatography. The third residue of the wild-type αs was mutated from a cysteine to an alanine by site-directed mutagenesis. This mutant was expressed in COS cells and localized to the particulate fraction as determined by immunoprecipitation of the [35S]methionine-labeled cells. The cysteine-3 mutant did not undergo radiolabeling with [3H]palmitate, indicating that this residue is crucial for the modification.

Original languageEnglish (US)
Pages (from-to)8057-8061
Number of pages5
JournalBiochemistry
Volume32
Issue number32
DOIs
StatePublished - Aug 17 1993
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

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