Cysteine 3 Is Not the Site of in Vitro Palmitoylation on Gsα

Several studies have examined the role of palmitoylation of G protein α subunits by nonenzymatic in vitro acylation using palmitoyl-CoA. Here, we investigated nonenzymatic palmitoylation of purified Gsαin vitro. GDP-bound Gsα was stoichiometrically autoacylated on cysteine residue(s) with micromolar concentrations of palmitoyl-CoA. The acylation led to a complete loss of steady-state GTPase activity and GTPγS binding to Gsα. Mutation of Cys 3 to Ala in Gsα did not prevent either palmitoylation or its consequent functional alterations. However, stoichiometric palmitoylation of His6-Gsα did not alter its GTPase activity or GTPγS binding. Isoelectric focusing of tryptic peptides from autoacylated wild type, His6-tagged, and C3A mutant of Gsα showed that Cys 160 is the site of in vitro palmitoylation. Therefore, we conclude that in vitro palmitoylation of Gsα occurs on Cys 160 and this modification decreases the ability of the protein to exchange GTP for GDP; N-terminus elongation of Gsα prevents this latter effect without altering palmitoylation.