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Abstract
Much in vivo and in vitro evidence has shown that the α subunits of heterotrimeric GTP-binding proteins (G proteins) exist as oligomers in their base state and disaggregate when being activated. In this article, the influence of palmitoylation modification of Gαo on its oligomerization was explored extensively. Gαo protein was expressed and purified from Escherichia coli strain JM109 cotransformed with pQE60(Gαo) and pBB131(N-myristoyltransferase). Non-denaturing gel electrophoresis analysis revealed that Gαo existed to a small extent as monomers but mostly as oligomers including dimers, trimers, tetramers and pentamers which could disaggregate completely into monomers by GTPγS stimulation. Palmitoylated Gαo, on the other hand, only present as oligomers that were difficult to disaggregate into monomers. The effect of palmitoylation on oligomerization of Gαo was further investigated by several other biochemical and biophysical methods including gel filtration chromatography, analytical ultracentrifugation and atomic force microscopy analysis. The results consistently demonstrated that palmitoylation facilitated oligomerization of the Gαo protein. Autoradiography indicated that [14C]-palmitoylated Gαo would in no case disaggregate into monomers after treatment with GTPγS. [35S]-GTPγS binding activity assay showed that palmitoylated Gαo was saturated at only 7.8 nmol/mg compared to 21.8 nmol/mg for non-palmitoylated Gαo. Fluorescent quenching studies using BODIPY FL-GTPγS as a probe showed that the conformation of GTP-binding domain of Gαo tended to become more compact after palmitoylation. These results implied that palmitoylation may regulate the GDP/GTP exchange of Gαo by influencing the oligomerization state of Gαo and thereby modulate the on-off switch of the G protein in G protein-coupled signal transduction.