N-TERMINAL FATTY ACYLATION OF THE ALPHA-SUBUNIT OF THE G-PROTEIN G(I)1 - ONLY THE MYRISTOYLATED PROTEIN IS A SUBSTRATE FOR PALMITOYLATION

The alpha-subunit of the G-protein G(1)1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha(i)1 we have generated an alpha(i)1-G2A mutant, carrying alanine instead of glycine at position 2, an alpha(i)1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha(i)1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha(i)1 monitoring metabolic labelling with [H-3]palmitate or [H-3]myristate. The disruption of the palmitoylation site in alpha(i)1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha(i)1G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for ail to be post-translationally palmitoylated. The non-palmitoylated alpha(i)l-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha(i)1-G2A and alpha(i)1-G2A/C3S mutants, indicating that the attachment of palmitic acid is neccessary for proper interaction with the membrane.