Thioacylation is required for targeting G-protein subunit G(o1)alpha to detergent-insoluble caveolin-containing membrane domains

alpha -Subunits of heterotrimeric G(i)-like proteins (alpha (i), alpha (o) and alpha (z)) associate with the cytoplasmic leaflet of the plasma membran e by means of N-terminally linked myristic acid and palmitic acid. An addit ional role for palmitate has been recently suggested by the observation tha t fusion with the palmitoylated N-terminus of alpha (i1), relocalizes cytos olic green-fluorescent-protein reporter to low buoyancy, Triton-insoluble m embrane domains (TIFF; Triton-insoluble floating Fraction), enriched with c aveolin-1 [Galbiati, Volonte, Meani, Milligan, Lublin, Lisanti and Parenti (1999) J. Biol. Chem 274, 5843-5850]. Here we show that, upon transient exp ression in transfected COS-7 cells, myristoylated and palmitoylated a, (or, Wt, where wt is wild-type) is exclusively found in TIFF, from where non-pal mitoylated alpha (o)wt and alpha (o)C3S (Cys(3) --> Ser) mutant are exclude d. Moreover, a, fused to N-terminally truncated human vasopressin V, recept or (V,TR-a,), lacking myristate and palmitate, still localizes at the plasm a membrane by means of first transmembrane helix of V,R, but is excluded fr om TIFF. Likewise, alpha (o)C3S does not partition into TIFF, even when its membrane avidity is enhanced by coexpression of beta gamma -subunits. Thus membrane association, in the absence of added palmitate, is not sufficient to confer partitioning of a, within TIFF, suggesting that palmitoylation i s a signal for membrane compartmentalization of dually acylated alpha -subu nits.