Thioacylation is required for targeting G-protein subunit G(o1alpha) 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 membrane by means of N-terminally linked myristic acid and palmitic acid. An additional role for palmitate has been recently suggested by the observation that fusion with the palmitoylated N-terminus of alpha(i1) relocalizes cytosolic green-fluorescent-protein reporter to low buoyancy, Triton-insoluble membrane domains (TIFF; Triton-insoluble floating fraction), enriched with caveolin-1 [Galbiati, Volonté, Meani, Milligan, Lublin, Lisanti and Parenti (1999) J. Biol. Chem 274, 5843-5850]. Here we show that, upon transient expression in transfected COS-7 cells, myristoylated and palmitoylated alpha(o) (alpha(o)wt, where wt is wild-type) is exclusively found in TIFF, from where non-palmitoylated alpha(o)wt and alpha(o)C3S (Cys(3)-->Ser) mutant are excluded. Moreover, alpha(o) fused to N-terminally truncated human vasopressin V2 receptor (V2TR-alpha(o)), lacking myristate and palmitate, still localizes at the plasma membrane by means of first transmembrane helix of V2R, but is excluded from TIFF. Likewise, alpha(o)C3S does not partition into TIFF, even when its membrane avidity is enhanced by co-expression of betagamma-subunits. Thus membrane association, in the absence of added palmitate, is not sufficient to confer partitioning of alpha(o) within TIFF, suggesting that palmitoylation is a signal for membrane compartmentalization of dually acylated alpha-subunits.
Guzzi
F
F
Department of Experimental and Environmental Medicine and Medical Biotechnologies, University of Milano-Bicocca, Via Cadore 48, 20052 Monza, Italy.
Zanchetta
D
D
Chini
B
B
Parenti
M
M
D000215:Acylation; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D011485:Protein Binding; D017483:Receptors, Vasopressin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013501:Surface-Active Agents / Q000737:chemistry*
D051242:Caveolin 1; D022461:Caveolins; D017931:DNA Primers; D017483:Receptors, Vasopressin; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D013501:Surface-Active Agents; D019308:Palmitic Acid; D011092:Polyethylene Glycols; D019204:GTP-Binding Proteins