The dually acylated NH2-terminal domain of Gi1α is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains -: Palmitoylation of caveolin-1 is required for the recognition of dually acylated G-protein α subunits in vivo

Here we investigate the molecular mechanisms that govern the targeting of G-protein cr subunits to the plasma membrane. For this purpose, we used G(i1 alpha), as a model dually acylated G-protein, We fused full-length G(i1 alpha) or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of G(i1 alpha) are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by cofractionation and co-immunoprecipitation with caveolin-1, Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1, Taken together, these results indicate that lipid modification of the NH2-terminal of G(i1 alpha) is essential for targeting to its correct destination and interaction with caveolin-1, Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions, Thus, dual acylation of the NH2-terminal domain of G(i1 alpha) and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules.