The Dually Acylated NH2-terminal Domain of Gi1α Is Sufficient to Target a Green Fluorescent Protein Reporter to Caveolin-enriched Plasma Membrane Domains

Abstract

Here we investigate the molecular mechanisms that govern the targeting of G-protein ␣ subunits to the plasma membrane. For this purpose, we used G i1␣ as a model dually acylated G-protein. We fused full-length G i1␣ or its extreme NH 2 -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␣ 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 caveolinrich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolincontaining 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 NH 2 -terminal of G i1␣ 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 NH 2 -terminal domain of G i1␣ 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.

Gβγ and Palmitate Target Newly Synthesized Gαzto the Plasma Membrane

“…The chimera was presumably unable to bind ␤␥, which precluded it from targeting to the PM and finding its palmitoyl transferase. By contrast, a fusion protein containing a longer N-terminal sequence (32 amino acids) that was derived from ␣ i1 connected to green fluorescent protein, associated with the PM and underwent palmitoylation (39). In the latter case, the N-terminal sequence furnished both a site for myristoylation and a sequence containing side chains that are known to interact directly with ␤␥; this N-terminal domain may have supplied sufficient affinity for ␤␥ to allow PM association and palmitoylation, thus anchoring the fusion protein at the PM.…”

Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase

“…We addressed whether C 390 in hTGH was acylated, because acylation can affect the subcellular localization of proteins and their association with lipid monolayers (54)(55)(56). The substrate pools for TGH are the lipid droplets associated with the endoplasmic reticulum (19).…”

Plasma membrane localization and function of the estrogen receptor α variant (ER46) in human endothelial cells

“…ER46 has 12 cysteines that could be acylated by palmitate. The chemical susceptibility of thioester bond formation between palmitate and cysteine residue(s) is known to permit a rapid and reversible acylation of proteins (24) that facilitate molecular shuttling (25) and membrane receptor clustering (26). There- fore, it is possible that by timely palmitoylation (27), ER46 is dynamically recruited from the cytoplasmic pool to plasmalemmal caveolae in which it could encounter other palmitoylated molecules including eNOS, the G protein ␣-subunit, Src kinases, Ha-Ras, and cav-1.…”