N-Myristoylation and βγ Play Roles beyond Anchorage in the Palmitoylation of the G Protein αoSubunit

Abstract

Many of the ␣ subunits of heterotrimeric GTP-binding regulatory proteins (G proteins) are palmitoylated, a modification proposed to play a key role in the stable anchorage of the subunits to the plasma membrane. Palmitoylation of ␣ subunits from the G i family is preceded by N-myristoylation, which alone or together with ␤␥ probably supports a reversible interaction of the ␣ subunit with membrane as a prerequisite to the eventual incorporation of palmitate. Previous studies have not addressed, however, the question of whether membrane association alone, carried out through N-myristoylation, interaction with ␤␥, or other events, is sufficient for palmitoylation. We report here for ␣ o that it is not. We found that N-myristoylation is required for palmitoylation at least in part because it supports events subsequent to membrane attachment. Mutants of ␣ o designed to target the subunit to membrane without an N-myristoyl group are unable to be palmitoylated as evaluated by incorporation of [ 3 H]palmitate. Mutants of ␣ o unable to interact normally with ␤␥ yet still attach to membrane demonstrate that ␤␥, in contrast, is not required for palmitoylation. ␤␥ becomes necessary, however, when the N-myristoyl group is absent. Our results suggest that N-myristoylation and ␤␥, while almost certainly relevant to the reversible interaction of ␣ o with membrane, also play at least partly overlapping, postanchorage roles in palmitoylation.Fatty acid acylation is one of the major covalent modifications of heterotrimeric GTP-binding regulatory protein (G protein) ␣ subunits (1-4). For most ␣ subunits of the G i family, including ␣ i , ␣ o , and ␣ z , two distinct acylation events occur. N-Myristoylation takes place at the amino terminus and represents the attachment of a myristate through an amide bond to Gly 2 following cleavage of the initiator methionine (5, 6). Palmitoylation occurs at the adjacent Cys 3 and represents the attachment of a palmitate (primarily) through a thioester bond (7,8). ␣ s , ␣ q , and ␣ 12 family members are palmitoylated but not N-myristoylated (8 -11), whereas ␣ t is N-myristoylated but not palmitoylated (12). The functional consequences of N-myristoylation and palmitoylation have been studied intensively. NMyristoylation plays an important role in the attachment of G i family ␣ subunits to membrane (5, 6), targeting of the subunits to caveolin-enriched membrane domains (13), and interactions with the ␤␥ heterodimer (14) and effectors such as adenylyl cyclase (15). Palmitoylation is relevant to the attachment of G protein ␣ subunits to membrane (10, 16) and interaction of subunits with ␤␥ (17) and RGS proteins (18).The palmitoylation of G protein ␣ subunits has drawn much attention recently, as this modification, unlike N-myristoylation, is reversible and subject to regulation. Several groups have demonstrated that the activation of G s is accompanied by an increased rate of palmitate exchange on ␣ s , probably reflecting depalmitoylation and repalmitoylation of the subunit released from ␤␥ (19 -21)....

Gβγ Isoforms Selectively Rescue Plasma Membrane Localization and Palmitoylation of Mutant Gαs and Gαq

“…The function of the ␤␥ complex in helping to localize the ␣ subunit to the PM has been demonstrated recently (6,33,37), but little is known about the localization of ␤␥ itself except that it is considered to be anchored at the PM by prenylation (38). Here we have demonstrated that ␤␥ localizes to the PM very poorly when overexpressed in HEK293 cells.…”

S-Nitrosocysteine Increases Palmitate Turnover on Ha-Ras in NIH 3T3 Cells

“…G s ␣ is then repalmitoylated and binds again to ␤␥ (72). Studies of palmitoylation of G o ␣ and G z ␣ have shown a similar relationship between palmitate modification and activation state (73). Palmitoylation of G z ␣ inhibits its interaction with the G z GAP and other RGS proteins that stimulate hydrolysis of G z ␣-bound GTP (74).…”

Live Cell Analysis of G Protein β5Complex Formation, Function, and Targeting

“…The ability of the ␤ subunit to influence targeting of the ␥ subunit was surprising, because the ␤ subunit is not known to have a membrane-targeting signal, such as the prenyl group on the ␥ subunit (Wedegaertner et al, 1995). Because ␣ o was reported recently to target ␤ 5 RGS7 to the plasma membrane (Takida et al, 2005), we hypothesized that plasma membrane targeting of ␤ 5 ␥ 2 also might require ␣ o , which is not expressed in HEK-293 cells (Wang et al, 1999b). Indeed, we found that coexpressed ␣ o did target both Cer-C-␤ 5 Cer-N-␥ 2 and Cer-C-␤ 5 Cer-N-RGS7 to the plasma membrane (Fig.…”