Plasma Membrane Localization of Gaz Requires Two Signals

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the a subunits are myristoylated and/or palmitoylated, whereas the g chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled a subunit, az. Wild-type az (az-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does az-WT. Removal of the myristoylation site produced a mutant az that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of bg with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on az-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on az-WT. Together these results suggest that myristate and bg promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on az specific localization at the plasma membrane.