Heterotrimeric G proteins couple cell surface receptors to intracellular effectors at the cytoplasmic face of membranes. Palmitoylation, the reversible addition of palmitic acid to cysteine residues, occurs on most alpha subunits and participates in their membrane localization. Since our previous studies indicated that depalmitoylation was the critical step in palmitate turnover, we have attempted to identify the G protein palmitoyl thioesterase. We found a DNA sequence, named APT2 that is highly homologous to an acyl protein thioesterase. The RNA for APT2 is widely distributed to a number of tissues. The protein also displays lysophospholipase and palmitoyl CoA hydrolase activity. Inactivation of an ortholog in C. elegans by the RNAi technique did not lead to gross changes in development. We are currently evaluating the intracellular function of this protein.Palmitoylation may be involved in targeting G proteins to membrane microdomains enriched in cholesterol and sphingolipids. We are investigating the functional significance of these domains by depleting cellular cholesterol that disrupts the localization of G protein alpha subunits to these domains. cAMP accumulation after isoproterenol stimulation was significantly greater in cholesterol-depleted cells compared to control cells. This result suggests that these membrane microdomains may inhibit signaling through the Gs pathway. We have studied whether palmitoylation directs proteins to specific intracellular membranes by transfecting cells with the wild-type or mutant forms of Xlalpha s, a splice variant of the alpha s subunit found on Golgi membranes. Mutation of cysteines in a cysteine-rich domain inhibited its palmitoylation but did not alter Golgi localization. In contrast, deletion of a proline-rich domain abolished Golgi localization. The proline-rich and cysteine-rich domains together were sufficient to target a GFP fusion protein to the Golgi. Our results suggest that proline-rich regions in conjunction with palmitoylation can be a Golgi targeting signal for G protein alpha subunits. - palmitoylation, heterotrimeric G proteins, signal transduction, protein thioesterase, membrane microdomains, Golgi targeting