Heterotrimeric G proteins sit at the cytoplasmic face of membranes and transduce signals from cell surface receptors to intracellular effectors. The heterotrimer consists of the alpha subunit that binds GTP and the beta and gamma subunits that form a tight complex. Most of the G alpha subunits undergo palmitoylation, the reversible, post-translational addition of palmitate to an amino-terminal cysteine residue. Palmitoylation increases the membrane affinity of the G alpha subunit as well as modulating protein interactions with the beta/gamma subunits, receptors and effectors. Activation of G proteins by a receptor increases the palmitate turnover on the alpha subunit. The enzymes responsible for palmitoylation and depalmitoylation of proteins have been elusive. To better understand the regulation of this cycle, we are investigating the enzyme responsible for G-protein palmitoylation. We have partially purified PAT activity from rat liver and human erythrocytes with a significantly higher degree of enrichment compared to previous reports by using affinity chromatography to an inhibitor of PAT activity and to palmitoyl CoA. In collaboration with Lewis Pannell (NIDDK), we analyzed the partially purified products by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and identified ~50 proteins. To test these candidates, we developed a novel expression system and assay that allows facile expression of properly folded proteins.
