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 enzymes responsible for reversible thioacylation. A strong candidate for the thioesterase that removes palmitate is the 25-kDa protein, acyl-protein thioesterase (APT1). Both APT1 and an isoform, APT2, (67% identical) are soluble proteins and expressed ubiquitously. The crystal structure of the human form of APT1 was solved to 1.8 angstrom resolution and showed that APT1 is a member of the alpha/beta hydrolase family, which includes other acylhydrolases such as the palmitoyl protein thioesterase. APT1 appears to be dimeric with the substrate-binding pocket and active site occluded by the dimer interface suggesting that dissociation occurs upon interaction with the substrate. The enzyme responsible for protein palmitoylation has not been identified though a number of investigators have worked on this problem. We are using a proteomic approach to identify potential candidates. We have obtained partially purified fractions of rat liver and human red blood cell membranes that retain their palmitoyl transferase activity. The proteins from these fractions are separated by SDS-PAGE and digested by trypsin. The trypsin fragments are analyzed by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry. We are currently analyzing a number of potential candidates from this method.
