Searching for the Protein Acyltransferase of Gpa1

Abstract

This project focuses on mechanisms that localize proteins to cell membranes. Specifically, we worked to identify the enzyme that catalyzes S-palmitoylation of Gpa1, a G-protein alpha subunit in yeast, which results in its localization to the plasma membrane. Enyzmes that catalyze palmitoylation are called protein acyltransferases (PATs). These proteins increase the membrane localization of their substrates through the addition of one or more lipids. Gpa1 is dually lipidated, undergoing palmitoylation by an unknown PAT but only after first being myristoylated by N-myristoyltransferase. Gpa1 resides in the inner leaflet of the plasma membrane via its N-terminal myristoyl and palmitoyl chains and functions in the yeast mating pheromone response cascade. Identified PATs have transmembrane domains, undergo autoacylation, and have a DHHC motif embedded in a cysteine-rich domain (CRD). The DHHC-CRD motif is essential for PAT activity. We produced a myristoylated Gpa1 fusion construct (Gpa1f) in E. coli and purified it by chromatography. Using myristoylated Gpa1f as substrate, we assayed five PAT candidates from the yeast DHHC-CRD family for in vitro PAT activity. According to these assays, a small level of palmitoylation of myristoylated Gpa1f occurs in the presence of Pfa3. This research was supported by the Washington University School of Medicine.