Enzymatic depalmitoylation of viral glycoproteins with acyl-protein thioesterase 1 in vitro

Many glycoproteins of enveloped viruses as well as cellular proteins are co valently modified with fatty acids. Palmitoylation is often reversible, but the enzymology of this hydrophobic protein modification Is not understood. Recently a cytosolic enzyme designated acyl-protein thioesterase 1 (APT1) was purified, which depalmitoylates several cellular proteins. Since hither to no transmembrane proteins have been tested as substrates for APT1 we hav e investigated whether palmitoylated viral membrane glycoproteins can be de acylated by use of this enzyme. Recombinant APT1 was purified from Escheric hia coli, and depalmitoylation of [H-3]palmitate-labeled glycoproteins pres ent in virus particles was measured by SDS-PAGE, fluorography, and scanning densitometry. We find that APT1 causes rapid and almost complete cleavage of fatty acids from the G-protein of vesicular stomatitis virus, hemaggluti nin proteins of influenza A and C virus, and E2 of Semliki Forest virus (SF V). In contrast, E1 of SFV is largely resistant against APT1 activity. This substrate specificity of APT1 was also observed using microsomes prepared from SFV-infected cells. Our data emphasize the potential of APT1 as a tool for functional analysis of protein-bound fatty acids. (C) 2001 Academic Pr ess.