Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP-43.

TOMATIS VANESA; TRENCHI, ALEJANDRA; GOMEZ GUILLERMO; DANIOTTI JOSE L.

PLOS ONE

PUBLIC LIBRARY SCIENCE

Lugar: San francistico; Año: 2010 vol. 5 p. 1 - 15

1932-6203

An acylation/deacylation cycle is necessary to maintain the steady-state distribution of S-acylated peripheral proteins, such as the N- and H-Ras, being this cycle necessary for their appropriate cellular distribution and biological activity. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases that deacylate proteins. In this work, we investigate the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in CHO-K1 and HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By reverse transcriptase-PCR and biochemical analysis we observed that acyl protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results reveal the existence of an acylation/deacylation cycle operating in GAP-43 subcellular distribution in which APT-2 is the protein thioesterase involved in the acylation cycle.