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

Abstract

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated 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 Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR 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 in 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 demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

Stable expression and function of the inositol 1,4,5-triphosphate receptor requires palmitoylation by a DHHC6/selenoprotein K complex

“…This is of interest because removal of the palmitate moieties from the IP3R may serve to increase the turnover of this Ca 2+ channel protein. S-palmitoylation is reversed by acyl protein thioesterases (APTs), mainly the cytosolic enzymes APT1 and APT2 (36,37). In this manner, palmitoylation/depalmitoylation through PATs/APTs may represent an enzymatic process that serves to fine tune the level of Ca 2+ in activated immune cells and thereby regulate immune responses.…”

Dynamic Palmitoylation Links Cytosol-Membrane Shuttling of Acyl-protein Thioesterase-1 and Acyl-protein Thioesterase-2 with That of Proto-oncogene H-Ras Product and Growth-associated Protein-43

“…Two of these thioesterases, acyl-protein thioesterase-1 (APT1) (11) and APT2 (12), are localized predominantly in the cytoplasm, and the other two, palmitoyl-protein thioesterase-1 (PPT1) (13,14) and PPT2 (15), are lysosomal enzymes (16,17). The first cytosolic thioesterase to be characterized was APT1, which cat-alyzed depalmitoylation of the ␣-subunit of G-proteins and proto-oncogene H-Ras product in vitro (11) as well as synaptosomal-associated protein 23 (SNAP-23) (18).…”

“…The first cytosolic thioesterase to be characterized was APT1, which cat-alyzed depalmitoylation of the ␣-subunit of G-proteins and proto-oncogene H-Ras product in vitro (11) as well as synaptosomal-associated protein 23 (SNAP-23) (18). More recently, a second cytosolic thioesterase, APT2, has been reported to depalmitoylate GAP-43 (growth-associated protein 43) (12). Although both H-Ras and GAP-43 undergo palmitoylation (3,4) for membrane localization, these proteins also require depalmitoylation catalyzed by APT1 and APT2, respectively, to detach from the membrane and to be recycled.…”

Emerging roles for protein S-palmitoylation in Toxoplasma biology

“…The preferred substrates of APT1 were identified as thioacylated proteins (Duncan and Gilman, 1998;Hirano et al, 2009) although APT1 was originally identified as a lysophospholipase (Sugimoto et al, 1996). Further characterisation of human APT1 (hAPT1) has shown that it is responsible for cleaving palmitate from substrates both in vitro and in vivo (Duncan and Gilman, 1998;Tomatis et al, 2010). It is possible that the distinct substrate specificities of APT1 and APT2 are due in part to their differential expression between cell types, although some overlap might exist (Tomatis et al, 2010).…”

“…Further characterisation of human APT1 (hAPT1) has shown that it is responsible for cleaving palmitate from substrates both in vitro and in vivo (Duncan and Gilman, 1998;Tomatis et al, 2010). It is possible that the distinct substrate specificities of APT1 and APT2 are due in part to their differential expression between cell types, although some overlap might exist (Tomatis et al, 2010). The palmitoylation cycle that controls the correct compartmentalisation of H-Ras and N-Ras is the most intensively studied example of an APT1 substrate (Baekkeskov and Kanaani, 2009).…”