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.
Highlights
Fatty-acylated peripheral proteins such as members of the small G-protein Ras family, heterotrimeric G-proteins, the neuronal proteins PSD-95 and growth-associated protein-43 (GAP-43) [1,2,3,4] are synthesized in the cytosol and posttranlationally modified by different lipid moieties [5,6,7,8,9]
The results demonstrate that acyl-protein thioesterases (APTs)-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution
We characterized the acylation, membrane association and intracellular trafficking of full-length (GAP-43full) and the acylation motif (MLCCMRRTKQVEK, N13GAP-43) of GAP-43 fused to spectral variants of green fluorescent protein (GFP) [cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP)] [34,42]
Summary
Fatty-acylated peripheral proteins such as members of the small G-protein Ras family, heterotrimeric G-proteins, the neuronal proteins PSD-95 and growth-associated protein-43 (GAP-43) [1,2,3,4] are synthesized in the cytosol and posttranlationally modified by different lipid moieties [5,6,7,8,9]. These lipid modifications govern their membrane association and membrane subdomain segregation as well as their trafficking, function and stability [8,10,11]. The deacylation step seems to occurs everywhere in the cell and no specific consensus sequence or substrate specificity has been described for this enzymatic reaction so far [21]
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