acyl-protein thioesterase 2 catalizes the deacylation of peripheral membrane-associated gap-43acyl-protein thioesterase 2 catalizes外围膜相关gap-43脱酰作用.pdfVIP

Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP-43 Vanesa M. Tomatis¤a, Alejandra Trenchi, Guillermo A. Gomez¤b, Jose L. Daniotti* ´ ´ ´ ´ ´ ´ Centro de Investigaciones en Quımica Biologica de Cordoba (CIQUIBIC, UNC-CONICET), Departamento de Quımica Biologica, Facultad de Ciencias Quımicas, Universidad ´ ´ Nacional de Cordoba, Cordoba, Argentina 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 palmitoyl- transferases (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