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

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

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A light-inducible organelle-targeting system for dynamically activating and inactivating signaling in budding yeast

Protein localization plays a central role in cell biology. Although powerful tools exist to assay the spatial and temporal dynamics of proteins in living cells, our ability to control these dynamics has been much more limited. We previously used the phytochrome B– phytochrome-interacting factor light-gated dimerization system to recruit proteins to the plasma membrane, enabling us to control the activation of intracellular signals in mammalian cells. Here we extend this approach to achieve rapid, reversible, and titratable control of protein localization for eight different organelles/positions in budding yeast. By tagging genes at the endogenous locus, we can recruit proteins to or away from their normal sites of action. This system provides a general strategy for dynamically activating or inactivating proteins of interest by controlling their localization and therefore their availability to binding partners and substrates, as we demonstrate for galactose signaling. More importantly, the temporal and spatial precision of the system make it possible to identify when and where a given protein's activity is necessary for function, as we demonstrate for the mitotic cyclin Clb2 in nuclear fission and spindle stabilization. Our light-inducible organelle-targeting system represents a powerful approach for achieving a better understanding of complex biological systems