The molecular machinery that catalyzes S-acylation reactions at the Golgi comprises zDHHC enzymes with major differences in substrate affinity and S-acylation activity. It is proposed that the coexistence of these two groups of enzymes in cells is important to allow the Golgi S-acylation machinery to modify a wide and diverse set of substrates.

Background: Specific mutations in the chaperone protein CSPα cause adult-onset neuronal ceroid lipofuscinosis.Results: These mutants form SDS-resistant aggregates in a palmitoylation-dependent manner in cell lines and brain samples from mutation carriers.Conclusion: Palmitoylation induces disease-causing CSPα mutants to form SDS-resistant aggregates.Significance: Formation of SDS-resistant CSPα aggregates may underlie development of adult-onset neuronal ceroid lipofuscinosis.

SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b. Despite the conspicuous differences within the palmitoylated domains of these secretory proteins, there is no information on their comparative interactions with palmitoyl transferases. We report that membrane association of all SNAP25/23 proteins is enhanced by Golgi-localized DHHC3, DHHC7, and DHHC17. In contrast, DHHC15 promoted a statistically significant increase in membrane association of only SNAP25b. To investigate the underlying cause of this differential specificity, we examined a SNAP23 point mutant (C79F) designed to mimic the cysteine-rich domain of SNAP25b. DHHC15 promoted a marked increase in membrane binding and palmitoylation of this SNAP23 mutant, demonstrating that the distinct cysteine-rich domains of SNAP25/23 contribute to differential interactions with DHHC15. The lack of activity of DHHC15 toward wild-type SNAP23 was not overcome by replacing its DHHC domain with that from DHHC3, suggesting that substrate specificity is not determined by the DHHC domain alone. Interestingly, DHHC2, which is closely related to DHHC15, associates with the plasma membrane in PC12 cells and can palmitoylate all SNAP25 isoforms. DHHC2 is, thus, a candidate enzyme to regulate SNAP25/23 palmitoylation dynamics at the plasma membrane. Finally, we demonstrate that overexpression of specific Golgi-localized DHHC proteins active against SNAP25/23 proteins perturbs the normal secretion of human growth hormone from PC12 cells. SNAP25a2 and SNAP25b are SNARE proteins that are highly expressed in the brain, where they perform essential functions in presynaptic neurotransmitter release. SNAP25a/b are present at the presynaptic plasma membrane and form a complex with syntaxin 1, an additional SNARE present at the plasma membrane, and the synaptic vesicle SNARE protein VAMP2(1). The formation of this trans-SNARE complex between the plasma membrane and the vesicle membrane is an essential step for subsequent membrane fusion (exocytosis) and secretion of neurotransmitters into the synaptic cleft. In addition to neurons, SNAP25a/b display a restricted tissue distribution and have limited functions outside the nervous system, most notably in regulated exocytosis pathways in pancreatic beta cells and adrenal medullary chromaffin cells (2, 3). SNAP25a and SNAP25b are derived from a single gene by differential splicing of exon 5, and the proteins differ by only 9 of 206 amino acids (4). Despite their high sequence conservation, the proteins are not functionally identical; SNAP25b supports more exocytosis than SNAP25a when overexpressed in embryonic adrenal medullary chromaffin cells (5), and replacement of SNAP25b with an extra copy of SNAP25a in mice leads to developmental defects, seizures, and impairment of ...

Synaptic vesicle exocytosisThe fusion of synaptic vesicles (SVs) with the pre-synaptic plasma membrane underlies synaptic transmission at chemical synapses. This fusion event, termed exocytosis, occurs spontaneously at a low and asynchronous rate, whereas an increase in pre-synaptic [Ca 2+ ] mediates a larger and synchronous fusion of SVs with the plasma membrane (Neher and Sakaba 2008). SV exocytosis results in the secretion of soluble vesicle cargoes (neurotransmitters) into the synaptic cleft, and the elevated release of transmitter in response to Ca 2+ influx increases the likelihood of achieving a robust post-synaptic response. Because of the essential and fundamental nature of SV exocytosis, there is considerable interest in the mechanisms that regulate this fusion pathway.Central to SV exocytosis is the formation of a protein complex between SNARE proteins present on the plasma membrane and SV membrane (Sollner et al. 1993a,b). In neurons, vesicle-associated membrane protein (VAMP2; also known as synaptobrevin 2) functions as the vesicle (or v) SNARE, whereas the plasma membrane proteins SNAP25 and syntaxin 1 act as target membrane (or t) SNAREs. These three SNARE proteins interact to form a trans-SNARE complex which is thought to represent the minimal membrane fusion machinery (Weber et al. 1998 Abbreviations used: ABE, acyl-biotin exchange; Apt1, acyl protein thioesterase 1; CSP, cysteine-string protein; DHHC, aspartate-histidinehistidine-cysteine; DRM, detergent-resistant membrane; EJP, excitatory junctional potential; Ppt, protein palmitoyl thioesterase; SNAP25, synaptosomal-associated protein of 25 kDa; SNARE, soluble NSF (Nethylmaleimide-sensitive factor) attachment protein receptor; TMD, transmembrane domain; VAMP, vesicle-associated membrane protein. AbstractThe fusion of synaptic vesicles with the pre-synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein-protein interactions. Of central importance are the soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre-synaptic plasma membrane and vesicle-associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca 2+ levels, and a major Ca 2+ sensor for vesicle fusion is synaptotagmin I.Synaptotagmin is likely to couple Ca 2+ entry to vesicle fusion via Ca 2+-dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulati...

Background: Mammalian genomes encode 24 "DHHC" S-palmitoyltransferases. Results: Sorting signals were mapped in DHHC4/6, and the localization of DHHC3 was shown not to impact substrate palmitoylation. Conclusion: Lysine-based signals target DHHC4/6 to the endoplasmic reticulum, and DHHC3 localization is a primary determinant of site of substrate palmitoylation. Significance: This work highlights how DHHC protein targeting is regulated and the relationship between DHHC targeting and substrate palmitoylation.