Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 295, Issue 21, Pages 7501-7515Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA120.012726
Keywords
protein palmitoylation; post-translational modification (PTM); protein acylation; protein chemical modification; protein domain; ankyrin repeat domain; mPEG-click; S-acylation; zDHHC enzyme; zDHHC17
Categories
Funding
- Biotechnology and Biological Sciences Research Council [BB/L022087/1]
- Medical Research Council [MR/R011842/1]
- BBSRC [BB/L022087/1] Funding Source: UKRI
- MRC [MR/R011842/1] Funding Source: UKRI
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S-Acylation of the SNARE protein SNAP25 (synaptosome-associated protein of 25 kDa) is mediated by a subset of Golgi zinc finger DHHC-type palmitoyltransferase (zDHHC) enzymes, particularly zDHHC17. The ankyrin repeat domain of zDHHC17 interacts with a short linear motif known as the zDHHC ankyrin repeat?binding motif (zDABM) in SNAP25 ((112)VVASQP(117)), which is downstream of its S-acylated, cysteine-rich domain ((85)CGLCVCPC(92)). Here, we investigated the importance of a flexible linker region (amino acids 93?111, referred to hereafter as the ?mini-linker? region) that separates the zDABM and S-acylated cysteines in SNAP25. Shortening the mini-linker did not affect the SNAP25?zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, but extended and rigid alanine-proline repeats perturbed it. A SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localization as WT SNAP25, indicating that the amino acid composition of the mini-linker is not important for SNAP25 localization. Using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher-affinity zDABM. This mutant interacted with zDHHC17 more strongly but was S-acylated with reduced efficiency in HEK293T cells, implying that a lower-affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. These results show that amino acids 93?111 in SNAP25 act as a flexible molecular spacer that ensures efficient coupling of the SNAP25?zDHHC17 interaction and S-acylation of SNAP25.
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