The linker domain of the SNARE protein SNAP25 acts as a flexible molecular spacer that ensures efficient S-acylation
| Autor: | Luke H. Chamberlain, Christine Salaün, Nicholas C. O. Tomkinson, Jennifer Greaves |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Synaptosomal-Associated Protein 25 post-translational modification (PTM) Acylation Amino Acid Motifs Protein domain PC12 Cells Biochemistry protein palmitoylation ankyrin repeat domain S-acylation RS 03 medical and health sciences Protein acylation Protein Domains Animals Humans Protein palmitoylation Short linear motif QD zDHHC17 protein acylation Molecular Biology Zinc finger 030102 biochemistry & molecular biology Chemistry protein domain zDHHC enzyme Cell Biology Rats HEK293 Cells 030104 developmental biology mPEG-click Biophysics Ankyrin repeat protein chemical modification Linker |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | S-Acylation of the SNARE protein SNAP25 (synaptosomeassociated 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 ( 112VVASQP 117), which is downstream of its S-acylated, cysteine-rich domain ( 85CGLCVCPC 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. |
| Databáze: | OpenAIRE |
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