气候变化领域集成服务门户(CCPortal): Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells

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DOI	10.1073/pnas.2025748118
	Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells
	Wan L.; Chen X.; Almers W.
发表日期	2021
ISSN	0027-8424
卷号	118 期号:49
英文摘要	Syntaxin helps in catalyzing membrane fusion during exocytosis. It also forms clusters in the plasma membrane, where both its transmembrane and SNARE domains are thought to homo-oligomerize. To study syntaxin clustering in live PC12 cells, we labeled granules with neuropeptide-Y-mCherry and syntaxin clusters with syntaxin-1a green fluorescent protein (GFP). Abundant clusters appeared under total internal reflection (TIRF) illumination, and some of them associated with granules (“on-granule clusters”). Syntaxin-1a-GFP or its mutants were expressed at low levels and competed with an excess of endogenous syntaxin for inclusion into clusters. On-granule inclusion was diminished by mutations known to inhibit binding to Munc18-1 in vitro. Knock-down of Munc18-1 revealed Munc18-dependent and -independent on-granule clustering. Clustering was inhibited by mutations expected to break salt bridges between syntaxin’s Hb and SNARE domains and was rescued by additional mutations expected to restore them. Most likely, syntaxin is in a closed conformation when it clusters on granules, and its SNARE and Hb domains approach to within atomic distances. Pairwise replacements of Munc18-contacting residues with alanines had only modest effects, except that the pair R114A/I115A essentially abolished on-granule clustering. In summary, an on-granule cluster arises from the specific interaction between a granule and a dense cluster of syntaxin-Munc18-1 complexes. Off-granule clusters, by contrast, were resistant to even the strongest mutations we tried and required neither Munc18-1 nor the presence of a SNARE domain. They may well form through the nonstoichiometric interactions with membrane lipids that others have observed in cell-free systems. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Exocytosis; Munc18; Protein clustering; Syntaxin
语种	英语
scopus关键词	Munc18 protein; syntaxin; syntaxin 1A; Munc18 protein; protein binding; Snap25 protein, rat; Stxbp1 protein, rat; synaptosomal associated protein 25; syntaxin; Article; cell granule; cell membrane; complex formation; conformational transition; controlled study; endocrine cell; exocytosis; Hb domain; membrane fusion; PC12 cell line (pheochromocytoma); protein domain; SNARE domain; total internal reflection fluorescence microscopy; adrenal gland; animal; cell membrane; chemistry; cytology; gene expression regulation; genetics; metabolism; molecular model; mutation; PC12 cell line; physiology; protein conformation; rat; Adrenal Glands; Animals; Cell Membrane; Gene Expression Regulation; Models, Molecular; Munc18 Proteins; Mutation; PC12 Cells; Protein Binding; Protein Conformation; Qa-SNARE Proteins; Rats; Synaptosomal-Associated Protein 25
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/250949
作者单位	Vollum Institute, Oregon Health and Science University, Portland, OR 97239, United States; Department of Chemical Physiology and Biochemistry, School of Medicine, Oregon Health and Science University, Portland, OR 97239, United States
推荐引用方式 GB/T 7714	Wan L.,Chen X.,Almers W.. Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells[J],2021,118(49).
APA	Wan L.,Chen X.,&Almers W..(2021).Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells.Proceedings of the National Academy of Sciences of the United States of America,118(49).
MLA	Wan L.,et al."Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells".Proceedings of the National Academy of Sciences of the United States of America 118.49(2021).