Protease resistance of syntaxin·SNAP-25-VAMP complexes: Implications for assembly and structure

Michelle A. Poirier, Joe C. Hao, Per N. Malkus, Charles Chan, Michael F. Moore, David S. King, Mark K. Bennett

Research output: Contribution to journalArticle

Abstract

A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A·SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and amino-and carboxyl-terminal regions of SNAP-25. Furthermore, separate amino- and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino- and carboxyl-terminal regions of SNAP-25 may function as independent domains.

Original languageEnglish (US)
Pages (from-to)11370-11377
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number18
DOIs
StatePublished - May 1 1998
Externally publishedYes

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Vesicle-Associated Membrane Protein 2
Syntaxin 1
Synaptosomes
Peptide Hydrolases
Proteins
Trypsin
Cytoplasmic Vesicles
Qa-SNARE Proteins
Proteolysis
Synaptic Vesicles
Exocytosis
Recombinant Proteins
Cell membranes
Blood Proteins
Digestion
Anchors
Mass Spectrometry
Membrane Proteins
Mass spectrometry
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

Cite this

Poirier, M. A., Hao, J. C., Malkus, P. N., Chan, C., Moore, M. F., King, D. S., & Bennett, M. K. (1998). Protease resistance of syntaxin·SNAP-25-VAMP complexes: Implications for assembly and structure. Journal of Biological Chemistry, 273(18), 11370-11377. https://doi.org/10.1074/jbc.273.18.11370

Protease resistance of syntaxin·SNAP-25-VAMP complexes : Implications for assembly and structure. / Poirier, Michelle A.; Hao, Joe C.; Malkus, Per N.; Chan, Charles; Moore, Michael F.; King, David S.; Bennett, Mark K.

In: Journal of Biological Chemistry, Vol. 273, No. 18, 01.05.1998, p. 11370-11377.

Research output: Contribution to journalArticle

Poirier, MA, Hao, JC, Malkus, PN, Chan, C, Moore, MF, King, DS & Bennett, MK 1998, 'Protease resistance of syntaxin·SNAP-25-VAMP complexes: Implications for assembly and structure', Journal of Biological Chemistry, vol. 273, no. 18, pp. 11370-11377. https://doi.org/10.1074/jbc.273.18.11370
Poirier, Michelle A. ; Hao, Joe C. ; Malkus, Per N. ; Chan, Charles ; Moore, Michael F. ; King, David S. ; Bennett, Mark K. / Protease resistance of syntaxin·SNAP-25-VAMP complexes : Implications for assembly and structure. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 18. pp. 11370-11377.
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