Protease resistance of syntaxin.SNAP-25.VAMP complexes. Implications for assembly and structure.
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.
Poirier
M A
MA
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Hao
J C
JC
Malkus
P N
PN
Chan
C
C
Moore
M F
MF
King
D S
DS
Bennett
M K
MK
D000818:Animals; D001704:Biopolymers; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D012967:Sodium Dodecyl Sulfate; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1
D001704:Biopolymers; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D012967:Sodium Dodecyl Sulfate; D010450:Endopeptidases