The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling.
Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.
Department of Pharmacology, Howard Hughes Medical Institute, New Haven, Connecticut 06510.
C
C
Walch-Solimena
J
J
Blasi
L
L
Edelmann
E R
ER
Chapman
G F
GF
von Mollard
R
R
Jahn
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001905:Botulinum Toxins / Q000494:pharmacology; D001921:Brain / Q000166:cytology / Q000378:metabolism; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D002458:Cell Fractionation; D002966:Clathrin; D018699:Coated Vesicles / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification; D007150:Immunohistochemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008562:Membrane Glycoproteins / Q000302:isolation & purification; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology; D015388:Organelles / Q000737:chemistry / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism*; D016708:Synaptophysin / Q000302:isolation & purification; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D050827:Syntaxin 1; D020692:rab3 GTP-Binding Proteins
D000954:Antigens, Surface; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D002966:Clathrin; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D016708:Synaptophysin; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D050857:Synaptotagmins; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000254:Sodium-Potassium-Exchanging ATPase