Tracking SNARE complex formation in live endocrine cells.
Syntaxin, synaptosome-associated protein of 25 kD (SNAP25), and vesicle-associated membrane protein/synaptobrevin are collectively called SNAP receptor (SNARE) proteins, and they catalyze neuronal exocytosis by forming a "core complex." The steps in core complex formation are unknown. Here, we monitored SNARE complex formation in vivo with the use of a fluorescent version of SNAP25. In PC12 cells, we found evidence for a syntaxin-SNAP25 complex that formed with high affinity, required only the amino-terminal SNARE motif of SNAP25, tolerated a mutation that blocks formation of other syntaxin-SNAP25 complexes, and assembled reversibly when Ca2+ entered cells during depolarization. The complex may represent a precursor to the core complex formed during a Ca2+-dependent priming step of exocytosis.
An
Seong J
SJ
Vollum Institute L-474, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97201, USA.
Almers
Wolfhard
W
D000313:Adrenal Medulla / Q000166:cytology; D000818:Animals; D001426:Bacterial Proteins; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology; D016716:PC12 Cells; D050765:Qa-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins / Q000502:physiology*
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; C494208:SNAP25 protein, human; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins