title: All three components of the neuronal SNARE complex contribute to secretory vesicle docking.
abstract: Before exocytosis, vesicles must first become docked to the plasma membrane. The SNARE complex was originally hypothesized to mediate both the docking and fusion steps in the secretory pathway, but previous electron microscopy (EM) studies indicated that the vesicular SNARE protein synaptobrevin (syb) was dispensable for docking. In this paper, we studied the function of syb in the docking of large dense-core vesicles (LDCVs) in live PC12 cells using total internal reflection fluorescence microscopy. Cleavage of syb by a clostridial neurotoxin resulted in significant defects in vesicle docking in unfixed cells; these results were confirmed via EM using cells that were prepared using high-pressure freezing. The membrane-distal portion of its SNARE motif was critical for docking, whereas deletion of a membrane-proximal segment had little effect on docking but diminished fusion. Because docking was also inhibited by toxin-mediated cleavage of the target membrane SNAREs syntaxin and SNAP-25, syb might attach LDCVs to the plasma membrane through N-terminal assembly of trans-SNARE pairs.
authors.affiliation: Howard Hughes Medical Institute, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Yao
authors.initials: Y
authors.lastname: Wu
authors.forename: Yiwen
authors.initials: Y
authors.lastname: Gu
authors.forename: Mary K
authors.initials: MK
authors.lastname: Morphew
authors.forename: Jun
authors.initials: J
authors.lastname: Yao
authors.forename: Felix L
authors.initials: FL
authors.lastname: Yeh
authors.forename: Min
authors.initials: M
authors.lastname: Dong
authors.forename: Edwin R
authors.initials: ER
authors.lastname: Chapman
mesh_terms: D000818:Animals; D005089:Exocytosis; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D009474:Neurons / Q000378:metabolism*; D009498:Neurotoxins / Q000737:chemistry; D016716:PC12 Cells; D011485:Protein Binding; D050683:R-SNARE Proteins / Q000737:chemistry; D051381:Rats; D050600:SNARE Proteins / Q000737:chemistry*; D022142:Secretory Vesicles / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D009498:Neurotoxins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins