Fusion pore dynamics are regulated by synaptotagmin*t-SNARE interactions.
Exocytosis involves the formation of a fusion pore that connects the lumen of secretory vesicles with the extracellular space. Exocytosis from neurons and neuroendocrine cells is tightly regulated by intracellular [Ca2+] and occurs rapidly, but the molecular events that mediate the opening and subsequent dilation of fusion pores remain to be determined. A putative Ca2+ sensor for release, synaptotagmin I (syt), binds directly to syntaxin and SNAP-25, which are components of a conserved membrane fusion complex. Here, we show that Ca2+-triggered syt*SNAP-25 interactions occur rapidly. The tandem C2 domains of syt cooperate to mediate binding to syntaxin/SNAP-25; lengthening the linker that connects C2A and C2B selectively disrupts this interaction. Expression of the linker mutants in PC12 cells results in graded reductions in the stability of fusion pores. Thus, the final step of Ca2+-triggered exocytosis is regulated, at least in part, by direct contacts between syt and SNAP-25/syntaxin.
Bai
Jihong
J
Department of Physiology, University of Wisconsin, Madison, WI 53706, USA.
Wang
Chih-Tien
CT
Richards
David A
DA
Jackson
Meyer B
MB
Chapman
Edwin R
ER
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000235:genetics; D002135:Calcium-Binding Proteins; D005089:Exocytosis / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D046911:Macromolecular Substances; D008561:Membrane Fusion / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D017729:Presynaptic Terminals / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D050765:Qa-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D013570:Synaptic Membranes / Q000378:metabolism* / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000235:genetics*; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D002118:Calcium