期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 115, 期 32, 页码 E7624-E7631出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1808792115
关键词
regulated exocytosis; synaptotagmin; SNARE protein; calcium; PC12 cells
资金
- NIH [DK027044]
- Wellcome Trust Strategic Award [104033]
- MRC [MC_UP_1502/1, MR/M013812/1, G0600089] Funding Source: UKRI
Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+. Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery.
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