All SNAP25 molecules in the vesicle–plasma membrane contact zone change conformation during vesicle priming

Zhao, YingNanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Göttingen D-37077, Germany (författare)

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25- based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild- type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR- FRET) imaging reveals a rapid FRET increase preceding indi-vidual fusion events by 65 ms. The experiments are performed under conditions of a steady- state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle–plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.

Fang, QinghuaNanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Göttingen D-37077, Germany (författare)
Sharma, SatyanUppsala universitet,Molekylär biofysik,Science for Life Laboratory, SciLifeLab,Nanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Göttingen D-37077, Germany(Swepub:uu)satsh421 (författare)
Jakhanwal, ShruteeDepartment of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen D-37077, Germany (författare)
Jahn, ReinhardDepartment of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen D-37077, Germany (författare)
Lindau, ManfredNanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Göttingen D-37077, Germany;Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, FL 33136 (författare)
Nanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Göttingen D-37077, GermanyMolekylär biofysik (creator_code:org_t)

Ingår i:Proceedings of the National Academy of Sciences of the United States of America: Proceedings of the National Academy of Sciences (PNAS)121:20027-84241091-6490

Av författaren/redakt...: Zhao, Ying; Fang, Qinghua; Sharma, Satyan; Jakhanwal, Shrut ...; Jahn, Reinhard; Lindau, Manfred

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