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Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane
Author(s) -
Tabata Kazuhito V,
Sato Ken,
Ide Toru,
Nishizaka Takayuki,
Nakano Akihiko,
Noji Hiroyuki
Publication year - 2009
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/emboj.2009.269
Subject(s) - biology , copii , visualization , planar , membrane , vesicular transport proteins , microbiology and biotechnology , biophysics , anatomy , biochemistry , endoplasmic reticulum , computer graphics (images) , golgi apparatus , intracellular , mechanical engineering , engineering , secretory pathway , endosome , computer science , vacuolar protein sorting
Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non‐cargo proteins during COPII vesicle formation using single‐molecule microscopy combined with an artificial planar lipid bilayer. Single‐molecule analysis showed that the Sar1p–Sec23/24p‐cargo complex, but not the Sar1p–Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non‐cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non‐cargo proteins from the COPII vesicles.