Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane.
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.
Tabata
Kazuhito V
KV
Department of Biomolecular Energetics, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan.
Sato
Ken
K
Ide
Toru
T
Nishizaka
Takayuki
T
Nakano
Akihiko
A
Noji
Hiroyuki
H
D022181:COP-Coated Vesicles / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000032:analysis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism
C107845:BET1 protein, S cerevisiae; D008051:Lipid Bilayers; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae