2 research outputs found
Immuno-electron tomography of ER exit sites reveals the existence of free COPII-coated transport carriers
Transport from the endoplasmic reticulum (ER) to the Golgi
complex requires assembly of the COPII coat complex at
ER exit sites. Recent studies have raised the question as to
whether in mammalian cells COPII coats give rise to COPIIcoated
transport vesicles or instead form ER sub-domains
that collect proteins for transport via non-coated carriers. To
establish whether COPII-coated vesicles do exist in vivo, we
developed approaches to combine quantitative immunogold
labelling (to identify COPII) and three-dimensional electron
tomography (to reconstruct entire membrane structures).
In tomograms of both chemically fixed and high-pressurefrozen
HepG2 cells, immuno-labelled COPII was found on
ER-associated buds as well as on free ~50-nm diameter
vesicles. In addition, we identified a novel type of COPII-coated
structure that consists of partially COPII-coated, 150–200-nm
long, dumb-bell-shaped tubules. Both COPII-coated carriers
also contain the SNARE protein Sec22b, which is necessary
for downstream fusion events. Our studies unambiguously
establish the existence of free, bona fide COPII-coated
transport carriers at the ER–Golgi interface, suggesting that
assembly of COPII coats in vivo can result in vesicle formation