Uncoating of COPII from ER exit site membranes precedes cargo accumulation and membrane fission

Abstract

COPII and COPI are considered to be analogous sets of vesicle coat protein heterocomplexes. Coupled to cargo selection, they mediate the formation of membrane vesicles translocating in opposite directions to differ rent destinations within the secretory pathway. Here, live cell and electron microscopy provided evidence for a different localization and mode of function of the COPII coat during protein export from the endoplasmic reticulum (ER). Pharmaceutical and genetic perturbations of ER-Golgi transport were used to demonstrate that COPII is recruited to membranes defining the boundary of ER-ER Exit Sites (ERES) where it facilitates selective cargo concentration.Uncoating of COPII membranes precedes cargo accumulation and fission of Golgi-bound carriers. Moreover, we report what may be direct transfer of cargo to the Golgi apparatus from Golgi-associated BFA sensitive ERESs. Finally, in ldlF cells the stably expressed functional e-COPI-EYFP labeled both ERESs and anterograde carriers. These findings change our understanding of the role of coat proteins in ER to Golgi transport.

Vesicular and uncoated Rab1-dependent cargo carriers facilitate ER to Golgi transport

“…The COPII pool remained stable on the ER at the ERES, even when cargo was released. These results are consistent with previous work suggesting that ERES are relatively long-lived on the ER ( Hammond and Glick, 2000 ; Shomron et al, 2019 preprint; Stephens et al, 2000 ). This could indicate an adaptive mechanism by which mammalian cells maximize secretory efficiency by retaining COPII coat proteins at stable ERES.…”

“…Additionally, temperature-sensitive mutants of the COPII coat are not available in animal cells. Instead, although various techniques have been used to study secretory cargo trafficking in animal cells, the majority of studies have utilized a single temperature-sensitive fluorescently tagged cargo, ts-VSVG ( Bonfanti et al, 1998 ; Gallione and Rose, 1983 ; Kreitzer et al, 2000 ; Shomron et al, 2019 preprint). At non-permissive temperature, GFP-ts-VSVG is stuck in the ER as an unfolded protein.…”

“…Studies monitoring the VSVG cargo system have shown that when the temperature is reduced, intermediate structures become enlarged, accumulate secretory cargo and form in the vicinity of COPII-labeled ERES ( Mironov et al, 2003 ; Presley et al, 1997 ; Stephens, 2003 ). These structures acquire the vesicular coat complex COPI ( Aridor et al, 1995 ; Lee et al, 2004 ; Shomron et al, 2019 preprint; Stephens et al, 2000 ) and, upon temperature release, the structures move towards the Golgi complex in a microtubule-dependent manner ( Presley et al, 1997 ; Shima et al, 1999 ; Stephens et al, 2000 ). More recent studies have keyed in on how these intermediate compartment structures rearrange to facilitate trafficking of large cargoes such as collagen ( McCaughey et al, 2019 ; Saito et al, 2009 ).…”

Positioning of endoplasmic reticulum exit sites around the Golgi depends on BicaudalD2 and Rab6 activity

“…Interestingly, although ERES can be found throughout the cytoplasm, they tend to gather around the central Golgi 5 . It has been proposed that the reduction of the distance between ERES and Golgi may facilitate the transport of secretory cargo from ER to Golgi 6,7 . However, the mechanisms of ERES‐Golgi association remain obscure.…”

“…Transport carriers (TCs) form and bud through a COPII‐dependent mechanism on ERES 15 . These TCs lose the COPII coat shortly after budding 6,16 and travel to the Golgi area at the center of the cell, moving long distances in the cytoplasm 11 . It is assumed that the TCs translocation is driven by dynein, 11 although the mechanism of dynein attraction to TC and its regulation requires further investigation.…”