ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat

Abstract

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPγS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.

ArfGAP1 promotes COPI vesicle formation by facilitating coatomer polymerization

“…While our results contrast with previously reported results 18,28,29,34,35,39,58 in which it was reported that Arf1 and coatomer were sufficient for vesicle formation, they do not necessarily contradict these studies. The population of LUVs generated by the extrusion method, which is a frequently used method that we used in this work, contains many vesicles with diameters between 30 and 150 nm.…”

“…It is possible that Arf1•GTPgS could slow coatomer polymerization, implying a mechanism in which release of Arf1•GTP is required to ensure that cargo associates with coatomer prior to coatomer polymerization. This interpretation is consistent with the results reported by Hsu and colleagues 35,36,68 in which they found that the [R50K] ArfGAP1 did not efficiently generate COPI vesicles.…”

“…31 Direct evidence from a mammalian model was the finding that ArfGAP1 induces vesicle formation from isolated Golgi membranes in the presence of coatomer and Arf1•GTP. [34][35][36] Note that this latter result directly contradicts results taken to support the model of ArfGAP1 as a negative regulator of Arf1•GTP, including experiments with isolated Golgi in which different methods were used to detect coated vesicles. Possible explanations have been offered for the discrepancies.…”

“…[54][55][56][57] Thus, our results are consistent with previous work identifying ArfGAP1 as a coat component. 35,36 Furthermore, the in vitro assembly will provide a basis for additional studies of the molecular determinants and regulation of COPI vesicle formation.…”

COPI Budding within the Golgi Stack

“…Among those, Brefeldin-A ADP-ribosylated Substrate (BARS) (Yang et al 2005), endophilin (Yang et al 2006), and ArfGAP1 (Yang et al 2002) are thought to play a role in membrane scission and/or act as a coat component. Some of these functions have been challenged by more recent investigations (Gallop et al 2005;Beck et al 2009b).…”