ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation

Abstract

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.

ArfGAP1 promotes COPI vesicle formation by facilitating coatomer polymerization

“…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.…”

“…Phosphatidic acid, used in as least two reports 58,61 induces membrane curvature, [62][63][64] which would favor vesicle budding. In other work, vesicles contained a peptide from the cargo protein p23, which does not bind ArfGAP1, 34,36 10% phosphatidylinositol 4,5-bisphosphate, which does not occur in the Golgi apparatus, and 30% phosphatidylserine, for the EM studies. 65 In contrast, we used a peptide from the cargo protein p25, which does bind to ArfGAP1, 36 linked to palmitic acid to anchor it to vesicles.…”

“…In other work, vesicles contained a peptide from the cargo protein p23, which does not bind ArfGAP1, 34,36 10% phosphatidylinositol 4,5-bisphosphate, which does not occur in the Golgi apparatus, and 30% phosphatidylserine, for the EM studies. 65 In contrast, we used a peptide from the cargo protein p25, which does bind to ArfGAP1, 36 linked to palmitic acid to anchor it to vesicles. The vesicles contained 1% phosphatidylinositol 4-phosphate, a phospholipid normally found in the Golgi apparatus.…”

“…[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.…”

Mint3/X11γ Is an ADP-Ribosylation Factor-dependent Adaptor that Regulates the Traffic of the Alzheimer's Precursor Protein from theTrans-Golgi Network

“…However, it later became evident that Arf GTPase activation proteins play essential roles in vesicle biogenesis and specificity of cargo selection (Goldberg, 1998;Lee et al, 2005), making GTP hydrolysis and dissociation of Arfs from the nascent bud or vesicle likely. The absence of stoichiometric levels of Arf on two different vesicle populations, containing different Arf-dependent coats, AP-3 and now Mint3, is consistent with models in which Arfs dissociate from membranes before scission of transport carriers or shortly thereafter.…”