ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

Abstract

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.

Recruitment of Arf1-GDP to Golgi by Glo3p-Type ArfGAPs Is Crucial for Golgi Maintenance and Plant Growth

“…These results suggest that normal AGD8 and AGD9 levels are crucial for the maintenance of the Golgi morphology. A previous study in animal cells demonstrated that knockdown of both ArfGAP2 and ArfGAP3 in animal cells caused similar alterations in Golgi morphology (Kartberg et al, 2010). Thus, it is possible that AGD8 and AGD9 are functional orthologs of the Glo3p-type ArfGAPs in animal cells, ArfGAP2/ArfGAP3.…”

“…If AGD8/AGD9 are Arabidopsis orthologs of Glo3p-type ArfGAPs, they are likely to be involved in a COPI-related process (i.e. in retrograde Golgi-toendoplasmic reticulum trafficking), as observed for Glo3p ArfGAPs (Frigerio et al, 2007;Kliouchnikov et al, 2009;Kartberg et al, 2010). However, it is technically very difficult to monitor retrograde trafficking in plant cells.…”

ArfGAP1 promotes COPI vesicle formation by facilitating coatomer polymerization

“…Future studies will determine if the interaction of ArfGAP1 with Arf1 and cargo affects vesicle formation and whether other ArfGAPs, such as ArfGAP2 and 3 and AGAPs, that bind to coat proteins and cargo 12,15,[69][70][71] function by a mechanism analogous to ArfGAP1.…”

“…The function of ArfGAP1, 2 and 3 is, at least in part, related to interaction with the substrate protein Arf1•GTP, vesicle coat protein and the cargo proteins that are being transported. [8][9][10][11][12][13][14][15] The molecular basis for retrograde traffic between the Golgi cisternae or from the Golgi apparatus to the ER has been extensively studied. 2,[4][5][6][7]16 In these pathways, cargo is transferred from trans-side cisternae to the more cis-side, or from the Golgi apparatus to the endoplasmic reticulum (ER).…”

COPI Budding within the Golgi Stack

“…ArfGAP2/3 followed the dynamics of membrane association of coatomer more closely than does ArfGAP1. Furthermore, ArfGAP2/3 knock down caused unstacking of the Golgi and cisternal shortening, similar to conditions in which vesicle uncoating was blocked (Kartberg et al 2010). Taken together, ArfGAP1, independent of coatomer, is likely to drive the cycle of activation and inactivation of Arf1 explained above, whereas the coatomer-dependent ArfGAPs2/3 are candidate-uncoating, GTPase-activating proteins.…”