Three Homologous ArfGAPs Participate in Coat Protein I-mediated Transport

Akina Saitoh,Hye-Won Shin,Akane Yamada,Satoshi Waguri,Kazuhisa Nakayama

doi:10.1074/jbc.m900749200

Akina Saitoh, Hye-Won Shin + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m900749200

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Abstract

ArfGAP1 is a prototype of GTPase-activating proteins for ADP-ribosylation factors (ARFs) and has been proposed to be involved in retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER) by regulating the uncoating of coat protein I (COPI)-coated vesicles. Depletion of ArfGAP1 by RNA interference, however, causes neither a discernible phenotypic change in the COPI localization nor a change in the Golgi-to-ER retrograde transport. Therefore, we also examined ArfGAP2 and ArfGAP3, closely related homologues of ArfGAP1. Cells in which ArfGAP1, ArfGAP2, and ArfGAP3 are simultaneously knocked down show an increase in the GTP-bound ARF level. Furthermore, in these cells proteins resident in or cycling through the cis-Golgi, including ERGIC-53, beta-COP, and GM130, accumulate in the ER-Golgi intermediate compartment, and Golgi-to-ER retrograde transport is blocked. The phenotypes observed in the triple ArfGAP knockdown cells are similar to those seen in beta-COP-depleted cells. Both the triple ArfGAP- and beta-COP-depleted cells accumulate characteristic vacuolar structures that are visible under electron microscope. Furthermore, COPI is concentrated at rims of the vacuolar structures in the ArfGAP-depleted cells. On the basis of these observations, we conclude that ArfGAP1, ArfGAP2, and ArfGAP3 have overlapping roles in regulating COPI function in Golgi-to-ER retrograde transport.

Highlights

Active state; in the latter form they recruit various effectors, including the coat protein I (COPI) coat [1, 2]
Additional evidence suggests roles of ArfGAP1 beyond that of a simple inactivator of ADP-ribosylation factors (ARFs); first, GTP hydrolysis on ARF is required for proper sorting of cargo molecules into COPI-coated vesicles [15,16,17]; second, ArfGAP1 promotes COPI-coated vesicle formation by coupling cargo sorting to vesicle formation (18 –20); third, imaging studies have suggested that ArfGAP1 undergoes ARF1dependent cycling between the cytosol and Golgi membranes independent of vesicle budding [21, 22]; Antonny and co-workers [23, 24] have proposed a model in which ArfGAP1 and Gcs1 sense the curvature of budding vesicles through a motif outside of their catalytic domain
To explore the role of ArfGAP1 in COPI-mediated transport, we looked for phenotypic changes in cells depleted of ArfGAP1 by RNA interference (RNAi)

Summary

EXPERIMENTAL PROCEDURES

Antibodies, Reagents, and Plasmids—Antiserum to human ArfGAP1 was raised in rabbits against a synthetic peptide (amino acid residues 377–390) conjugated to keyhole limpet hemocyanin and affinity-purified using the immunized peptide immobilized on Sulfolink beads (Pierce). Polyclonal rabbit and monoclonal mouse anti-␤-COP antibodies were from Affinity Bioreagents and Sigma-Aldrich, respectively. The transfected cells were transferred to a culture dish containing coverslips, further incubated for up to 120 and 48 h in the case of ArfGAP knockdown and ␤-COP knockdown, respectively, and processed for immunofluorescence and immunoblot analyses and transport assays. Electron Microscopy—For conventional electron microscopy, control and siRNA-treated cells were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer They were post-fixed with 1% OsO4, embedded in Epon812, and sectioned as previously described [42]. Immunoelectron microscopy on ultrathin cryosections was performed as described previously [35, 42]

RESULTS

The punctate staining for

DISCUSSION