Three homologous ArfGAPs participate in coat protein imediated transport

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 byRNA 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, β-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 β-COP-depleted cells. Both the triple ArfGAP- and β-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 Arf-GAP3 have overlapping roles in regulating COPI function in Golgi-to-ER retrograde transport. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.