Three homologous ArfGAPs participate in coat protein I-mediated transport.
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.
Saitoh
Akina
A
Graduate School of Pharmaceutical Sciences and Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Shin
Hye-Won
HW
Graduate School of Pharmaceutical Sciences and Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan; Career-Path Promotion Unit for Young Life Scientists, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Yamada
Akane
A
Department of Anatomy and Histology, Fukushima Medical University, School of Medicine, Fukushima City, Fukushima 960-1295, Japan.
Waguri
Satoshi
S
Department of Anatomy and Histology, Fukushima Medical University, School of Medicine, Fukushima City, Fukushima 960-1295, Japan.
Nakayama
Kazuhisa
K
Graduate School of Pharmaceutical Sciences and Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address: kazunaka@pharm.kyoto-u.ac.jp.
D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D022181:COP-Coated Vesicles / Q000235:genetics / Q000378:metabolism*; D020756:Coatomer Protein / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D037241:Mannose-Binding Lectins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism
D001324:Autoantigens; D020756:Coatomer Protein; D020690:GTPase-Activating Proteins; C097741:Golgin subfamily A member 2; C084004:LMAN1 protein, human; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins