Saitoh Akina, Shin Hye-Won, Yamada Akane, Waguri Satoshi, Nakayama Kazuhisa
Graduate School of Pharmaceutical Sciences and Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
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.
J Biol Chem. 2009 May 15;284(20):13948-13957. doi: 10.1074/jbc.M900749200. Epub 2009 Mar 19.
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.
ArfGAP1是一种ADP核糖基化因子（ARFs）的GTP酶激活蛋白的原型，有人提出它通过调节衣被蛋白I（COPI）包被囊泡的脱衣被作用参与从高尔基体到内质网（ER）的逆行运输。然而，通过RNA干扰使ArfGAP1缺失，既不会导致COPI定位出现明显的表型变化，也不会使高尔基体到内质网的逆行运输发生改变。因此，我们还研究了ArfGAP1的密切相关同源物ArfGAP2和ArfGAP3。ArfGAP1、ArfGAP2和ArfGAP3同时被敲低的细胞中，结合GTP的ARF水平升高。此外，在这些细胞中，驻留在顺面高尔基体或在其中循环的蛋白质，包括内质网-高尔基体中间腔蛋白53（ERGIC-53）、β-COP和GM130，在内质网-高尔基体中间腔中积累，高尔基体到内质网的逆行运输被阻断。在三重ArfGAP敲低细胞中观察到的表型与β-COP缺失细胞中看到的表型相似。三重ArfGAP和β-COP缺失的细胞都积累了在电子显微镜下可见的特征性液泡结构。此外，在ArfGAP缺失的细胞中，COPI集中在液泡结构的边缘。基于这些观察结果，我们得出结论，ArfGAP1、ArfGAP2和ArfGAP3在调节高尔基体到内质网逆行运输中COPI功能方面具有重叠作用。
