ADP-ribosylation factors (ARFs) are a family of small GTPases that are involved in various aspects of membrane trafficking events. These include ARF1—ARF6, which are divided into three classes on the basis of similarity in the primary structure: Class I, ARF1—ARF3; Class II, ARF4 and ARF5; and Class III, ARF6. Previous studies identified a novel family of potential ARF effectors, termed GGA1—GGA3, which interact specifically with GTP-bound ARF1 and ARF3 and are localized to the trans-Golgi network (TGN) or its related compartment(s) (GGA is an abbreviation for Golgi-localizing, γ-adaptin ear homology domain, ARF-binding protein). In the present study we have shown that ARF proteins belonging to the three classes, ARF1, ARF5 and ARF6, can interact with all GGA proteins in a yeast two-hybrid assay, in vitro and in vivo. Segmentation of GGA proteins and isolation of GGA mutants defective in ARF binding have revealed that a limited region within the GGA homology domain, which is conserved in the GGA family, is essential for ARF binding. Expression in cells of GTPase-restricted mutants of ARF1 and ARF5 blocks dissociation of GGA proteins from membranes induced by brefeldin A. However, neither of the ARF mutants recruits GGA mutants defective in ARF binding. On the basis of these observations, we conclude that at least ARF1 (Class I) and ARF5 (Class II) in their GTP-bound state cause recruitment of GGA proteins on to TGN membranes. In contrast, on the basis of similar experiments, ARF6 (Class III) may be involved in recruitment of GGA proteins to other compartments, possibly early endosomes.
- brefeldin A
- trans-Golgi network
- yeast two-hybrid system
↵1 Present address: Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan.
↵2 Present address: Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan.
↵3 Present address: Division of Chem-Bio Informatics, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan.
↵4 Present address and address for correspondence: Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan (e-mail ).
Abbreviations used: ER, endoplasmic reticulum; AP, adaptor protein; TGN, trans-Golgi network; ARF, ADP-ribosylation factor; GGA, Golgi-localizing, AGEH (γ-adaptin ear homology) domain, ARF-binding protein; VHS, Vps27p/Hrs/STAM; GGAH, GGA homology; GST, glutathione S-transferase; HA, haemagglutinin; BFA, brefeldin A; Q71L mutation (etc.), Gln71→Leu (etc.); GTP[S], guanosine 5′-[γ-thio]triphosphate; WT, wild-type; EEA1, early endosomal autoantigen 1; Flag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys.
- The Biochemical Society, London ©2002