The Rab5 guanine nucleotide exchange factor Rabex-5 binds ubiquitin (Ub) and functions as a Ub ligase through an atypical Ub-interacting motif and a zinc finger domain.
Rabex-5, the mammalian orthologue of yeast Vps9p, is a guanine nucleotide exchange factor for Rab5. Rabex-5 forms a tight complex with Rabaptin-5, a multivalent adaptor protein that also binds to Rab4, Rab5, and to domains present in gamma-adaptins and the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). Rabaptin-5 augments the Rabex-5 exchange activity, thus generating GTP-bound, membrane-associated Rab5 that, in turn, binds Rabaptin-5 and stabilizes the Rabex-5.Rabaptin-5 complex on endosomes. Although the Rabex-5.Rabaptin-5 complex is critical to the regulation of endosomal fusion, the structural determinants of this interaction are unknown. Likewise, the possible binding and covalent attachment of ubiquitin to Rabex-5, two modifications that are critical to the function of yeast Vps9p in endosomal transport, have not been studied. In this study, we identify the 401-462 and 551-661 coiled-coils as the regions in Rabex-5 and Rabaptin-5, respectively, that interact with one another. We also demonstrate that Rabex-5 undergoes ubiquitination and binds ubiquitin, though not via its proposed C-terminal CUE-like domain. Instead, the N-terminal region of Rabex-5 (residues 1-76), comprising an A20-like Cys2/Cys2 zinc finger and an adjacent alpha-helix, is important for ubiquitin binding and ubiquitination. Importantly, we demonstrate that the Rabex-5 zinc finger displays ubiquitin ligase (E3) activity. These observations extend our understanding of the regulation of Rabex-5 by Rabaptin-5. Moreover, the demonstration that Rabex-5 is a ubiquitin ligase that binds ubiquitin and undergoes ubiquitination indicates that its role in endosome fusion may be subject to additional regulation by ubiquitin-dependent modifications.
Mattera
Rafael
R
Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892.
Tsai
Yien Che
YC
Weissman
Allan M
AM
Bonifacino
Juan S
JS
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D002417:Cattle; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism* / Q000502:physiology; D016335:Zinc Fingers / Q000502:physiology*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D020662:Guanine Nucleotide Exchange Factors; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D020691:rab GTP-Binding Proteins