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Vol. 18, Issue 10, 4119-4128, October 2007

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Rabaptin-5-independent Membrane Targeting and Rab5 Activation by Rabex-5 in the Cell

Huaiping Zhu^*, Guangyu Zhu, Jay Liu^*, Zhimin Liang^*, Xuejun C. Zhang, and Guangpu Li^*

*Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and Crystallography Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104

Submitted February 5, 2007; Revised July 12, 2007; Accepted August 2, 2007
Monitoring Editor: Patrick Brennwald

Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135–399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81–135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135–230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5–independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Guangpu Li (guangpu-li{at}ouhsc.edu)

Abbreviations used: BHK, baby hamster kidney; EET, early endosomal targeting; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GST, glutathione S-transferase; HB, helical bundle; MBM, membrane-binding motif; MBP, maltose-binding protein; PBS, phosphate-buffered saline; R5BD, Rab5-binding domain; RFP, red fluorescent protein; ZnF, Zn²⁺ finger; UIM, ubiquitin-interacting motif.