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

Abstract

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.

Rabex-5 Is a Rab22 Effector and Mediates a Rab22-Rab5 Signaling Cascade in Endocytosis

“…Baby hamster kidney (BHK)-21 cells were cultured as described previously (Zhu et al, 2007). The Rabex-5-deficient NF73 cells, which were mouse embryo fibroblasts isolated from Rabex-5 knockout mice (Kalesnikoff et al, 2007), were kindly provided by Dr. S. J. Galli's laboratory at Stanford University (Stanford, CA).…”

“…Rabex-5 contains no transmembrane domain but can target to early endosomal membrane for Rab5 activation by two mechanisms. An indirect mechanism is mediated by the Rabaptin-5-binding domain immediately downstream of the GEF domain, which leads to the formation of Rabex-5/Rabaptin-5 complexes (Lippe et al, 2001;Mattera et al, 2006;Delprato and Lambright, 2007;Kalesnikoff et al, 2007;Zhu et al, 2007). Rabaptin-5 is a Rab5 effector that binds to Rab5-GTP on early endosomes (Stenmark et al, 1995;Zhu et al, 2004) and brings Rabex-5 to the proximity of more substrates (Rab5-GDP) on the target membrane, which may lead to further Rab5 activation in a positive feedback loop.…”

“…Rabaptin-5 is a Rab5 effector that binds to Rab5-GTP on early endosomes (Stenmark et al, 1995;Zhu et al, 2004) and brings Rabex-5 to the proximity of more substrates (Rab5-GDP) on the target membrane, which may lead to further Rab5 activation in a positive feedback loop. In addition, a direct membrane targeting mechanism is mediated by the early endosomal targeting (EET) domain that is composed of a membrane-binding motif immediately upstream of the GEF domain and the following HB domain, thus overlapping with the GEF domain (Zhu et al, 2007). The EET domain-mediated direct membrane targeting allows Rabex-5 to associate with early endosomes and activate Rab5 independent of Rabaptin-5, which may generate a relatively high basal level of Rab5-GTP on early endosomes to facilitate recruitment of Rabex-5/Rabaptin-5 complexes for further Rab5 activation and establishment of functional Rab5-GTP domains in endosome fusion.…”

Rabaptin5 is recruited to endosomes by Rab4 and Rabex5 to regulate endosome maturation

“…In this manner, high Rab5 activity is maintained by a positive-feedback loop. Free Rabex5 has also been shown to bind to endosomes directly through an early-endosome-targeting sequence (Zhu et al, 2007) or a ubiquitin-binding domain (Mattera and Bonifacino, 2008), independently of its GEF activity and of Rab5. This has been proposed as a mechanism to initiate the Rab5 activation loop (Zhu et al, 2007(Zhu et al, , 2010.…”

“…Free Rabex5 has also been shown to bind to endosomes directly through an early-endosome-targeting sequence (Zhu et al, 2007) or a ubiquitin-binding domain (Mattera and Bonifacino, 2008), independently of its GEF activity and of Rab5. This has been proposed as a mechanism to initiate the Rab5 activation loop (Zhu et al, 2007(Zhu et al, , 2010. The feedback loop is finally broken in the process of maturation from Rab5-to Rab7-dominated endosomes (Huotari and Helenius, 2011;Poteryaev et al, 2010;Rink et al, 2005).…”