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

Abstract

Rab GTPases control membrane identity, fusion and transport by interaction with effector proteins. Effectors that influence the activation-inactivation cycle of their own or other Rab proteins contribute to the timely conversion of Rab membrane identities. Rab5 and its effector rabaptin5 (Rbpt5, also known as RABEP1) are generally considered the prime example for a positive-feedback loop in which Rab5-GTP recruits Rbpt5 in complex with Rabex5 (also known as RABGEF1), the GDP/GTP exchange factor of Rab5, to early endosomes, thus maintaining the Rab5 membrane identity. By deletion analysis, we found that the membrane recruitment of Rabaptin5 required binding to Rab4 and Rabex5, but not Rab5. Deletion of either one of the two Rab5-binding domains or silencing of Rab5 expression did not affect Rabaptin5 recruitment, but produced giant endosomes with early and late endosomal characteristics. The results contradict the model of feedback activation of Rab5 and instead indicate that Rbpt5 is recruited by both Rabex5 recognizing ubiquitylated cargo and by Rab4 to activate Rab5 in a feed-forward manner.

Variants of Rab GTPase–Effector Binding Protein-2 Cause Variation in the Collateral Circulation and Severity of Stroke

“…12 Endosomes traffic receptors such as VEGFR2 to sites involved in downstream signaling, lysosomes, or back to the cell membrane. 13–15 We thus examined EE in pial membrane of Rabep2 − / − mice at E14.5 when collaterogenesis is underway. Endosome diameter was larger in Rabep2 − / − while density was similar (Figure 5A–C).…”

A fresh look at the function of Rabaptin5 on endosomes

“…20 Their function was proposed to be the regulation of AP-1 or GGA dependent vesicle transport at endosomes. 16,20 In the recent study by K€ alin et al, 21 …”

“…The contribution of Rabex5 to Rabaptin5 recruitment could be localized to its ubiquitin interaction domain, which recognizes ubiquitinated endocytic cargo. 10,22 The second requirement for Rabaptin5 EE recruitment was found to be Rab4-GTP 21 : siRNA silencing of Rab4 or deletion of both CC1-1 and CC1-2 abolished membrane binding, while individual deletion of either one coiled-coil domain did not. In vitro, Rabaptin5 lacking CC1-1 was still able to bind Rab4-GTP, whereas no interaction could be detected without CC1-2, suggesting a low affinity site at CC1-1 or interaction with another component that itself is dependent on Rab4.…”

“…In vitro, this was shown to bind Rab5-GTP, but with lower affinity than the C-terminal site. 16,21 Overexpression of Rabaptin5 mutants lacking either one of the Rab5 binding sites or of wild-type Rabaptin5 in Rab5 silenced cells induced the formation of giant, often deformed structures that were positive for both EE and LE markers. This might suggest that Rabaptin5 has the ability to promote uncontrolled progression to late endosomal characteristics, unless Rab5 binding to its 2 interaction sites keeps it in check.…”

Multiple Types of Guanine Nucleotide Exchange Factors (GEFs) for Rab Small GTPases

“…In mammalian cells, Rabex-5 is recruited to early endosomes through its N-terminal ubiquitin-binding domain, where Rabaptin5, a Rabex-5-binding partner, relieves autoinhibition of Rabex-5 thereby yielding an active form of Rab5 (Mattera and Bonifacino, 2008;Zhang et al, 2014). Although a feedback mechanism in which Rab5 recruits Rabaptin5 that is associated with Rabex-5 and enhances Rab5 activation has been suggested, a recent study has shown that the membrane localization of Rabaptin5 depends on Rab4 and Rabex-5, not on Rab5 (Kälin et al, 2015). On the other hand, Gapex-5 (also called hRME-6), which was originally identified in C. elegance rme-6 mutants and exhibits GEF activity toward Rab5 and Rab22B/31, localizes on clathrincoated vesicles and regulates Rab5-mediated uncoating of AP2 from clathrin-coated vesicles in mammalian cells Lodhi et al, 2007;Semerdjieva et al, 2008).…”