Rabankyrin‐5 Interacts with EHD1 and Vps26 to Regulate Endocytic Trafficking and Retromer Function

Abstract

Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein EHD1. We have demonstrated this interaction by GST-pulldown, yeast two-hybrid assay, isothermal calorimetry, and co-immunoprecipitation and found that the binding occurs between the EH-domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 co-localizes and interacts with components of the retromer complex such as Vps26, suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor (M6PR) transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as over-expression of a wild-type Rank-5-siRNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of VSV-G. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.

The retromer complex – endosomal protein recycling and beyond

“…EHD1 has also been shown to associate with the cargo-selective retromer complex, and loss of function of either EHD1, or its closely related paralogue EHD3, results in an endosome-to-Golgi retrieval defect (Gokool et al, 2007;Naslavsky et al, 2009). Along with EHD1, recent evidence has now shown that the EHD1-interacting protein rabankyrin-5 (Rank-5, also known as ANKFY1) associates with the cargo-selective retromer complex and has a regulatory role in retromer-mediated endosome-to-Golgi retrieval, although the nature of that role remains to be determined (Zhang et al, 2012).…”

VEGF induces signalling and angiogenesis by directing VEGFR2 internalisation through macropinocytosis

“…Additionally, VEGFR2 colocalised with rabankyrin-5 (Fig. 3A), an endosomal protein that, besides being localised to diverse endocytic vesicles (Fabrowski et al, 2013;Ishii et al, 2003;Schnatwinkel et al, 2004;Zhang et al, 2012), also localises to macropinosomes (Schnatwinkel et al, 2004). We also tested the colocalisation of internalised VEGFR2 with EEA1, a marker of the early endosomes (Mu et al, 1995).…”

RME-8 coordinates the WASH complex with the retromer SNX-BAR dimer to control endosomal tubulation

“…The SNX dimer, currently understood to comprise a combination of SNX1 or SNX2 with SNX5 or SNX6 (Wassmer et al, 2007), binds to phosphoinositol 3-phosphate through its phox homology domains and induces membrane curvature by the action of its C-terminal BAR (BinAmphiphysin-Rvs) domains (Kurten et al, 2001;Cheever et al, 2001;Yu and Lemmon, 2001;Cozier et al, 2002;Carlton et al, 2004). The tubules generated by the SNX dimer are stabilised by EHD1 (also known as RME-1), which associates with the CSC (Gokool et al, 2007b;Zhang et al, 2012). Although SNX5 and SNX6 do not drive membrane tubulation (van Weering et al, 2012), both interact with the p150Glued component of dynactin (Wassmer et al, 2009;Hong et al, 2009) and, therefore, link retromer-mediated protein sorting with microtubules through dynein.…”