Rabankyrin‐5 Interacts with EHD 1 and Vps 26 to Regulate Endocytic Trafficking and Retromer Function

Abstract

Rabankyrin‐5 (Rank‐5) has been implicated as an effector of the small GTPase Rab 5 and plays an important role in macropinocytosis. We have now identified Rank‐5 as an interaction partner for the recycling regulatory protein, Eps 15 homology domain 1 (EHD 1). We have demonstrated this interaction by glutathione S‐transferase‐pulldown, yeast two‐hybrid assay, isothermal calorimetry and co‐immunoprecipitation, and found that the binding occurs between the EH domain of EHD 1 and the NPFED motif of Rank‐5. Similar to EHD 1, we found that Rank‐5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps 26), suggesting a role for Rank‐5 in retromer‐based transport. Indeed, depletion of Rank‐5 causes mislocalization of Vps 26 and affects both the retrieval of mannose 6‐phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank‐5 is required for normal retromer distribution, as overexpression of a wild‐type Rank‐5‐small interfering RNA‐resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank‐5 or EHD 1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank‐5 and EHD 1, and novel endocytic regulatory roles that include retromer‐based transport and secretion.