Retromer is a membrane-associated heteropentameric coat complex that functions in the endosome-to-Golgi retrieval of the cation-independent mannose-6-phosphate receptor, the Wntless protein and other membrane proteins of physiological significance. Retromer comprises two functional subcomplexes: the cargo-selective subcomplex is a trimer of the VPS35, VPS29, VPS26 proteins, whereas the sorting nexin proteins, Snx1 and Snx2 function to tubulate the endosomal membrane. Unlike the sorting nexins, which contain PtdIns3P-binding PX domains, the cargo-selective VPS35/29/26 complex has no lipid-binding domains and its recruitment to the endosomal membrane remains mechanistically uncharacterised. In this study we show that the VPS35/29/26 complex interacts with the small GTPase Rab7 and requires Rab7 for its recruitment to the endosome. We show that the Rab7K157N mutant that causes the peripheral neuropathy, Charcot-Marie-Tooth disease, does not interact with the VPS35/29/26 complex, resulting in a weakened association with the membrane. We have also identified a novel retromer-interacting protein, TBC1D5, which is a member of the Rab GAP family of proteins that negatively regulates VPS35/29/26 recruitment and causes Rab7 to dissociate from the membrane. We therefore propose that recruitment of the cargo-selective VPS35/29/26 complex is catalysed by Rab7 and inhibited by the Rab-GAP protein, TBC1D5.

Endosome-to-Golgi retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR) requires the function of the retromer complex. Retromer is localized to endosomes and comprises two distinct sub complexes: the vacuolar protein sorting 35/29/26 sub complex that binds cargo and the sorting nexin (SNX)1/2 sub complex that tubulates endosomal membranes. To identify up-or down-stream regulatory factors of retromer, a comparative proteomic strategy was employed. Protein profiles of endosomally enriched membranes, from either wild-type or retromer-deficient mouse cells, were compared to identify proteins with either elevated or reduced expression levels. Eps15 homology domain-containing protein-1 (EHD1) was identified in endosomally enriched membrane fractions from retromer-deficient cells and was found to be approximately threefold upregulated in the absence of retromer. EHD1 is localized to tubular and vesicular endosomes, partially colocalizes with retromer and is associated with retromer in vivo. Mutation of the nucleotide-binding P-loop of EHD1 results in a dominantnegative effect upon retromer localization and endosome-to-Golgi retrieval, while loss of EHD1 expression by RNA interference destabilizes SNX1-positive tubules and inhibits endosome-to-Golgi retrieval. The interaction between EHD1 and retromer and the requirement for EHD1 to stabilize SNX1-tubules establish EHD1 as a novel facilitating component of endosome-to-Golgi retrieval.