Retromer/WASH dependent sorting of nutrient transporters requires a multivalent interaction network with ANKRD50

Retromer and the associated actin polymerizing WASH-complex are essential for the endocytic recycling of a wide range of integral membrane proteins. A hereditary Parkinson's disease causing point mutation (D620N) in the retromer subunit VPS35 perturbs retromer's association with the WASH-complex and also with the uncharacterized protein Ankyrin Repeat Domain Containing Protein 50 (ANKRD50). Here, we firmly establish ANKRD50 as a novel and essential component of the SNX27-retromer-WASH supercomplex. Depletion of ANKRD50 in HeLa or U2OS cells phenocopied the loss of endosome to cell surface recycling of multiple transmembrane proteins seen upon suppression of SNX27, retromer or WASH-complex components. Mass spectrometric quantification of the cell surface proteome of ANKRD50 depleted cells identified amino acid transporters of the SLC1A family, among them SLC1A4, as additional cargo molecules that depend on ANKRD50 and retromer for their endocytic recycling. Mechanistically, we show that ANKRD50 simultaneously engages multiple parts of the SNX27-retromer-WASH complex machinery in a direct and co-operative interaction network that is needed to efficiently recycle the nutrient transporters GLUT1, SLC1A4 and potentially many other surface proteins.