Disruption of Retriever Function Impacts Retrograde Trafficking From Endosomes

Within endosomes cargo proteins are sorted and packaged into endosomal-transport carriers (ETCs) enabling their delivery to other intracellular compartments. Retromer, a conserved multimeric protein complex, has defined functions in sorting cargo mediating formation of ETCs for both retrograde trafficking back to the trans-Golgi network (TGN) and recycling of cargo to the cell surface. Recent studies have identified the retriever complex, which is structurally like retromer, that also can function in the recycling of cargo from endosomes. However, retriever's function in retrograde trafficking from endosomes has not been investigated. CrispR mediated knock-out cell models for retromer and retriever in A549 lung carcinoma epithelial cells were generated. Retriever's role in recycling of established cargo Integrin β1 in A549 cells was confirmed. Cation-independent mannose 6-phosphate receptor and TGN46, two well-established retrograde cargos showed a redistribution from the TGN to early endosomes in retromer knockout (KO) and retriever KO cells which is consistent with decreased retrograde trafficking. Application of a ETC redirection assay in A549 cells identified that ETCs dependent on either retromer or retriever can be tethered by Golgin97 and Golgin245, but not GCC88. Overall, the presence of retriever is required for efficient endosomal retrograde trafficking. Within A549 cells, the requirement of retromer for Wiskott-Aldrich Syndrome Protein and SCAR Homolog (WASH) endosomal recruitment was confirmed while recruitment of WASH in the absence of retriever was not reduced. Furthermore, the efficient recruitment of retriever to endosomes was dependent on retromer. The underlying mechanism by which these complexes initiate the formation of retrograde ETCs therefore seems distinct.