SummaryEarly endosome-to-trans-Golgi network (TGN) transport is organized by the retromer complex. Consisting of cargo-selective and membrane-bound subcomplexes, retromer coordinates sorting with membrane deformation and carrier formation. Here, we describe four mammalian retromers whose membrane-bound subcomplexes contain specific combinations of the sorting nexins (SNX), SNX1, SNX2, SNX5, and SNX6. We establish that retromer requires a dynamic spatial organization of the endosomal network, which is regulated through association of SNX5/SNX6 with the p150glued component of dynactin, an activator of the minus-end directed microtubule motor dynein; an association further defined through genetic studies in C. elegans. Finally, we also establish that the spatial organization of the retromer pathway is mediated through the association of SNX1 with the proposed TGN-localized tether Rab6-interacting protein-1. These interactions describe fundamental steps in retromer-mediated transport and establish that the spatial organization of the retromer network is a critical element required for efficient retromer-mediated sorting.

SNX-BAR proteins are a sub-family of sorting nexins implicated in endosomal sorting. Here, we establish that through its phox homology (PX) and Bin-Amphiphysin-Rvs (BAR) domains, sorting nexin-4 (SNX4) is associated with tubular and vesicular elements of a compartment that overlaps with peripheral early endosomes and the juxtanuclear endocytic recycling compartment (ERC). Suppression of SNX4 perturbs transport between these compartments and causes lysosomal degradation of the transferrin receptor (TfnR). Through an interaction with KIBRA, a protein previously shown to bind dynein light chain 1, we establish that SNX4 associates with the minus end-directed microtubule motor dynein. Although suppression of KIBRA and dynein perturbs early endosome-to-ERC transport, TfnR sorting is maintained. We propose that by driving membrane tubulation, SNX4 coordinates iterative, geometric-based sorting of the TfnR with the long-range transport of carriers from early endosomes to the ERC. Finally, these data suggest that by associating with molecular motors, SNX-BAR proteins may coordinate sorting with carrier transport between donor and recipient membranes.

The mammalian retromer is a multimeric protein complex involved in mediating endosome-to-trans-Golgi-network retrograde transport of the cation-independent mannose-6-phosphate receptor. The retromer is composed of two subcomplexes, one containing SNX1 and forming a membrane-bound coat, the other comprising VPS26, VPS29 and VPS35 and being cargo-selective. In yeast, an additional sorting nexin - Vps17p - is a component of the membrane bound coat. It remains unclear whether the mammalian retromer requires a functional equivalent of Vps17p. Here, we have used an RNAi loss-of-function screen to examine whether any of the other 30 mammalian sorting nexins are required for retromer-mediated endosome-to-trans-Golgi-network retrieval of the cation-independent mannose-6-phosphate receptor. Using this screen, we identified two proteins, SNX5 and SNX6, that, when suppressed, induced a phenotype similar to that observed upon suppression of known retromer components. Whereas SNX5 and SNX6 colocalised with SNX1 on early endosomes, in immunoprecipitation experiments only SNX6 appeared to exist in a complex with SNX1. Interestingly, suppression of SNX5 and/or SNX6 resulted in a significant loss of SNX1, an effect that seemed to result from post-translational regulation of the SNX1 level. Such data suggest that SNX1 and SNX6 exist in a stable, endosomally associated complex that is required for retromer-mediated retrieval of the cation-independent mannose-6-phosphate receptor. SNX5 and SNX6 may therefore constitute functional equivalents of Vps17p in mammals.

Supplementary material available online at

No abstract