A loss-of-function screen reveals SNX5 and SNX6 as potential components of the mammalian retromer

Abstract

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.

The retromer component SNX6 interacts with dynactin p150Glued and mediates endosome-to-TGN transport

“…Depletion of SNX6 did not have any effect on the membrane localization of SNX2, indicating that SNX2's association with membranes does not require SNX6 (Figure 3C, m-p). In contrast, consistent with previous reports [26], very weak if any SNX1 signals were detected in SNX6-depleted cells ( Figure 3C, e-h), suggesting that SNX1 might be stabilized via complexing with SNX6. Moreover, although depletion of PI(3)P caused dissociation of SNX1, SNX2 and SNX6 from the membrane, it did not weaken SNX1's interaction with SNX5 or SNX6 ( Figure 3D), indicating that the interactions between SNXs do not require their membrane association.…”

“…Consistent with previous reports [26], we observed colocalization of SNX6 with SNX1-labeled vesicles and tubulovesicular structures (Supplementary information, Movie S1). To confirm that SNX6 localizes to early endosomes, we also performed live imaging of HeLa cells co-expressing YFP-EEA1 and mCherry-SNX6.…”

“…Confocal immunofluorescence microscopy showed that CI-MPR distribution was unchanged upon SNX6 R68A mutant expression ( Figure 5A, d-f), possibly because the cytosolic mutant's affinity for SNX1 is much lower than that of WT SNX6 ( Figure 4B), which is abundant in cells. Since SNX6 binds to SNX1, and depletion of both proteins causes CI-MPR dispersal [26], we reasoned that suppressing protein levels of both SNX1 and SNX6 would decrease the intracellular concentrations of these retromer components to such a low level that they become limiting factors for the transport machinery required for CI-MPR retrieval (Supplementary information, Figure S5). To examine SNX6's role in endosome-to-TGN transport, we depleted SNX1/6 (Supplementary information, Figure S5) in HeLa cells by RNAi and attempted to rescue the disruption of CI-MPR retrieval by WT or mutant SNX6 proteins expressed at low levels.…”

“…SNX1 and SNX2 are mammalian orthologs of Vps5p [15,25], whereas there is no obvious sequence homolog of Vps17p in mammals. Most recently, an RNAi loss-of-function screen in HeLa cells identified SNX5 and SNX6 as putative components of the SNX subcomplex [26].…”

Opposing Activities of the Snx3-Retromer Complex and ESCRT Proteins Mediate Regulated Cargo Sorting at a Common Endosome

“…Since then, retromer has been implicated in a broad range of protein-sorting pathways and has most recently been shown to be required for the maintenance of proper Wnt secretion in the development of both Caenorhabditis elegans and Drosophila melanogaster (Belenkaya et al, 2008;Franch-Marro et al, 2008;Pan et al, 2008;Port et al, 2008;Yang et al, 2008), underscoring its importance in retrograde transport. In yeast retromer is composed of five proteins that assemble into two subcomplexes: Vps5 and Vps17 (Snx1/ Snx2 and Snx5/Snx6 in mammals) and Vps26, Vps29, and Vps35 (and the homologous proteins in metazoans; Seaman et al, 1998;Haft et al, 2000;Wassmer et al, 2007). Vps5 and Vps17 are members of the sorting nexin family of proteins and are responsible for localizing the retromer complex to tubular endosomal membranes via the concerted action of their BAR (Bin/Amphiphysin/RVS) and PX (phox homology) domains (Carlton et al, 2004;Seet and Hong, 2006).…”