Sequence-dependent cargo recognition by SNX-BARs mediates retromer-independent transport of CI-MPR.
ABSTRACT: Endosomal recycling of transmembrane proteins requires sequence-dependent recognition of motifs present within their intracellular cytosolic domains. In this study, we have reexamined the role of retromer in the sequence-dependent endosome-to-trans-Golgi network (TGN) transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Although the knockdown or knockout of retromer does not perturb CI-MPR transport, the targeting of the retromer-linked sorting nexin (SNX)-Bin, Amphiphysin, and Rvs (BAR) proteins leads to a pronounced defect in CI-MPR endosome-to-TGN transport. The retromer-linked SNX-BAR proteins comprise heterodimeric combinations of SNX1 or SNX2 with SNX5 or SNX6 and serve to regulate the biogenesis of tubular endosomal sorting profiles. We establish that SNX5 and SNX6 associate with the CI-MPR through recognition of a specific WLM endosome-to-TGN sorting motif. From validating the CI-MPR dependency of SNX1/2-SNX5/6 tubular profile formation, we provide a mechanism for coupling sequence-dependent cargo recognition with the biogenesis of tubular profiles required for endosome-to-TGN transport. Therefore, the data presented in this study reappraise retromer's role in CI-MPR transport.
Project description:The retromer complex, which recycles the cation-independent mannose 6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), is thought to consist of a cargo-selective VPS26-VPS29-VPS35 trimer and a membrane-deforming subunit of sorting nexin (SNX)-Bin, Amphyphysin, and Rvs (BAR; SNX-BAR) proteins. In this study, we demonstrate that heterodimers of the SNX-BAR proteins, SNX1, SNX2, SNX5, and SNX6, are the cargo-selective elements that mediate the retrograde transport of CI-MPR from endosomes to the TGN independently of the core retromer trimer. Using quantitative proteomics, we also identify the IGF1R, among more potential cargo, as another SNX5 and SNX6 binding receptor that recycles through SNX-BAR heterodimers, but not via the retromer trimer, in a ligand- and activation-dependent manner. Overall, our data redefine the mechanics of retromer-based sorting and call into question whether retromer indeed functions as a complex of SNX-BAR proteins and the VPS26-VPS29-VPS35 trimer.
Project description:Early 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 p150(glued) 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.
Project description:Endocytic recycling of internalized transmembrane proteins is essential for many important physiological processes. Recent studies have revealed that retromer-related Sorting Nexin family (SNX)-Bin/Amphiphysin/Rvs (BAR) proteins can directly recognize cargoes like cation-independent mannose 6-phosphate receptor (CI-MPR) and Insulin-like growth factor 1 receptor (IGF1R); however, it remains poorly understood how SNX-BARs select specific cargo proteins and whether they recognize additional ligands. Here, we discovered that the binding between SNX-BARs and CI-MPR or IGF1R is mediated by the phox-homology (PX) domain of SNX5 or SNX6 and a bipartite motif, termed SNX-BAR-binding motif (SBM), in the cargoes. Using this motif, we identified over 70 putative SNX-BAR ligands, many of which play critical roles in apoptosis, cell adhesion, signal transduction, or metabolite homeostasis. Remarkably, SNX-BARs could cooperate with both SNX27 and retromer in the recycling of ligands encompassing the SBM, PDZ-binding motif, or both motifs. Overall, our studies establish that SNX-BARs function as a direct cargo-selecting module for a large set of transmembrane proteins transiting the endosome, in addition to their roles in phospholipid recognition and biogenesis of tubular structures.
Project description:DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.
Project description:The retromer is a heteropentameric complex that associates with the cytosolic face of endosomes and mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network. The mammalian retromer complex comprises a sorting nexin dimer composed of a still undefined combination of SNX1, SNX2, SNX5 and SNX6, and a cargo-recognition trimer composed of Vps26, Vps29 and Vps35. The SNX subunits contain PX and BAR domains that allow binding to PI(3)P enriched, highly curved membranes of endosomal vesicles and tubules, while Vps26, Vps29 and Vps35 have arrestin, phosphoesterase and alpha-solenoid folds, respectively. Recent studies have implicated retromer in a broad range of physiological, developmental and pathological processes, underscoring the critical nature of retrograde transport mediated by this complex.
Project description:Sorting nexin 1 (SNX1), a peripheral membrane protein, has previously been shown to regulate the cell-surface expression of the human epidermal growth factor receptor [Kurten, Cadena and Gill (1996) Science 272, 1008-1010]. Searches of human expressed sequence tag databases with SNX1 revealed eleven related human cDNA sequences, termed SNX2 to SNX12, eight of them novel. Analysis of SNX1-related sequences in the Saccharomyces cerevisiae genome clearly shows a greatly expanded SNX family in humans in comparison with yeast. On the basis of the predicted protein sequences, all members of this family of hydrophilic molecules contain a conserved 70-110-residue Phox homology (PX) domain, referred to as the SNX-PX domain. Within the SNX family, subgroups were identified on the basis of the sequence similarities of the SNX-PX domain and the overall domain structure of each protein. The members of one subgroup, which includes human SNX1, SNX2, SNX4, SNX5 and SNX6 and the yeast Vps5p and YJL036W, all contain coiled-coil regions within their large C-terminal domains and are found distributed in both membrane and cytosolic fractions, typical of hydrophilic peripheral membrane proteins. Localization of the human SNX1 subgroup members in HeLa cells transfected with the full-length cDNA species revealed a similar intracellular distribution that in all cases overlapped substantially with the early endosome marker, early endosome autoantigen 1. The intracellular localization of deletion mutants and fusions with green fluorescent protein showed that the C-terminal regions of SNX1 and SNX5 are responsible for their endosomal localization. On the basis of these results, the functions of these SNX molecules are likely to be unique to endosomes, mediated in part by interactions with SNX-specific C-terminal sequences and membrane-associated determinants.
Project description:The endosomal trafficking pathways are essential for many cellular activities. They are also important targets by many intracellular pathogens. Key regulators of the endosomal trafficking include the retromer complex and sorting nexins (SNXs). Chlamydia trachomatis effector protein IncE directly targets the retromer components SNX5 and SNX6 and suppresses retromer-mediated transport, but the exact mechanism has remained unclear. We present the crystal structure of the PX domain of SNX5 in complex with IncE, showing that IncE binds to a highly conserved hydrophobic groove of SNX5. The unique helical hairpin of SNX5/6 is essential for binding, explaining the specificity of SNX5/6 for IncE. The SNX5/6-IncE interaction is required for cellular localization of IncE and its inhibitory function. Mechanistically, IncE inhibits the association of CI-MPR cargo with retromer-containing endosomal subdomains. Our study provides new insights into the regulation of retromer-mediated transport and illustrates the intricate competition between host and pathogens in controlling cellular trafficking.
Project description:Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.
Project description:Protein dimerization and oligomerization is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to transfer information in signaling, transcriptional, and membrane trafficking events. Here we show that a combination of cell-free protein expression, a proximity based interaction assay (AlphaScreen), and single-molecule fluorescence allow rapid mapping of homo- and hetero-oligomerization of proteins. We have applied this approach to the family of BAR domain-containing sorting nexin (SNX-BAR) proteins, which are essential regulators of membrane trafficking and remodeling in all eukaryotes. Dimerization of BAR domains is essential for creating a concave structure capable of sensing and inducing membrane curvature. We have systematically mapped 144 pairwise interactions between the human SNX-BAR proteins and generated an interaction matrix of preferred dimerization partners for each family member. We find that while nine SNX-BAR proteins are able to form homo-dimers, several including the retromer-associated SNX1, SNX2, and SNX5 require heteromeric interactions for dimerization. SNX2, SNX4, SNX6, and SNX8 show a promiscuous ability to bind other SNX-BAR proteins and we also observe a novel interaction with the SNX3 protein which lacks the BAR domain structure.
Project description:Sorting nexin 5 (SNX5), a member of sorting nexin family, plays an important role in membrane trafficking, including the retrograde trafficking of the cation independent mannose 6-phosphate receptor (CI-M6PR) and macropinocytosis. Using ESI-LCMS/MS analysis, we confirmed that SNX5 serine 226 is phosphorylated. Since SNX5 forms heterodimers with SNX1 or SNX2, we examined the effect of phosphorylation at S226 on the heterodimer formations. Wild-type and mutants of SNX5, in which S226 was mutated to a glutamic acid or an alanine, were expressed in 8505C cells. In pull-down assays using SNX5 as bait, only the S226E mutant failed to precipitate both SNX1 and SNX2. Confocal microscopy data indicated that the wild type and S226A mutant were colocalized with SNX1 and SNX2 in endosomes, but the S226E was not. SNX5 and SNX6 support each other's functions and are involved with CI-M6PR retrograde trafficking. In SNX5 and SNX6 double knockdown cells, CI-M6PR was dispersed and colocalized with the endosomal marker EEA1. In a rescue experiment using SNX5 mutants, the S226A rescued CI-M6PR localization, similar to control cells, but S226E did not. Furthermore, the decrease in the uptake of dextran by macropinocytosis in SNX5 knockdown cells was recovered by the expression of rescue-wild type or S226A mutant, but not by the rescue-S226E mutant. These observations indicate that SNX5 constitutive phosphorylation that mimics the mutant S226E decreases the active SNX5 in these cells. The phosphorylation of SNX5 regulates the dimerization with SNX1 or SNX2, and this suggests that it controls membrane trafficking and protein sorting.