A global analysis of SNX27–retromer assembly and cargo specificity reveals a function in glucose and metal ion transport
TL;DR: The data identify the SNX27–retromer as a major endosomal recycling hub required to maintain cellular nutrient homeostasis and establish that direct interaction of theSNX27 PDZ domain with the retromer subunit VPS26 is necessary and sufficient to prevent lysosomal entry of SNX 27 cargo.
Abstract: The PDZ-domain-containing sorting nexin 27 (SNX27) promotes recycling of internalized transmembrane proteins from endosomes to the plasma membrane by linking PDZ-dependent cargo recognition to retromer-mediated transport. Here, we employed quantitative proteomics of the SNX27 interactome and quantification of the surface proteome of SNX27- and retromer-suppressed cells to dissect the assembly of the SNX27 complex and provide an unbiased global view of SNX27-mediated sorting. Over 100 cell surface proteins, many of which interact with SNX27, including the glucose transporter GLUT1, the Menkes disease copper transporter ATP7A, various zinc and amino acid transporters, and numerous signalling receptors, require SNX27-retromer to prevent lysosomal degradation and maintain surface levels. Furthermore, we establish that direct interaction of the SNX27 PDZ domain with the retromer subunit VPS26 is necessary and sufficient to prevent lysosomal entry of SNX27 cargo. Our data identify the SNX27-retromer as a major endosomal recycling hub required to maintain cellular nutrient homeostasis.
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TL;DR: The current understanding of the organization and functions of the endocytic pathway, differences across species, and the process of endosome maturation are summarized.
390 citations
TL;DR: The increasing body of evidence that implicates an ancient evolutionary conserved complex, termed "retromer," as a master conductor in the complex orchestration of multiple cargo-sorting events within the endosomal network is discussed.
Abstract: The endosomal network comprises an interconnected network of membranous compartments whose primary function is to receive, dissociate, and sort cargo that originates from the plasma membrane and the biosynthetic pathway. A major challenge in cell biology is to achieve a thorough molecular description of how this network operates, and in so doing, how defects contribute to the etiology and pathology of human disease. We discuss the increasing body of evidence that implicates an ancient evolutionary conserved complex, termed "retromer," as a master conductor in the complex orchestration of multiple cargo-sorting events within the endosomal network.
371 citations
Cites background from "A global analysis of SNX27–retromer..."
...2012), and other proteins that associate with CSC, including sorting nexin-3 (SNX3), SNX27, and the retromer SNX-BARs, have been implicated in cargo recognition (Parks et al. 2001; Heydorn et al. 2004; Strochlic et al. 2007; Temkin et al. 2011; Steinberg et al. 2013)....
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...On the endosome membrane, the WASH complex activates the Arp2/3 complex, which mediates the nucleation of actin branching to a preexisting actin filament (Rotty et al. 2013)....
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...70 cargos require the presence of SNX27 for their recycling, many of which contain PDZ ligands, whereas others contain NPxY/NxxY sorting motifs (Steinberg et al. 2013)....
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...Consistent with this, SNX27 lies at the core of a multiprotein complex defined by the binding to retromer, and the independent association with the retromer SNX-BARs and the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) (Temkin et al. 2011; Steinberg et al. 2013), which by regulating the activity of the Arp2/3 complex mediates the formation of branched actin filaments on the endosomal network (Derivery et al. 2009; Gomez and Billadeau 2009)....
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...Through WASH-mediated actin polymerization a cargo-enriched endosome subdomain is assembled thereby avoiding ESCRT-mediated sorting into forming intraluminal vesicles....
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TL;DR: The PD-causing D620N mutation in VPS35 restricts Wash complex recruitment to endosomes, and reveals a novel role for the WASH complex in autophagosome formation, which is linked to several neurodegenerative diseases.
Abstract: Endosomal protein sorting controls the localization of many physiologically important proteins and is linked to several neurodegenerative diseases. VPS35 is a component of the retromer complex, which mediates endosome-to-Golgi retrieval of membrane proteins such as the cation-independent mannose 6-phosphate receptor. Furthermore, retromer is also required for the endosomal recruitment of the actin nucleation promoting WASH complex. The VPS35 D620N mutation causes a rare form of autosomal-dominant Parkinson's disease (PD). Here we show that this mutant associates poorly with the WASH complex and impairs WASH recruitment to endosomes. Autophagy is impaired in cells expressing PD-mutant VPS35 or lacking WASH. The autophagy defects can be explained, at least in part, by abnormal trafficking of the autophagy protein ATG9A. Thus, the PD-causing D620N mutation in VPS35 restricts WASH complex recruitment to endosomes, and reveals a novel role for the WASH complex in autophagosome formation.
366 citations
TL;DR: The discovery of sequence-specific sorting motifs and the identification of cargo adaptors and associated coat complexes have begun to uncover the highly orchestrated nature of endosomal cargo recycling, thereby providing new insight into the function and (patho)physiology of this process.
Abstract: Newly endocytosed integral cell surface proteins are typically either directed for degradation or subjected to recycling back to the plasma membrane The sorting of integral cell surface proteins, including signalling receptors, nutrient transporters, ion channels, adhesion molecules and polarity markers, within the endolysosomal network for recycling is increasingly recognized as an essential feature in regulating the complexities of physiology at the cell, tissue and organism levels Historically, endocytic recycling has been regarded as a relatively passive process, where the majority of internalized integral proteins are recycled via a nonspecific sequence-independent 'bulk membrane flow' pathway Recent work has increasingly challenged this view The discovery of sequence-specific sorting motifs and the identification of cargo adaptors and associated coat complexes have begun to uncover the highly orchestrated nature of endosomal cargo recycling, thereby providing new insight into the function and (patho)physiology of this process
309 citations
TL;DR: An overview of the ultrastructural characteristics and membrane organization of endosomal compartments, along with their organizing machineries is provided.
Abstract: Live-cell imaging reveals the endolysosomal system as a complex and highly dynamic network of interacting compartments. Distinct types of endosomes are discerned by kinetic, molecular, and morphological criteria. Although none of these criteria, or combinations thereof, can capture the full complexity of the endolysosomal system, they are extremely useful for experimental purposes. Some membrane domain specializations and specific morphological characteristics can only be seen by ultrastructural analysis after preparation for electron microscopy (EM). Immuno-EM allows a further discrimination of seemingly identical compartments by their molecular makeup. In this review we provide an overview of the ultrastructural characteristics and membrane organization of endosomal compartments, along with their organizing machineries.
278 citations
Cites background from "A global analysis of SNX27–retromer..."
...SNX27 has been recently shown to interact with the retromer subunit VPS26 via its PDZ domain, and such interaction is necessary and sufficient to prevent lysosomal entry of SNX27 cargo (such as, e.g., GLUT1, the copper transporter ATP7A) (Steinberg et al. 2013)....
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..., GLUT1, the copper transporter ATP7A) (Steinberg et al. 2013)....
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References
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TL;DR: The hypothesis that retromer performs a selective function in endosome-to-Golgi transport, mediating retrieval of the cation-independent MPR (CI-MPR), but not furin is supported.
Abstract: fEndosome-to-Golgi retrieval of the mannose 6-phosphate receptor (MPR) is required for lysosome biogenesis. Currently, this pathway is poorly understood. Analyses in yeast identified a complex of proteins called “retromer” that is essential for endosome-to-Golgi retrieval of the carboxypeptidase Y receptor Vps10p. Retromer comprises five distinct proteins: Vps35p, 29p, 26p, 17p, and 5p, which are conserved in mammals. Here, we show that retromer is required for the efficient retrieval of the cation-independent MPR (CI-MPR). Cells lacking mammalian VPS26 fail to retrieve the CI-MPR, resulting in either rapid degradation of or mislocalization to the plasma membrane. We have localized mVPS26 to multivesicular body endosomes by electron microscopy, and through the use of CD8 reporter protein constructs have examined the effect of loss of mVPS26 upon the trafficking of membrane proteins that cycle between the endosome and the Golgi. The data presented here support the hypothesis that retromer performs a selective function in endosome-to-Golgi transport, mediating retrieval of the CI-MPR, but not furin.
603 citations
TL;DR: Observations indicate that retromer prevents the delivery of the CI-MPR to lysosomes, probably by sequestration into endosome-derived tubules from where the receptor returns to the TGN.
Abstract: The cation-independent mannose 6-phosphate receptor (CI-MPR) mediates sorting of lysosomal hydrolase precursors from the TGN to endosomes. After releasing the hydrolase precursors into the endosomal lumen, the unoccupied receptor returns to the TGN for further rounds of sorting. Here, we show that the mammalian retromer complex participates in this retrieval pathway. The hVps35 subunit of retromer interacts with the cytosolic domain of the CI-MPR. This interaction probably occurs in an endosomal compartment, where most of the retromer is localized. In particular, retromer is associated with tubular–vesicular profiles that emanate from early endosomes or from intermediates in the maturation from early to late endosomes. Depletion of retromer by RNA interference increases the lysosomal turnover of the CI-MPR, decreases cellular levels of lysosomal hydrolases, and causes swelling of lysosomes. These observations indicate that retromer prevents the delivery of the CI-MPR to lysosomes, probably by sequestration into endosome-derived tubules from where the receptor returns to the TGN.
590 citations
TL;DR: This review reports both the genomic structure and function of each transporter as well as intra-species comparative genomic analysis of some of these transporters and introduces some pathophysiological roles of thesetransporters in human.
Abstract: Glucose is the major energy source for mammalian cells as well as an important substrate for protein and lipid synthesis. Mammalian cells take up glucose from extracellular fluid into the cell through two families of structurallyrelated glucose transporters. The facilitative glucose transporter family (solute carriers SLC2A, protein symbol GLUT) mediates a bidirectional and energy-independent process of glucose transport in most tissues and cells, while the NaM(+)/glucose cotransporter family (solute carriers SLC5A, protein symbol SGLT) mediates an active, Na(+)-linked transport process against an electrochemical gradient. The GLUT family consists of thirteen members (GLUT1-12 and HMIT). Phylogenetically, the members of the GLUT family are split into three classes based on protein similarities. Up to now, at least six members of the SGLT family have been cloned (SGLT1-6). In this review, we report both the genomic structure and function of each transporter as well as intra-species comparative genomic analysis of some of these transporters. The affinity for glucose and transport kinetics of each transporter differs and ranges from 0.2 to 17mM. The ability of each protein to transport alternative substrates also differs and includes substrates such as fructose and galactose. In addition, the tissue distribution pattern varies between species. There are different regulation mechanisms of these transporters. Characterization of transcriptional control of some of the gene promoters has been investigated and alternative promoter usage to generate different protein isoforms has been demonstrated. We also introduce some pathophysiological roles of these transporters in human.
531 citations
TL;DR: A reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes) is presented.
Abstract: The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.
486 citations
TL;DR: The data support an evolutionarily conserved function for SNX1 from yeast to mammals and provide functional insight into the molecular mechanisms underlying lipid-mediated protein targeting and tubular-based protein sorting.
451 citations