Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health.

Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson's and Alzheimer's diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform an integrated multi-omics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify widespread changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide a holistic view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.

SNX27-Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling.

Coat complexes coordinate cargo recognition through cargo adaptors with biogenesis of transport carriers during integral membrane protein trafficking. Here, we combine biochemical, structural, and cellular analyses to establish the mechanistic basis through which SNX27-Retromer, a major endosomal cargo adaptor, couples to the membrane remodeling endosomal SNX-BAR sorting complex for promoting exit 1 (ESCPE-1). In showing that the SNX27 FERM (4.1/ezrin/radixin/moesin) domain directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the SNX1/SNX2 subunits of ESCPE-1, we propose a handover model where SNX27-Retromer captured cargo proteins are transferred into ESCPE-1 transport carriers to promote endosome-to-plasma membrane recycling. By revealing that assembly of the SNX27:Retromer:ESCPE-1 coat evolved in a stepwise manner during early metazoan evolution, likely reflecting the increasing complexity of endosome-to-plasma membrane recycling from the ancestral opisthokont to modern animals, we provide further evidence of the functional diversification of yeast pentameric Retromer in the recycling of hundreds of integral membrane proteins in metazoans.

SNX3-retromer requires an evolutionary conserved MON2:DOPEY2:ATP9A complex to mediate Wntless sorting and Wnt secretion.

Wntless transports Wnt morphogens to the cell surface and is required for Wnt secretion and morphogenic gradients formation. Recycling of endocytosed Wntless requires the sorting nexin-3 (SNX3)-retromer-dependent endosome-to-Golgi transport pathway. Here we demonstrate the essential role of SNX3-retromer assembly for Wntless transport and report that SNX3 associates with an evolutionary conserved endosome-associated membrane re-modelling complex composed of MON2, DOPEY2 and the putative aminophospholipid translocase, ATP9A. In vivo suppression of Ce-mon-2, Ce-pad-1 or Ce-tat-5 (respective MON2, DOPEY2 and ATP9A orthologues) phenocopy a loss of SNX3-retromer function, leading to enhanced lysosomal degradation of Wntless and a Wnt phenotype. Perturbed Wnt signalling is also observed upon overexpression of an ATPase-inhibited TAT-5(E246Q) mutant, suggesting a role for phospholipid flippase activity during SNX3-retromer-mediated Wntless sorting. Together, these findings provide in vitro and in vivo mechanistic details to describe SNX3-retromer-mediated transport during Wnt secretion and the formation of Wnt-morphogenic gradients.

Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin.

The endo-lysosomal network serves an essential role in determining the fate of endocytosed transmembrane proteins and their associated proteins and lipids. Sorting nexins (SNXs) play a central role in the functional organisation of this network. Comprising over 30 proteins in humans, SNXs are classified into sub-groups based on the presence of additional functional domains. Sorting nexin-20 (SNX20) and sorting nexin-21 (SNX21) comprise the SNX-PXB proteins. The presence of a predicted protein-protein interaction domain, termed the PX-associated B (PXB) domain, has led to the proposal that they function as endosome-associated scaffolds. Here, we used unbiased quantitative proteomics to define the SNX21 interactome. We reveal that the N-terminal extension of SNX21 interacts with huntingtin (Htt) whereas the PXB domain appears to associate with septins, a family of cytoskeletal- and membrane-associated proteins. In establishing that these interactions are sufficient for SNX21 to recruit Htt and septins on to an endosomal population, we reveal a scaffolding function for this sorting nexin. Our work paves the way for a more-detailed mechanistic analysis of the role(s) of the SNX-PXB proteins in endosomal biology.

Retromer associates with the cytoplasmic amino-terminus of polycystin-2.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic human disease, with around 12.5 million people affected worldwide. ADPKD results from mutations in either PKD1 or PKD2, which encode the atypical G-protein coupled receptor polycystin-1 (PC1) and the transient receptor potential channel polycystin-2 (PC2), respectively. Although altered intracellular trafficking of PC1 and PC2 is an underlying feature of ADPKD, the mechanisms which govern vesicular transport of the polycystins through the biosynthetic and endosomal membrane networks remain to be fully elucidated. Here, we describe an interaction between PC2 and retromer, a master controller for the sorting of integral membrane proteins through the endo-lysosomal network. We show that association of PC2 with retromer occurs via a region in the PC2 cytoplasmic amino-terminal domain, independently of the retromer-binding Wiskott-Aldrich syndrome and scar homologue (WASH) complex. Based on observations that retromer preferentially interacts with a trafficking population of PC2, and that ciliary levels of PC1 are reduced upon mutation of key residues required for retromer association in PC2, our data are consistent with the identification of PC2 as a retromer cargo protein.This article has an associated First Person interview with the first author of the paper.

Sequence-dependent cargo recognition by SNX-BARs mediates retromer-independent transport of CI-MPR.

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.

Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling.

Following endocytosis into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multiprotein complex that orchestrates cargo retrieval and recycling and, importantly, is biochemically and functionally distinct from the established retromer pathway. We have called this complex 'retriever'; it is a heterotrimer composed of DSCR3, C16orf62 and VPS29, and bears striking similarity to retromer. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to CCC (CCDC93, CCDC22, COMMD) and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5ß1 integrin. Through quantitative proteomic analysis, we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, that require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major endosomal retrieval and recycling pathway.

Atypical parkinsonism-associated retromer mutant alters endosomal sorting of specific cargo proteins.

The retromer complex acts as a scaffold for endosomal protein complexes that sort integral membrane proteins to various cellular destinations. The retromer complex is a heterotrimer of VPS29, VPS35, and VPS26. Two of these paralogues, VPS26A and VPS26B, are expressed in humans. Retromer dysfunction is associated with neurodegenerative disease, and recently, three VPS26A mutations (p.K93E, p.M112V, and p.K297X) were discovered to be associated with atypical parkinsonism. Here, we apply quantitative proteomics to provide a detailed description of the retromer interactome. By establishing a comparative proteomic methodology, we identify how this interactome is perturbed in atypical parkinsonism-associated VPS26A mutants. In particular, we describe a selective defect in the association of VPS26A (p.K297X) with the SNX27 cargo adaptor. By showing how a retromer mutant leads to altered endosomal sorting of specific PDZ ligand-containing cargo proteins, we reveal a new mechanism for perturbed endosomal cargo sorting in atypical parkinsonism.

A defect in the retromer accessory protein, SNX27, manifests by infantile myoclonic epilepsy and neurodegeneration.

The composition of the neuronal cell surface dictates synaptic plasticity and thereby cognitive development. This remodeling of the synapses is governed by the endocytic network which internalize transmembrane proteins, then sort them back to the cell surface or carry them to the lysosome for degradation. The multi-protein retromer complex is central to this selection, capturing specific transmembrane proteins and remodeling the cell membrane to form isolated cargo-enriched transport carriers. We investigated a consanguineous family with four patients who presented in infancy with intractable myoclonic epilepsy and lack of psychomotor development. Using exome analysis, we identified a homozygous deleterious mutation in SNX27, which encodes sorting nexin 27, a retromer cargo adaptor. In western analysis of patient fibroblasts, the encoded mutant protein was expressed at an undetectable level when compared with a control sample. The patients' presentation and clinical course recapitulate that reported for the SNX27 knock-out mouse. Since the cargo proteins for SNX27-mediated sorting include subunits of ionotropic glutamate receptors and endosome-to-cell surface synaptic insertion of AMPA receptors is severely perturbed in SNX27(-/-) neurons, it is proposed that at least part of the neurological aberrations observed in the patients is attributed to defective sorting of ionotropic glutamate receptors. SNX27 deficiency is now added to the growing list of neurodegenerative disorders associated with retromer dysfunction.

Microtubule motors mediate endosomal sorting by maintaining functional domain organization.

Many microtubule motors have been shown to couple to endosomal membranes. These motors include dynein in addition to many different kinesin family members. Sorting nexins (SNXs) are central to the organization and function of endosomes. These proteins can actively shape endosomal membranes and couple directly or indirectly to the minus-end microtubule motor dynein. Motor proteins acting on endosomes drive their motility, dictate their morphology and affect cargo segregation. We have used well-characterized members of the SNX family to elucidate motor coupling using high-resolution light microscopy coupled with depletion of specific microtubule motors. Endosomal domains labelled with SNX1, SNX4 and SNX8 couple to discrete combinations of dynein and kinesin motors. These specific combinations govern the structure and motility of each SNX-coated membrane in addition to the segregation of distinct functional endosomal subdomains. Taken together, our data show that these key features of endosome dynamics are governed by the same set of opposing microtubule motors. Thus, microtubule motors help to define the mosaic layout of endosomes that underpins cargo sorting.