Global phosphoproteomics pinpoints uncharted Gcn2-mediated mechanisms of translational control.

The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the ß-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.

Spatially Distinct Pools of TORC1 Balance Protein Homeostasis.

The eukaryotic TORC1 kinase is a homeostatic controller of growth that integrates nutritional cues and mediates signals primarily from the surface of lysosomes or vacuoles. Amino acids activate TORC1 via the Rag GTPases that combine into structurally conserved multi-protein complexes such as the EGO complex (EGOC) in yeast. Here we show that Ego1, which mediates membrane-anchoring of EGOC via lipid modifications that it acquires while traveling through the trans-Golgi network, is separately sorted to vacuoles and perivacuolar endosomes. At both surfaces, it assembles EGOCs, which regulate spatially distinct pools of TORC1 that impinge on functionally divergent effectors: vacuolar TORC1 predominantly targets Sch9 to promote protein synthesis, whereas endosomal TORC1 phosphorylates Atg13 and Vps27 to inhibit macroautophagy and ESCRT-driven microautophagy, respectively. Thus, the coordination of three key regulatory nodes in protein synthesis and degradation critically relies on a division of labor between spatially sequestered populations of TORC1.

The autophagy machinery interacts with EBV capsids during viral envelope release.

Autophagy serves as a defense mechanism against intracellular pathogens, but several microorganisms exploit it for their own benefit. Accordingly, certain herpesviruses include autophagic membranes into their infectious virus particles. In this study, we analyzed the composition of purified virions of the Epstein-Barr virus (EBV), a common oncogenic γ-herpesvirus. In these, we found several components of the autophagy machinery, including membrane-associated LC3B-II, and numerous viral proteins, such as the capsid assembly proteins BVRF2 and BdRF1. Additionally, we showed that BVRF2 and BdRF1 interact with LC3B-II via their common protein domain. Using an EBV mutant, we identified BVRF2 as essential to assemble mature capsids and produce infectious EBV. However, BdRF1 was sufficient for the release of noninfectious viral envelopes as long as autophagy was not compromised. These data suggest that BVRF2 and BdRF1 are not only important for capsid assembly but together with the LC3B conjugation complex of ATG5-ATG12-ATG15L1 are also critical for EBV envelope release.

The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation.

Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.

Histone deacetylase inhibition mitigates fibrosis-driven disease progression in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disease caused by mutations in the gene encoding type VII collagen (C7). RDEB is associated with fibrosis, which is responsible for severe complications. The phenotypic variability observed in siblings with RDEB suggests that epigenetic modifications contribute to disease severity. Identifying epigenetic changes may help to uncover molecular mechanisms underlying RDEB pathogenesis and new therapeutic targets. OBJECTIVES: To investigate histone acetylation in RDEB skin and to explore histone deacetylase inhibitors (HDACi) as therapeutic molecules capable of counteracting fibrosis and disease progression in RDEB mice. METHODS: Acetylated histone levels were detected in human skin by immunofluorescence and in RDEB fibroblasts by enzyme-linked immunosorbent assay (ELISA). The effects of givinostat and valproic acid (VPA) on RDEB fibroblast fibrotic behaviour were assessed by a collagen-gel contraction assay, Western blot and immunocytofluorescence for α-smooth muscle actin, and ELISA for released transforming growth factor (TGF)-ß1. RNA sequencing was performed in HDACi- and vehicle-treated RDEB fibroblasts. VPA was systemically administered to RDEB mice and effects on overt phenotype were monitored. Fibrosis was investigated in the skin using histological and immunofluorescence analyses. Eye and tongue defects were examined microscopically. Mass spectrometry proteomics was performed on skin protein extracts from VPA-treated RDEB and control mice. RESULTS: Histone acetylation decreases in RDEB skin and primary fibroblasts. RDEB fibroblasts treated with HDACi lowered fibrotic traits, including contractility, TGF-ß1 release and proliferation. VPA administration to RDEB mice mitigated severe manifestations affecting the eyes and paws. These effects were associated with fibrosis inhibition. Proteomic analysis of mouse skin revealed that VPA almost normalized protein sets involved in protein synthesis and immune response, processes linked to the increased susceptibility to cancer and bacterial infections seen in people with RDEB. CONCLUSIONS: Dysregulated histone acetylation contributes to RDEB pathogenesis by facilitating the progression of fibrosis. Repurposing of HDACi could be considered for disease-modifying treatments in RDEB.

Recessive dystrophic epidermolysis bullosa (or 'RDEB') is a rare skin disease that affects fewer than 5,000 people in the USA. A similar number of people in Europe are affected. RDEB is caused by mutations in the gene that controls the production of a protein called 'type VII collagen' (or 'C7'). A shortage of C7 causes fragile skin that blisters. In severe forms of RDEB, wounds heal slowly and can even affect a person's life expectancy. Differences in the disease are common in people (even identical twins) with RDEB who have similar levels of C7. This suggests that how severe the disease is could be affected by molecular processes that control other genes. Understanding these processes may help us to find treatments for RDEB. This study was done in Italy, in collaboration with centres in Germany and Switzerland. We wanted to see whether a chemical modification called 'histone acetylation' (which influences gene activity) is different in RDEB and whether it can be targeted by a specific treatment. We found that histone acetylation is reduced in RDEB skin and in skin cells grown in the lab called 'fibroblasts'. When we increased histone acetylation in fibroblasts with two drugs called givinostat and valproic acid, the amount of scar tissue produced decreased. This is important because scar tissue can lead to severe symptoms. We carried out more experiments to study the effects of givinostat and valproic acid in mice with RDEB. We found that valproic acid reduces the severity of RDEB by decreasing the disease's harmful effects and reducing the amount of scar tissue. Our findings suggest that abnormal histone acetylation contributes to the scar tissue seen in RDEB. Our study shows that valproic acid could be useful in treating the scarring seen in RDEB and in reducing the effects of the disease. As this drug is used to treat other diseases, there could be potential for rapid repurposing of it for RDEB.

The mTOR pathway controls phosphorylation of BRAF at T401.

BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.

Downregulation of autophagy by Met30-mediated Atg9 ubiquitination.

Macroautophagy/autophagy is a highly conserved eukaryotic molecular process that facilitates the recycling of superfluous cytoplasmic materials, damaged organelles, and invading pathogens, resulting in proper cellular homeostasis and survival during stress conditions. Autophagy is stringently regulated at multiple stages, including control at transcriptional, translational, and posttranslational levels. In this work, we identified a mechanism by which regulation of autophagy is achieved through the posttranslational modification of Atg9. Here, we show that, in order to limit autophagy to a low, basal level during normal conditions, Atg9 is ubiquitinated and subsequently targeted for degradation in a proteasome-dependent manner through the action of the E3 ligase Met30. When cells require increased autophagy flux to respond to nutrient deprivation, the proteolysis of Atg9 is significantly reduced. Overall, this work reveals an additional layer of mechanistic regulation that allows cells to further maintain appropriate levels of autophagy and to rapidly induce this process in response to stress.

Control of RAB7 activity and localization through the retromer-TBC1D5 complex enables RAB7-dependent mitophagy.

Retromer is an endosomal multi-protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans-Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer-associated RAB7-specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer-dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin-mediated mitophagy.

Cilia-localized LKB1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney.

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.

Phosphorylation of mitochondrial matrix proteins regulates their selective mitophagic degradation.

Mitophagy is an important quality-control mechanism in eukaryotic cells, and defects in mitophagy correlate with aging phenomena and neurodegenerative disorders. It is known that different mitochondrial matrix proteins undergo mitophagy with very different rates but, to date, the mechanism underlying this selectivity at the individual protein level has remained obscure. We now present evidence indicating that protein phosphorylation within the mitochondrial matrix plays a mechanistic role in regulating selective mitophagic degradation in yeast via involvement of the Aup1 mitochondrial protein phosphatase, as well as 2 known matrix-localized protein kinases, Pkp1 and Pkp2. By focusing on a specific matrix phosphoprotein reporter, we also demonstrate that phospho-mimetic and nonphosphorylatable point mutations at known phosphosites in the reporter increased or decreased its tendency to undergo mitophagy. Finally, we show that phosphorylation of the reporter protein is dynamically regulated during mitophagy in an Aup1-dependent manner. Our results indicate that structural determinants on a mitochondrial matrix protein can govern its mitophagic fate, and that protein phosphorylation regulates these determinants.

A Dual-Acting Nitric Oxide Donor and Phosphodiesterase 5 Inhibitor Activates Autophagy in Primary Skin Fibroblasts.

Wound healing pathologies are an increasing problem in ageing societies. Chronic, non-healing wounds, which cause high morbidity and severely reduce the quality of life of affected individuals, are frequently observed in aged individuals and people suffering from diseases affected by the Western lifestyle, such as diabetes. Causal treatments that support proper wound healing are still scarce. Here, we performed expression proteomics to study the effects of the small molecule TOP-N53 on primary human skin fibroblasts and keratinocytes. TOP-N53 is a dual-acting nitric oxide donor and phosphodiesterase-5 inhibitor increasing cGMP levels to support proper wound healing. In contrast to keratinocytes, which did not exhibit global proteome alterations, TOP-N53 had profound effects on the proteome of skin fibroblasts. In fibroblasts, TOP-N53 activated the cytoprotective, lysosomal degradation pathway autophagy and induced the expression of the selective autophagy receptor p62/SQSTM1. Thus, activation of autophagy might in part be responsible for beneficial effects of TOP-N53.

Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition.

As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC-PCP-SILAC, we analyzed protein-protein interactions of hyperactive BRAFV600E and wild-type BRAF (BRAFWT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAFV600E resides in large complexes of higher molecular mass and activity, while BRAFWT is confined to smaller, slightly less active complexes. However, expression of oncogenic K-RasG12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAFWT into large, active complexes, whereas pharmacological inhibition of BRAFV600E has the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors.

Impaired lymphoid extracellular matrix impedes antibacterial immunity in epidermolysis bullosa.

Genetic loss of collagen VII causes recessive dystrophic epidermolysis bullosa (RDEB), a skin fragility disorder that, unexpectedly, manifests also with elevated colonization of commensal bacteria and frequent wound infections. Here, we describe an unprecedented systemic function of collagen VII as a member of a unique innate immune-supporting multiprotein complex in spleen and lymph nodes. In this complex, collagen VII specifically binds and sequesters the innate immune activator cochlin in the lumen of lymphoid conduits. In genetic mouse models, loss of collagen VII increased bacterial colonization by diminishing levels of circulating cochlin LCCL domain. Intraperitoneal injection of collagen VII, which restored cochlin in the spleen, but not in the skin, reactivated peripheral innate immune cells via cochlin and reduced bacterial skin colonization. Systemic administration of the cochlin LCCL domain was alone sufficient to diminish bacterial supercolonization of RDEB mouse skin. Human validation demonstrated that RDEB patients displayed lower levels of systemic cochlin LCCL domain with subsequently impaired macrophage response in infected wounds. This study identifies an intrinsic innate immune dysfunction in RDEB and uncovers a unique role of the lymphoid extracellular matrix in systemic defense against bacteria.

Skin proteomics - analysis of the extracellular matrix in health and disease.

INTRODUCTION: The skin protects the human body from external insults and regulates water and temperature homeostasis. A highly developed extracellular matrix (ECM) supports the skin and instructs its cell functions. Reduced functionality of the ECM is often associated with skin diseases that cause physical impairment and also have implications on social interactions and quality of life of affected individuals. AREAS COVERED: With a focus on the skin ECM we discuss how mass spectrometry (MS)-based proteomic approaches first contributed to establishing skin protein inventories and then facilitated elucidation of molecular functions and disease mechanisms. EXPERT OPINION: MS-based proteomic approaches have significantly contributed to our understanding of skin pathophysiology, but also revealed the challenges in assessing the skin ECM. The numerous posttranslational modifications of ECM proteins, like glycosylation, crosslinking, oxidation, and proteolytic maturation in disease settings can be difficult to tackle and remain understudied. Increased ease of handling of LC-MS/MS systems and automated/streamlined data analysis pipelines together with the accompanying increased usage of LC-MS/MS approaches will ensure that in the coming years MS-based proteomic approaches will continue to play a vital part in skin disease research. They will facilitate the elucidation of molecular disease mechanisms and, ultimately, identification of new druggable targets.

Combinatorial Omics Analysis Reveals Perturbed Lysosomal Homeostasis in Collagen VII-deficient Keratinocytes.

The extracellular matrix protein collagen VII is part of the microenvironment of stratified epithelia and critical in organismal homeostasis. Mutations in the encoding gene COL7A1 lead to the skin disorder dystrophic epidermolysis bullosa (DEB), are linked to skin fragility and progressive inflammation-driven fibrosis that facilitates aggressive skin cancer. So far, these changes have been linked to mesenchymal alterations, the epithelial consequences of collagen VII loss remaining under-addressed. As epithelial dysfunction is a principal initiator of fibrosis, we performed a comprehensive transcriptome and proteome profiling of primary human keratinocytes from DEB and control subjects to generate global and detailed images of dysregulated epidermal molecular pathways linked to loss of collagen VII. These revealed downregulation of interaction partners of collagen VII on mRNA and protein level, but also increased abundance of S100 pro-inflammatory proteins in primary DEB keratinocytes. Increased TGF-ß signaling because of loss of collagen VII was associated with enhanced activity of lysosomal proteases in both keratinocytes and skin of collagen VII-deficient individuals. Thus, loss of a single structural protein, collagen VII, has extra- and intracellular consequences, resulting in inflammatory processes that enable tissue destabilization and promote keratinocyte-driven, progressive fibrosis.

Influenza A Virus Induces Autophagosomal Targeting of Ribosomal Proteins.

Seasonal epidemics of influenza A virus are a major cause of severe illness and are of high socio-economic relevance. For the design of effective antiviral therapies, a detailed knowledge of pathways perturbed by virus infection is critical. We performed comprehensive expression and organellar proteomics experiments to study the cellular consequences of influenza A virus infection using three human epithelial cell lines derived from human lung carcinomas: A549, Calu-1 and NCI-H1299. As a common response, the type I interferon pathway was up-regulated upon infection. Interestingly, influenza A virus infection led to numerous cell line-specific responses affecting both protein abundance as well as subcellular localization. In A549 cells, the vesicular compartment appeared expanded after virus infection. The composition of autophagsomes was altered by targeting of ribosomes, viral mRNA and proteins to these double membrane vesicles. Thus, autophagy may support viral protein translation by promoting the clustering of the respective molecular machinery in autophagosomes in a cell line-dependent manner.

The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier.

Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrix-dependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression.

Cyclin O (Ccno) functions during deuterosome-mediated centriole amplification of multiciliated cells.

Mucociliary clearance and fluid transport along epithelial surfaces are carried out by multiciliated cells (MCCs). Recently, human mutations in Cyclin O (CCNO) were linked to severe airway disease. Here, we show that Ccno expression is restricted to MCCs and the genetic deletion of Ccno in mouse leads to reduced numbers of multiple motile cilia and characteristic phenotypes of MCC dysfunction including severe hydrocephalus and mucociliary clearance deficits. Reduced cilia numbers are caused by compromised generation of centrioles at deuterosomes, which serve as major amplification platform for centrioles in MCCs. Ccno-deficient MCCs fail to sufficiently generate deuterosomes, and only reduced numbers of fully functional centrioles that undergo maturation to ciliary basal bodies are formed. Collectively, this study implicates CCNO as first known regulator of deuterosome formation and function for the amplification of centrioles in MCCs.

The balance of Id3 and E47 determines neural stem/precursor cell differentiation into astrocytes.

Adult neural stem/precursor cells (NSPCs) of the subventricular zone (SVZ) are an endogenous source for neuronal replacement in CNS disease. However, adult neurogenesis is compromised after brain injury in favor of a glial cell fate, which is mainly attributed to changes in the NSPC environment. Yet, it is unknown how this unfavorable extracellular environment translates into a transcriptional program altering NSPC differentiation. Here, we show that genetic depletion of the transcriptional regulator Id3 decreased the number of astrocytes generated from SVZ-derived adult NSPCs in the cortical lesion area after traumatic brain injury. Cortical brain injury resulted in rapid BMP-2 and Id3 up-regulation in the SVZ stem cell niche. Id3(-/-) adult NSPCs failed to differentiate into BMP-2-induced astrocytes, while NSPCs deficient for the Id3-controlled transcription factor E47 readily differentiated into astrocytes in the absence of BMP-2. Mechanistically, E47 repressed the expression of several astrocyte-specific genes in adult NSPCs. These results identify Id3 as the BMP-2-induced transcriptional regulator, promoting adult NSPC differentiation into astrocytes upon CNS injury and reveal a molecular link between environmental changes and NSPC differentiation in the CNS after injury.

Retromer- and WASH-dependent sorting of nutrient transporters requires a multivalent interaction network with ANKRD50.

Retromer and the associated actin-polymerizing WASH complex are essential for the endocytic recycling of a wide range of integral membrane proteins. A hereditary Parkinson's-disease-causing point mutation (D620N) in the retromer subunit VPS35 perturbs retromer's association with the WASH complex and also with the uncharacterized protein ankyrin-repeat-domain-containing protein 50 (ANKRD50). Here, we firmly establish ANKRD50 as a new and essential component of the SNX27-retromer-WASH super complex. Depletion of ANKRD50 in HeLa or U2OS cells phenocopied the loss of endosome-to-cell-surface recycling of multiple transmembrane proteins seen upon suppression of SNX27, retromer or WASH-complex components. Mass-spectrometry-based quantification of the cell surface proteome of ANKRD50-depleted cells identified amino acid transporters of the SLC1A family, among them SLC1A4, as additional cargo molecules that depend on ANKRD50 and retromer for their endocytic recycling. Mechanistically, we show that ANKRD50 simultaneously engages multiple parts of the SNX27-retromer-WASH complex machinery in a direct and co-operative interaction network that is needed to efficiently recycle the nutrient transporters GLUT1 (also known as SLC2A1) and SLC1A4, and potentially many other surface proteins.