Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome.

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two sub-assemblies: Retriever composed of VPS35L, VPS26C, and VPS29; and the CCC complex which contains twelve subunits: COMMD1-COMMD10 and the coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy, and in silico predictions, we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilized by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a 16th subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG-Syt1-SV2 plasma membrane nanocluster for synaptic vesicle entry.

The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live-cell super-resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor-binding-deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG-synaptotagmin-1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1-SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A- and BoNT/E-induced neurointoxication as quantified by SNAP-25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.

FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.

To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.

PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4.

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both steady-state and induced mitophagy. Disruption of the phosphatase activity of PPTC7 does not influence BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homoeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-BNIP3/NIX and PPTC7-FBXL4 interactions and their disruption interferes with both BNIP3/NIX degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts BNIP3/NIX-induced mitophagy.

Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2.

Munc18-interacting proteins (Mints) are multidomain adaptors that regulate neuronal membrane trafficking, signaling, and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We found that a short acidic α-helical motif within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells, mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a (Syntaxin1a) proteins simultaneously in vitro, we find that they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ADP-ribosylation factor (ARF3/ARF4) small GTPases and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 (tight junction-associated protein 1) provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provide insights into the diversity of interactions mediated by the Mint family and show that Mints may help facilitate a key trigger point in SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) complex assembly and vesicle fusion.

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.

A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.

The AP2 adaptor complex (alpha, beta2, sigma2, and mu2 subunits) crosslinks the endocytic clathrin scaffold to PtdIns4,5P(2)-containing membranes and transmembrane protein cargo. In the "locked" cytosolic form, AP2's binding sites for the two endocytic motifs, YxxPhi on the C-terminal domain of mu2 (C-mu2) and [ED]xxxL[LI] on sigma2, are blocked by parts of beta2. Using protein crystallography, we show that AP2 undergoes a large conformational change in which C-mu2 relocates to an orthogonal face of the complex, simultaneously unblocking both cargo-binding sites; the previously unstructured mu2 linker becomes helical and binds back onto the complex. This structural rearrangement results in AP2's four PtdIns4,5P(2)- and two endocytic motif-binding sites becoming coplanar, facilitating their simultaneous interaction with PtdIns4,5P(2)/cargo-containing membranes. Using a range of biophysical techniques, we show that the endocytic cargo binding of AP2 is driven by its interaction with PtdIns4,5P(2)-containing membranes.

ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.

Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.

The PDLIM family of actin-associated proteins and their emerging role in membrane trafficking.

The PDZ and LIM domain (PDLIM) proteins are associated with the actin cytoskeleton and have conserved in roles in metazoan actin organisation and function. They primarily function as scaffolds linking various proteins to actin and its binding partner α-actinin via two conserved domains; an N-terminal postsynaptic density 95, discs large and zonula occludens-1 (PDZ) domain, and either single or multiple C-terminal LIN-11, Isl-1 and MEC-3 (LIM) domains in the actinin-associated LIM protein (ALP)- and Enigma-related proteins, respectively. While their role in actin organisation, such as in stress fibres or in the Z-disc of muscle fibres is well known, emerging evidence also suggests a role in actin-dependent membrane trafficking in the endosomal system. This is mediated by a recently identified interaction with the sorting nexin 17 (SNX17) protein, an adaptor for the trafficking complex Commander which is itself intimately linked to actin-directed formation of endosomal recycling domains. In this review we focus on the currently understood structural basis for PDLIM function. The PDZ domains mediate direct binding to distinct classes of PDZ-binding motifs (PDZbms), including α-actinin and other actin-associated proteins, and a highly specific interaction with the type III PDZbm such as the one found in the C-terminus of SNX17. The structures of the LIM domains are less well characterised and how they engage with their ligands is completely unknown. Despite the lack of experimental structural data, we find that recently developed machine learning-based structure prediction methods provide insights into their potential interactions and provide a template for further studies of their molecular functions.

EhC2B, a C2 domain-containing protein, promotes erythrophagocytosis in Entamoeba histolytica via actin nucleation.

Remodelling of the actin cytoskeleton in response to external stimuli is obligatory for many cellular processes in the amoebic cell. A rapid and local rearrangement of the actin cytoskeleton is required for the development of the cellular protrusions during phagocytosis, trogocytosis, migration, and invasion. Here, we demonstrated that EhC2B, a C2 domain-containing protein, is an actin modulator. EhC2B was first identified as an effector of EhRab21 from E. histolytica. In vitro interaction studies including GST pull-down, fluorescence-based assay and ITC also corroborated with our observation. In the amoebic trophozoites, EhC2B accumulates at the pseudopods and the tips of phagocytic cups. FRAP based studies confirmed the recruitment and dynamics of EhC2B at the phagocytic cup. Moreover, we have shown the role of EhC2B in erythrophagocytosis. It is well known that calcium-dependent signal transduction is essential for the cytoskeletal dynamics during phagocytosis in the amoebic parasite. Using liposome pelleting assay, we demonstrated that EhC2B preferentially binds to the phosphatidylserine in the presence of calcium. The EhC2B mutants defective in calcium or lipid-binding failed to localise beneath the plasma membrane. The cells overexpressing these mutants have also shown a significant reduction in erythrophagocytosis. The role of EhC2B in erythrophagocytosis and pseudopod formation was also validated by siRNA-based gene knockdown approach. Finally, with the help of in vitro nucleation assay using fluorescence spectroscopy and total internal reflection fluorescence microscopy, we have established that EhC2B is an actin nucleator. Collectively, based on the results from the study, we propose that EhC2B acts like a molecular bridge which promotes membrane deformation via its actin nucleation activity during the progression of the phagocytic cup in a calcium-dependent manner.

Towards a molecular understanding of endosomal trafficking by Retromer and Retriever.

Endosomes are dynamic intracellular compartments that control the sorting of a constant stream of different transmembrane cargos either for ESCRT-mediated degradation or for egress and recycling to compartments such as the Golgi and the plasma membrane. The recycling of cargos occurs within tubulovesicular membrane domains and is facilitated by peripheral membrane protein machineries that control both membrane remodelling and selection of specific transmembrane cargos. One of the primary sorting machineries is the Retromer complex, which controls the recycling of a large array of different cargo molecules in cooperation with various sorting nexin (SNX) adaptor proteins. Recently a Retromer-like complex was also identified that controls plasma membrane recycling of cargos including integrins and lipoprotein receptors. Termed "Retriever," this complex uses a different SNX family member SNX17 for cargo recognition, and cooperates with the COMMD/CCDC93/CCDC22 (CCC) complex to form a larger assembly called "Commander" to mediate endosomal trafficking. In this review we focus on recent advances that have begun to provide a molecular understanding of these two distantly related transport machineries.

Rational Design of Potent Peptide Inhibitors of the PD-1:PD-L1 Interaction for Cancer Immunotherapy.

Peptides have potential to be developed into immune checkpoint inhibitors, but the target interfaces are difficult to inhibit. Here, we explored an approach to mimic the binding surface of PD-1 to design inhibitors. Mimicking native PD-1 resulted in a mimetic with no activity. However, mimicking an affinity-optimized PD-1 resulted in the peptide mimetic MOPD-1 that displayed nanomolar affinity to PD-L1 and could inhibit PD-1:PD-L1 interactions in both protein- and cell-based assays. Mutagenesis and structural characterization using NMR spectroscopy and X-ray crystallography revealed that binding residues from the high affinity PD-1 are crucial for the bioactivity of MOPD-1. Furthermore, MOPD-1 was extremely stable in human serum and inhibited tumor growth in vivo, suggesting it has potential for use in cancer immunotherapy. The successful design of an inhibitor of PD-1:PD-L1 using the mimicry approach described herein illustrates the value of placing greater emphasis on optimizing the target interface before inhibitor design and is an approach that could have broader utility for the design of peptide inhibitors for other complex protein-protein interactions.

Enabling Efficient Folding and High-Resolution Crystallographic Analysis of Bracelet Cyclotides.

Cyclotides have attracted great interest as drug design scaffolds because of their unique cyclic cystine knotted topology. They are classified into three subfamilies, among which the bracelet subfamily represents the majority and comprises the most bioactive cyclotides, but are the most poorly utilized in drug design applications. A long-standing challenge has been the very low in vitro folding yields of bracelets, hampering efforts to characterize their structures and activities. Herein, we report substantial increases in bracelet folding yields enabled by a single point mutation of residue Ile-11 to Leu or Gly. We applied this discovery to synthesize mirror image enantiomers and used quasi-racemic crystallography to elucidate the first crystal structures of bracelet cyclotides. This study provides a facile strategy to produce bracelet cyclotides, leading to a general method to easily access their atomic resolution structures and providing a basis for development of biotechnological applications.

A variable undecad repeat domain in cavin1 regulates caveola formation and stability.

Caveolae are plasma membrane invaginations involved in transport, signalling and mechanical membrane sensing in metazoans. Their formation depends upon multiple interactions between membrane-embedded caveolins, lipids and cytosolic cavin proteins. Of the four cavin family members, only cavin1 is strictly required for caveola formation. Here, we demonstrate that an eleven residue (undecad) repeat sequence (UC1) exclusive to cavin1 is essential for caveolar localization and promotes membrane remodelling through binding to phosphatidylserine. In the notochord of mechanically stimulated zebrafish embryos, the UC1 domain is required for caveolar stability and resistance to membrane stress. The number of undecad repeats in the cavin1 UC1 domain varies throughout evolution, and we find that an increased number also correlates with increased caveolar stability. Lastly, we show that the cavin1 UC1 domain induces dramatic remodelling of the plasma membrane when grafted into cavin2 suggesting an important role in membrane sculpting. Overall, our work defines a novel conserved cavin1 modular domain that controls caveolar assembly and stability.

SNT-1 Functions as the Ca2+ Sensor for Tonic and Evoked Neurotransmitter Release in Caenorhabditis Elegans.

Synaptotagmin-1 (Syt1) binds Ca2+ through its tandem C2 domains (C2A and C2B) and triggers Ca2+-dependent neurotransmitter release. Here, we show that snt-1, the homolog of mammalian Syt1, functions as the Ca2+ sensor for both tonic and evoked neurotransmitter release at the Caenorhabditis elegans neuromuscular junction. Mutations that disrupt Ca2+ binding in double C2 domains of SNT-1 significantly impaired tonic release, whereas disrupting Ca2+ binding in a single C2 domain had no effect, indicating that the Ca2+ binding of the two C2 domains is functionally redundant for tonic release. Stimulus-evoked release was significantly reduced in snt-1 mutants, with prolonged release latency as well as faster rise and decay kinetics. Unlike tonic release, evoked release was triggered by Ca2+ binding solely to the C2B domain. Moreover, we showed that SNT-1 plays an essential role in the priming process in different subpopulations of synaptic vesicles with tight or loose coupling to Ca2+ entry.SIGNIFICANCE STATEMENT We showed that SNT-1 in Caenorhabditis elegans regulates evoked neurotransmitter release through Ca2+ binding to its C2B domain in a similar way to Syt1 in the mouse CNS and the fly neuromuscular junction. However, the largely decreased tonic release in snt-1 mutants argues SNT-1 has a clamping function. Indeed, Ca2+-binding mutations in the C2 domains in SNT-1 significantly reduced the frequency of the miniature EPSC, indicating that SNT-1 also acts as a Ca2+ sensor for tonic release. Therefore, revealing the differential mechanisms between invertebrates and vertebrates will provide significant insights into our understanding how synaptic vesicle fusion is regulated.

Sorting nexin 27 (SNX27) regulates the trafficking and activity of the glutamine transporter ASCT2.

Alanine-, serine-, cysteine-preferring transporter 2 (ASCT2, SLC1A5) is responsible for the uptake of glutamine into cells, a major source of cellular energy and a key regulator of mammalian target of rapamycin (mTOR) activation. Furthermore, ASCT2 expression has been reported in several human cancers, making it a potential target for both diagnostic and therapeutic purposes. Here we identify ASCT2 as a membrane-trafficked cargo molecule, sorted through a direct interaction with the PDZ domain of sorting nexin 27 (SNX27). Using both membrane fractionation and subcellular localization approaches, we demonstrate that the majority of ASCT2 resides at the plasma membrane. This is significantly reduced within CrispR-mediated SNX27 knockout (KO) cell lines, as it is missorted into the lysosomal degradation pathway. The reduction of ASCT2 levels in SNX27 KO cells leads to decreased glutamine uptake, which, in turn, inhibits cellular proliferation. SNX27 KO cells also present impaired activation of the mTOR complex 1 (mTORC1) pathway and enhanced autophagy. Taken together, our data reveal a role for SNX27 in glutamine uptake and amino acid-stimulated mTORC1 activation via modulation of ASCT2 intracellular trafficking.

The Phox Homology (PX) Domain.

The phox-homology (PX) domain is a phosphoinositide-binding domain conserved in all eukaryotes and present in 49 human proteins. Proteins containing PX domains, many of which are also known as sorting nexins (SNXs), have a large variety of functions in membrane trafficking, cell signaling, and lipid metabolism in association with membranes of the secretory and endocytic system. In this review we discuss the structural basis for both canonical lipid interactions with the endosome-enriched lipid phosphatidylinositol-3-phosphate (PtdIns3P) as well as non-canonical lipids that promote membrane association. We also describe recent advances in defining the diverse mechanisms by which PX domains interact with other proteins including the retromer trafficking complex and proteins secreted by bacterial pathogens. Like other membrane interacting domains, the attachment of PX domain proteins to specific membranes is often facilitated by additional interactions that contribute to binding avidity, and we discuss this coincidence detection for several known examples.

Origins of PDZ Binding Specificity. A Computational and Experimental Study Using NHERF1 and the Parathyroid Hormone Receptor.

Na+/H+ exchanger regulatory factor-1 (NHERF1) is a scaffolding protein containing two PSD95/discs large protein/ZO1 (PDZ) domains that modifies the signaling, trafficking, and function of the parathyroid hormone receptor (PTHR), a family B G-protein-coupled receptor. PTHR and NHERF1 bind through a PDZ-ligand-recognition mechanism. We show that PTH elicits phosphorylation of Thr591 in the canonical -ETVM binding motif of PTHR. Conservative substitution of Thr591 with Cys does not affect PTH(1-34)-induced cAMP production or binding of PTHR to NHERF1. The findings suggested the presence of additional sites upstream of the PDZ-ligand motif through which the two proteins interact. Structural determinants outside the canonical NHERF1 PDZ-PTHR interface that influence binding have not been characterized. We used molecular dynamics (MD) simulation to predict residues involved in these interactions. Simulation data demonstrate that the negatively charged Glu side chains at positions -3, -5, and -6 upstream of the PDZ binding motif are involved in PDZ-PTHR recognition. Engineered mutant peptides representing the PTHR C-terminal region were used to measure the binding affinity with NHERF1 PDZ domains. Comparable micromolar affinities for peptides of different length were confirmed by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance. Binding affinities measured for Ala variants validate MD simulations. The linear relation between the change in enthalpy and entropy following Ala substitutions at upstream positions -3, -5, and -6 of the PTHR peptide provides a clear example of the thermodynamic compensation rule. Overall, our data highlight sequences in PTHR that contribute to NHERF1 interaction and can be altered to prevent phosphorylation-mediated inhibition.

Parkinson Disease-linked Vps35 R524W Mutation Impairs the Endosomal Association of Retromer and Induces α-Synuclein Aggregation.

Endosomal sorting is a highly orchestrated cellular process. Retromer is a heterotrimeric complex that associates with endosomal membranes and facilitates the retrograde sorting of multiple receptors, including the cation-independent mannose 6-phosphate receptor for lysosomal enzymes. The cycling of retromer on and off the endosomal membrane is regulated by a network of retromer-interacting proteins. Here, we find that Parkinson disease-associated Vps35 variant, R524W, but not P316S, is a loss-of-function mutation as marked by a reduced association with this regulatory network and dysregulation of endosomal receptor sorting. Expression of Vps35 R524W-containing retromer results in the accumulation of intracellular α-synuclein-positive aggregates, a hallmark of Parkinson disease. Overall, the Vps35 R524W-containing retromer has a decreased endosomal association, which can be partially rescued by R55, a small molecule previously shown to stabilize the retromer complex, supporting the potential for future targeting of the retromer complex in the treatment of Parkinson disease.