Following endocytosis and entry into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are alternatively retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multi-protein complex which orchestrates cargo retrieval and recycling and importantly, is biochemically and functionally distinct to the established retromer pathway. Composed of a heterotrimer of DSCR3, C16orf62 and VPS29, and bearing striking similarity with retromer, we have called this complex ‘retriever’. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to the CCC and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5β1-integrin. Through quantitative proteomic analysis we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, which require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major new endosomal retrieval and recycling pathway.

The sorting nexin 27 (SNX27)-retromer complex is a major regulator of endosome-to-plasma membrane recycling of transmembrane cargos that contain a PSD95, Dlg1, zo-1 (PDZ)-binding motif. Here we describe the core interaction in SNX27-retromer assembly and its functional relevance for cargo sorting. Crystal structures and NMR experiments reveal that an exposed β-hairpin in the SNX27 PDZ domain engages a groove in the arrestin-like structure of the vacuolar protein sorting 26A (VPS26A) retromer subunit. The structure establishes how the SNX27 PDZ domain simultaneously binds PDZ-binding motifs and retromer-associated VPS26. Importantly, VPS26A binding increases the affinity of the SNX27 PDZ domain for PDZ-binding motifs by an order of magnitude, revealing cooperativity in cargo selection. With disruption of SNX27 and retromer function linked to synaptic dysfunction and neurodegenerative disease, our work provides the first step, to our knowledge, in the molecular description of this important sorting complex, and more broadly describes a unique interaction between a PDZ domain and an arrestin-like fold.

ORP5 and ORP8, members of the oxysterol-binding protein (OSBP)-related proteins (ORP) family, are endoplasmic reticulum membrane proteins implicated in lipid trafficking. ORP5 and ORP8 are reported to localize to endoplasmic reticulum–plasma membrane junctions via binding to phosphatidylinositol-4-phosphate (PtdIns(4)P), and act as a PtdIns(4)P/phosphatidylserine counter exchanger between the endoplasmic reticulum and plasma membrane. Here we provide evidence that the pleckstrin homology domain of ORP5/8 via PtdIns(4,5)P 2, and not PtdIns(4)P binding mediates the recruitment of ORP5/8 to endoplasmic reticulum–plasma membrane contact sites. The OSBP-related domain of ORP8 can extract and transport multiple phosphoinositides in vitro, and knocking down both ORP5 and ORP8 in cells increases the plasma membrane level of PtdIns(4,5)P 2 with little effect on PtdIns(4)P. Overall, our data show, for the first time, that phosphoinositides other than PtdIns(4)P can also serve as co-exchangers for the transport of cargo lipids by ORPs.

Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr-sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins.endosome | protein crystallography | X-ray scattering | membrane trafficking T he cell-surface levels of signaling and adhesion receptors, nutrient transporters, ion channels, and many other proteins are tightly regulated by opposing endocytic, exocytic, and endosomal recycling transport pathways. The selective sorting of these transmembrane proteins is the consequence of their interaction with essential adaptor proteins via conserved motifs present in their cytosolic tails, generally based on short linear amino acid sequences such as the Yxxϕ, DxxLL, and [DE]xxxL [LI] motifs (where ϕ is any bulky hydrophobic side-chain, and x is any residue) recognized by clathrin adaptors (1-3). The first identified sorting signal was the Asn-Pro-Xaa-Tyr (NPxY) motif, initially described in the LDL receptor (LDLR) isolated from patients suffering from familial hypercholesterolemia, where mutation of the sequence results in defective internalization and cholesterol uptake (4). The recent structure of the autosomal recessive hypercholesterolemia protein (ARH) phosphotyrosinebinding (PTB) domain in complex with the LDLR intracellular domain (ICD) provides the molecular basis of LDLR recognition and internalization within clathrin-coated pits by endocytic adaptor proteins (5).Although little is known about the sorting sequences and mechanisms required for competing endosome-to-cell surface recycling pathways, emerging evidence shows that the NPxY motif not only is vital to internalization but also aids cargo recycling via organelle-specific recognition by the endosomal protein, sorting nexin 17 (SNX17). SNX17 is a member of the Phox-homology (PX) domain-containing protein family and has been shown to be a critical regulator of endosomal sorting and cell-surface recycling of several essential cargo molecules. These include P-selectin (6-8), amyloid precursor protein (APP) (9, 10), integrins (11,12), and members of the LDL receptor family (13-17) ...

Following endocytosis, the fates of receptors, channels, and other transmembrane proteins are decided via specific endosomal sorting pathways, including recycling to the cell surface for continued activity. Two distinct phox-homology (PX)-domain-containing proteins, sorting nexin (SNX) 17 and SNX27, are critical regulators of recycling from endosomes to the cell surface. In this study we demonstrate that SNX17, SNX27, and SNX31 all possess a novel 4.1/ ezrin/radixin/moesin (FERM)-like domain. SNX17 has been shown to bind to Asn-Pro-Xaa-Tyr (NPxY) sequences in the cytoplasmic tails of cargo such as LDL receptors and the amyloid precursor protein, and we find that both SNX17 and SNX27 display similar affinities for NPxY sorting motifs, suggesting conserved functions in endosomal recycling. Furthermore, we show for the first time that all three proteins are able to bind the Ras GTPase through their FERM-like domains. These interactions place the PX-FERM-like proteins at a hub of endosomal sorting and signaling processes. Studies of the SNX17 PX domain coupled with cellular localization experiments reveal the mechanistic basis for endosomal localization of the PX-FERM-like proteins, and structures of SNX17 and SNX27 determined by small angle X-ray scattering show that they adopt non-self-assembling, modular structures in solution. In summary, this work defines a novel family of proteins that participate in a network of interactions that will impact on both endosomal protein trafficking and compartment specific Ras signaling cascades. P hox-homology (PX) domain-containing proteins are a diverse family of proteins implicated in many protein trafficking processes, and there is emerging recognition of their importance in cell signaling (1, 2). The PX domain binds phosphatidylinositol phospholipids (PIPs) to mediate localization to subcellular membranous compartments for regulation of cargo transport and processing. Most PX proteins also contain a variety of other functional modules including Ras-association (RA) and PSD-95/discs large/zona occludens (PDZ) domains. Thus PX proteins can function as scaffolds that facilitate spatiotemporal assembly of membrane trafficking and signaling complexes.The PX-protein sorting nexin 17 (SNX17) is important for endosomal sorting of transmembrane proteins from endosomes to the cell surface. Identified cargo molecules include the lowdensity lipoprotein receptor (LDLR), and other members of the LDLR family including LDLR-related protein 1 (LRP1), suggesting an important role in lipid metabolism (3-5). SNX17 also regulates the trafficking of P-selectin (6) and FEEL-1 (7) and associates with cytosolic factors Krit1 (8) and Kif1B (9). All of these proteins have been found to bind SNX17 via a conserved AsnPro-Xaa-Tyr (NPxY) sequence motif, but the molecular basis of this interaction is unknown. Recent data indicate an important role for SNX17 in trafficking of the amyloid precursor protein (APP) central to Alzheimer's disease (AD) (10). As the LDLR family, in particular LRP1, have also bee...

Isothermal titration calorimetry (ITC) is a biophysical technique for measuring the formation and dissociation of molecular complexes and has become an invaluable tool in many branches of science from cell biology to food chemistry. By measuring the heat absorbed or released during bond formation, ITC provides accurate, rapid, and label-free measurement of the thermodynamics of molecular interactions. In this review, we survey the recent literature reporting the use of ITC and have highlighted a number of interesting studies that provide a flavour of the diverse systems to which ITC can be applied. These include measurements of protein-protein and protein-membrane interactions required for macromolecular assembly, analysis of enzyme kinetics, experimental validation of molecular dynamics simulations, and even in manufacturing applications such as food science. Some highlights include studies of the biological complex formed by Staphylococcus aureus enterotoxin C3 and the murine T-cell receptor, the mechanism of membrane association of the Parkinson's disease-associated protein α-synuclein, and the role of non-specific tannin-protein interactions in the quality of different beverages. Recent developments in automation are overcoming limitations on throughput imposed by previous manual procedures and promise to greatly extend usefulness of ITC in the future. We also attempt to impart some practical advice for getting the most out of ITC data for those researchers less familiar with the method.

Background: RGS-PX proteins are regulators of signaling and trafficking within the endosomal system. Results: A structural basis for membrane interactions of RGS-PX proteins is established. Conclusion: The four mammalian paralogues display different membrane interaction properties. Significance: RGS-PX proteins possess a conserved functional architecture in all eukaryotes.

The COMMD proteins are a conserved family of proteins with central roles in intracellular membrane trafficking and transcription. They form oligomeric complexes with each other and act as components of a larger assembly called the CCC complex, which is localized to endosomal compartments and mediates the transport of several transmembrane cargos. How these complexes are formed however is completely unknown. Here, we have systematically characterised the interactions between human COMMD proteins, and determined structures of COMMD proteins using X-ray crystallography and X-ray scattering to provide insights into the underlying mechanisms of homo- and heteromeric assembly. All COMMD proteins possess an α-helical N-terminal domain, and a highly conserved C-terminal domain that forms a tightly interlocked dimeric structure responsible for COMMD-COMMD interactions. The COMM domains also bind directly to components of CCC and mediate non-specific membrane association. Overall these studies show that COMMD proteins function as obligatory dimers with conserved domain architectures.