Retromer is a membrane-associated heteropentameric coat complex that functions in the endosome-to-Golgi retrieval of the cation-independent mannose-6-phosphate receptor, the Wntless protein and other membrane proteins of physiological significance. Retromer comprises two functional subcomplexes: the cargo-selective subcomplex is a trimer of the VPS35, VPS29, VPS26 proteins, whereas the sorting nexin proteins, Snx1 and Snx2 function to tubulate the endosomal membrane. Unlike the sorting nexins, which contain PtdIns3P-binding PX domains, the cargo-selective VPS35/29/26 complex has no lipid-binding domains and its recruitment to the endosomal membrane remains mechanistically uncharacterised. In this study we show that the VPS35/29/26 complex interacts with the small GTPase Rab7 and requires Rab7 for its recruitment to the endosome. We show that the Rab7K157N mutant that causes the peripheral neuropathy, Charcot-Marie-Tooth disease, does not interact with the VPS35/29/26 complex, resulting in a weakened association with the membrane. We have also identified a novel retromer-interacting protein, TBC1D5, which is a member of the Rab GAP family of proteins that negatively regulates VPS35/29/26 recruitment and causes Rab7 to dissociate from the membrane. We therefore propose that recruitment of the cargo-selective VPS35/29/26 complex is catalysed by Rab7 and inhibited by the Rab-GAP protein, TBC1D5.

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.

Research Article 3703 IntroductionEndosomal protein sorting has a vital role in a number of physiologically important processes including antigen presentation, macromolecular nutrient uptake, growth factor receptor signaling and downregulation, autophagy and lysosome biogenesis (for reviews, see Sadowski et al., 2009;Saksena and Emr, 2009;Sann et al., 2009;Seaman, 2008;Lee et al., 2008). Recent studies of inherited diseases have identified several examples of genes encoding proteins that function in endosomal protein sorting that, when mutated, result in a range of pathologies. A notable example is hereditary spastic paraplegias (HSP), the hallmark of which is a selective distal axonopathy. There is a striking localisation of many of the HSP-encoded proteins to the endosome, including the microtubule-severing protein spastin, the ubiquitin-ligase-interacting protein spartin, and NIPA1, a membrane protein that mediates bone morphogenic protein signaling at the endosome (Tsang et al., 2009; for a review, see Salinas et al., 2008). Despite this concentration of HSP proteins at endosomes, in most cases their function is unknown.Much of the core machinery that carries out endosomal protein sorting is conserved in evolution, for example, the retromer complex (for reviews, see Attar and Cullen, 2009;Verges, 2008;Collins, 2008;Bonifacino and Hurley, 2008). Retromer mediates endosometo-Golgi retrieval of lysosomal and vacuolar hydrolase receptors (e.g. the cation-independent mannose 6 phosphate receptor, CIMPR) along with other physiologically significant membrane proteins including wntless, which functions in WNT secretion, and SORL1, a protein that is genetically linked to late-onset Alzheimer's disease (Arighi et al., 2004;Seaman, 2004;Eaton, 2008;Nielsen et al., 2007;Rogaeva et al., 2007).The retromer complex was first identified in yeast where it comprises five proteins encoded by vacuolar protein sorting (VPS) genes. The heteropentameric retromer complex can be functionally dissected into two subcomplexes: a cargo-selective complex formed from a conserved trimer of Vps35p, Vps29p and Vps26p and a 'structural complex' formed from a dimer of the sorting nexin (SNX) proteins Vps5p and Vps17p (Seaman et al., 1998). In mammals, SNX1, SNX2 with SNX5 and SNX6 provide the 'structural' role and can tubulate membranes through the C-terminal Bin, amphiphysin and Rvs (BAR) domains present in these proteins (Carlton et al., 2004;Wassmer et al., 2007). Additionally, SNX5 and SNX6 interact with the microtubule cytoskeleton via the p150glued protein that binds to dynein, thereby linking endosomal protein sorting to microtubules (Wassmer et al., 2009;Hong et al., 2009).The interaction between the SNX component of retromer and p150glued is an example of how retromer-interacting proteins facilitate retromer in mediating endosome-to-Golgi retrieval. In yeast, the SNX3 homologue Grd19p binds to Ftr1p to sort Ftr1p into the retromer pathway (Strochlic et al., 2008). In mammalian cells, the EPS15 homology domain protein, EHD1, interacts ...

The retromer complex is a conserved endosomal protein sorting complex that sorts membrane proteins into nascent endosomal tubules. The recognition of membrane proteins is mediated by the cargo-selective retromer complex, a stable trimer of the Vps35 (vacuolar protein sorting 35), Vps29 and Vps26 proteins. We have recently reported that the cargo-selective retromer complex associates with the WASH (Wiskott-Aldrich syndrome homologue) complex, a multimeric protein complex that regulates tubule dynamics at endosomes. In the present study, we show that the retromer-WASH complex interaction occurs through the long unstructured 'tail' domain of the WASH complex-Fam21 protein binding to Vps35, an interaction that is necessary and sufficient to target the WASH complex to endosomes. The Fam21-tail also binds to FKBP15 (FK506-binding protein 15), a protein associated with ulcerative colitis, to mediate the membrane association of FKBP15. Elevated Fam21-tail expression inhibits the association of the WASH complex with retromer, resulting in increased cytoplasmic WASH complex. Additionally, overexpression of the Fam21-tail results in cell-spreading defects, implicating the activity of the WASH complex in regulating the mobilization of membrane into the endosome-to-cell surface pathway.

Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids.

SummaryVARP is a Rab32/38 effector that also binds to the endosomal/lysosomal R-SNARE VAMP7. VARP binding regulates VAMP7 participation in SNARE complex formation and can therefore influence VAMP7-mediated membrane fusion events. Mutant versions of VARP that cannot bind Rab32:GTP, designed on the basis of the VARP ankyrin repeat/Rab32:GTP complex structure described here, unexpectedly retain endosomal localization, showing that VARP recruitment is not dependent on Rab32 binding. We show that recruitment of VARP to the endosomal membrane is mediated by its direct interaction with VPS29, a subunit of the retromer complex, which is involved in trafficking from endosomes to the TGN and the cell surface. Transport of GLUT1 from endosomes to the cell surface requires VARP, VPS29, and VAMP7 and depends on the direct interaction between VPS29 and VARP. Finally, we propose that endocytic cycling of VAMP7 depends on its interaction with VARP and, consequently, also on retromer.

Clathrin-coated vesicles (CCVs) facilitate the transport of cargo between the trans-Golgi network, endosomes, and the plasma membrane. This study presents the first comparative proteomics investigation of CCVs. A CCV-enriched fraction was isolated from HeLa cells and a “mock CCV” fraction from clathrin-depleted cells. We used a combination of 2D difference gel electrophoresis and isobaric tags for relative and absolute quantification (iTRAQ) in conjunction with mass spectrometry to analyze and compare the two fractions. In total, 63 bona fide CCV proteins were identified, including 28 proteins whose association with CCVs had not previously been established. These include numerous post-Golgi SNAREs; subunits of the AP-3, retromer, and BLOC-1 complexes; lysosomal enzymes; CHC22; and five novel proteins of unknown function. The strategy outlined in this paper should be widely applicable as a means of distinguishing genuine organelle components from contaminants.

Tapasin is an integral component of the peptide-loading complex (PLC) important for efficient peptide loading onto MHC class I molecules. We investigated the function of the tapasin-related protein, TAPBPR. Like tapasin, TAPBPR is widely expressed, IFN-γ-inducible, and binds to MHC class I coupled with β2-microglobulin in the endoplasmic reticulum. In contrast to tapasin, TAPBPR does not bind ERp57 or calreticulin and is not an integral component of the PLC. β2-microglobulin is essential for the association between TAPBPR and MHC class I. However, the association between TAPBPR and MHC class I occurs in the absence of a functional PLC, suggesting peptide is not required. Expression of TAPBPR decreases the rate of MHC class I maturation through the secretory pathway and prolongs the association of MHC class I on the PLC. The TAPBPR: MHC class I complex trafficks through the Golgi apparatus, demonstrating a function of TAPBPR beyond the endoplasmic reticulum/ cis-Golgi. The identification of TAPBPR as an additional component of the MHC class I antigen-presentation pathway demonstrates that mechanisms controlling MHC class I expression remain incompletely understood.