Wnt proteins are lipid modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a novel retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective sub-complex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.

SummaryRetromer is a protein assembly that plays a central role in orchestrating export of transmembrane-spanning cargo proteins from endosomes into retrieval pathways destined for the Golgi apparatus and the plasma membrane [1]. Recently, a specific mutation in the retromer component VPS35, VPS35(D620N), has linked retromer dysfunction to familial autosomal dominant and sporadic Parkinson disease [2, 3]. However, the effect of this mutation on retromer function remains poorly characterized. Here we established that in cells expressing VPS35(D620N) there is a perturbation in endosome-to-TGN transport but not endosome-to-plasma membrane recycling, which we confirm in patient cells harboring the VPS35(D620N) mutation. Through comparative stable isotope labeling by amino acids in cell culture (SILAC)-based analysis of wild-type VPS35 versus the VPS35(D620N) mutant interactomes, we establish that the major defect of the D620N mutation lies in the association to the actin-nucleating Wiskott-Aldrich syndrome and SCAR homolog (WASH) complex. Moreover, using isothermal calorimetry, we establish that the primary defect of the VPS35(D620N) mutant is a 2.2 ± 0.5-fold decrease in affinity for the WASH complex component FAM21. These data define the primary molecular defect in retromer assembly that arises from the VPS35(D620N) mutation and, by revealing functional effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in Parkinson disease.

In order to achieve coordinated growth and patterning during development, cells must communicate with one another, sending and receiving signals that regulate their activities. Such developmental signals can be soluble, bound to the extracellular matrix, or tethered to the surface of adjacent cells. Cells can also signal by releasing exosomes -extracellular vesicles containing bioactive molecules such as RNA, DNA and enzymes. Recent work has suggested that exosomes can also carry signalling proteins, including ligands of the Notch receptor and secreted proteins of the Hedgehog and WNT families. Here, we describe the various types of exosomes and their biogenesis. We then survey the experimental strategies used so far to interfere with exosome formation and critically assess the role of exosomes in developmental signalling.

The endosomal network is an organized array of intracellular, membranous compartments that function as sorting sites for endosomal and biosynthetic cargo. The fate of endocytic cargo is reliant upon interactions with a number of molecularly distinct sorting complexes, which tightly control the relationship between sorting of their respective cargo and the physical process of membrane re-scuplturing required for the formation of transport carries. One such complex, retromer, mediates retrograde transport from endosomes to the trans-Golgi network (TGN). Disregulation of retromer has been implicated in a host of disease states including late-onset Alzheimer's. Rather than give a broad overview of retromer biology, here we aim to outline the recent advances in understanding this complex, focussing on the involvement of both clathrin and the cytoskeleton in retromer function. The retrograde trafficking complex retromer was first identified in yeast (1). Yeast retromer mediates the retrieval of numerous cargos from a pre-vacuole compartment to the trans-Golgi network (TGN; 2-7), including Vps10p (3), a vacuolar hydrolase receptor that delivers carboxypeptidase Y to the vacuole, and via an association with the cargo adaptor Grd19/sorting nexin 3p (Snx3p), the iron transporter Fet3p-Ftr1p (7). The yeast retromer comprises five subunits and is composed of two subcomplexes (1). Vps26p, Vps29p and Vps35p form one subcomplex, which recruits cargo via an association between Vps35p and a sorting motif located within the cytoplasmic tail of cargo -this has led to this subcomplex being referred to as the 'cargo-selective subcomplex'. The second subcomplex comprises the SNXs, Vps5p and Vps17p, which dimerize via interactions in their C-termini (8). The N-terminus of Vps5p interacts with the cargo-selective subcomplex, and the association of Vps17p with Vps5p enhances this interaction.The mammalian retromer is more complex and comprises a VPS26-VPS29-VPS35 trimer (including two isoforms of VPS26), and gene duplication appears to have resulted in two Vps5p's, SNX1 and SNX2, and two (possibly three) Vps17p's, SNX5 and SNX6 (and possibly SNX32) (9-12). All the SNXs share a common domain architecture defined by an N-terminal phosphoinositidebinding PX domain (which in most cases binds the early endosomal phosphoinositide phosphatidylinositol 3-phosphate (PtdIns(3)P) but can also associate with other phosphoinositides, e.g. phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)) (13,14). Thirty-three mammalian SNXs have been annotated, subdivided into three families -SNX-BARs, SNX-PXs and SNX-others (15). The retromer SNXs belong to the SNX-BAR subfamily, and are capable of sensing and driving membrane curvature resulting in the formation of membrane tubules. This suggests that the association of SNX1/SNX2 and SNX5/SNX6/SNX32 with the cargo-selective subcomplex couples cargo recognition with membrane deformation. That said, the low affinity of this interaction has raised questions of whether during retromer evolution the pentameric com...

Retromer is a protein assembly that orchestrates the sorting of transmembrane cargo proteins into endosome-to-Golgi and endosome-to-plasma-membrane transport pathways. Here, we have employed quantitative proteomics to define the interactome of human VPS35, the core retromer component. This has identified a number of new interacting proteins, including ankyrin-repeat domain 50 (ANKRD50), seriologically defined colon cancer antigen 3 (SDCCAG3) and VPS9-ankyrin-repeat protein (VARP, also known as ANKRD27). Depletion of these proteins resulted in trafficking defects of retromer-dependent cargo, but differential and cargo-specific effects suggested a surprising degree of functional heterogeneity in retromer-mediated endosome-to-plasma-membrane sorting. Extending this, suppression of the retromer-associated WASH complex did not uniformly affect retromer cargo, thereby confirming cargo-specific functions for retromer-interacting proteins. Further analysis of the retromer–VARP interaction identified a role for retromer in endosome-to-melanosome transport. Suppression of VPS35 led to mistrafficking of the melanogenic enzymes, tyrosinase and tryrosine-related protein 1 (Tyrp1), establishing that retromer acts in concert with VARP in this trafficking pathway. Overall, these data reveal hidden complexities in retromer-mediated sorting and open up new directions in our molecular understanding of this essential sorting complex.