The retromer complex 1,2 is required for the sorting of acid hydrolases to lysosomes 3-7, transcytosis of the polymeric Ig receptor 8, Wnt gradient formation 9,10, iron transporter recycling 11, and processing of the amyloid precursor protein 12. Human retromer consists of two smaller complexes, the cargo recognition Vps26:Vps29:Vps35 heterotrimer, and a membrane-targeting heterodimer or homodimer of SNX1 and/or SNX2 13. The crystal structure of a Vps29:Vps35 subcomplex shows how the metallophosphoesterase-fold subunit Vps29 14,15 acts as a scaffold for the C-terminal half of Vps35. Vps35 forms a horseshoe-shaped right-handed α-helical solenoid whose concave face completely covers the metal-binding site of Vps29 and whose convex face exposes a series of hydrophobic interhelical grooves. Electron microscopy shows that the intact Vps26:Vps29:Vps35 complex is a stick-shaped, somewhat flexible, structure, ∼ 21 nm long. A hybrid structural model derived from crystal structures, electron microscopy, interaction studies, and bioinformatics shows that the α-solenoid fold extends the full length of Vps35, and that Vps26 is bound at the opposite end from Vps29. This extended structure presents multiple binding sites for the SNX complex and receptor cargo, and appears capable of flexing to conform to curved vesicular membranes.The retromer cargo recognition complex consists of the 38-kDa Vps26, 20-kDa Vps29, and 92-kDa Vps35 subunits. The structures of the two smaller subunits have been determined in isolation. Vps26 is a structural cousin of the arrestins 16, a family of trafficking proteins that directly bind to cell surface receptors and direct their internalization. Vps29 has a metallophosphoesterase fold 14,15 that can bind two metal ions. Compared to functional metallophosphoesterases, a key His that serves as a catalytic base in the 6To whom correspondence should be addressed:James H. Hurley, (301) 402−4703, fax (301) 480−0639; E-mail: hurley@helix.nih.gov. 2 These authors contributed equally. Author contributions A. L. R. and A. H. expressed and purified protein complexes, crystallized the Vps29:Vps35 C-terminal subcomplex, collected crystallographic data, and determined and refined the crystal structure; A. H. carried out phosphatase assays; R. R. and N. M. carried out immunoprecipitation and optical microscopy studies; G. E. and A. C. S. carried out and interpreted electron microscopy studies; A. V. K. carried out sequence analysis; and J. H. H., J. S. B., and A. C. S. designed the study. A. H. and A. L. R. contributed equally to this study. Here we take a structural approach to gain insight into the function of retromer. Author informationThe crystal structure of a Vps29:Vps35 subcomplex, containing the C-terminal 40% of the large Vps35 subunit, was determined at 2.8 Å resolution (Fig. 1a, Fig. S1, Table S1). The Cterminal portion of Vps35 consists of a single right-handed superhelix with a pitch of 12 Å and a total of 13 helices (Fig. 1a, S2). Vps35 resembles many other helical solenoid proteins, includ...

SUMMARY Retromer is a multi-protein complex that recycles transmembrane cargo from endosomes to the trans-Golgi network and the plasma membrane. Defects in retromer impair various cellular processes, and underlie some forms of Alzheimer’s disease and Parkinson’s disease. Although retromer was discovered over 15 years ago, the mechanisms for cargo recognition and recruitment to endosomes have remained elusive. Here we present an X-ray crystallographic analysis of a four-component complex comprising the VPS26 and VPS35 subunits of retromer, the sorting nexin SNX3, and a recycling signal from the divalent cation transporter DMT1-II. This analysis identifies a binding site for canonical recycling signals at the interface between VPS26 and SNX3. In addition, the structure highlights a network of cooperative interactions among the VPS subunits, SNX3 and cargo that couple signal-recognition to membrane recruitment.

Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs.