Functional architecture of the retromer cargo-recognition complex

Abstract

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...

A mechanism for retromer endosomal coat complex assembly with cargo

“…3D), and the second peptide identified spans residues 25-44 (Fig. 3D) and is contained within the minimal segment of VPS35 that assembles with VPS26 (19,20). These results confirm and extend a recent yeast two-hybrid study that reported that SNX3 can interact with the N-terminal half of VPS35 (21).…”

“…In solution, retromer is a ∼210-Å-long flexible rod (19), and our data demonstrate that it becomes initially anchored to the endosome membrane by interactions involving SNX3 and RAB7 at the N-terminal proximal region of VPS35. Mutations in yeast Vps35 that result in a cargo-specific sorting defect of Vps10 map to the C-terminal region of Vps35 (30), and our data show that this region in human retromer is responsible for recognition of DMT1-II (Fig.…”

“…The mechanism of retromer coat recruitment to the endosome is not known. RNAi-mediated knockdown of SNX3 and RAB7, and expression dominant interfering RAB7 proteins in cells, results in decreased endosome association of retromer (6,13,14,19), implicating them in this process. We began investigating the requirements for retromer membrane recruitment by assaying sedimentation of retromer with liposomes containing PtdIns3P, to which SNX3 binds (22).…”

Opposing Activities of the Snx3-Retromer Complex and ESCRT Proteins Mediate Regulated Cargo Sorting at a Common Endosome

“…Vps5 and Vps17 are members of the sorting nexin family of proteins and are responsible for localizing the retromer complex to tubular endosomal membranes via the concerted action of their BAR (Bin/Amphiphysin/RVS) and PX (phox homology) domains (Carlton et al, 2004;Seet and Hong, 2006). Vps26p, Vps29, and Vps35 comprise the cargo-recognition subcomplex of retromer, and the structure of the Vps29-Vps35 interface has recently been solved (Hierro et al, 2007). We and others have shown that another sorting nexin, Snx3/Grd19, associates with retromer and is necessary for retromer-dependent trafficking of a subset of retrograde cargo (Hettema et al, 2003;Restrepo et al, 2007;Strochlic et al, 2007).…”

Re-mention of an old neurodegenerative disease: Alzheimer’s disease

“…In addition, recent studies have shown that a missense mutation (D620N) of the VPS35 gene causes Parkinson's disease (PD) [75,76]. According to the previous crystal structure research, this mutation localizes near the interface between VPS35 and VPS29 [77]. Therefore, we speculate that this mutation causes PD potentially by disrupting the sorting efficiency of Retromer.…”