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Vps29 has a phosphoesterase fold that acts as a protein interaction scaffold for retromer assembly

Nature Structural & Molecular Biology volume 12, pages 594–602 (2005)Cite this article

Abstract

The retromer complex is responsible for the retrieval of mannose 6-phosphate receptors from the endosomal system to the Golgi. Here we present the crystal structure of the mammalian retromer subunit mVps29 and show that it has structural similarity to divalent metal-containing phosphoesterases. mVps29 can coordinate metals in a similar manner but has no detectable phosphoesterase activity in vitro, suggesting a unique specificity or function. The mVps29 and mVps26 subunits bind independently to mVps35 and together form a high-affinity heterotrimeric subcomplex. Mutagenesis reveals the structural basis for the interaction of mVps29 with mVps35 and subsequent association with endosomal membranes in vivo. A conserved hydrophobic surface distinct from the primary Vps35p binding site mediates assembly of the Vps29p–Vps26p–Vps35p subcomplex with sorting nexins in yeast, and mutation of either site results in a defect in retromer-dependent membrane trafficking.

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Figure 1: Structure of mVps29.
Figure 2: Comparison of mVps29 with related phosphoesterases.
Figure 3: Mammalian retromer assembly in vitro.
Figure 4: mVps29-GFP expression in mammalian cells.
Figure 5: Retromer assembly in vivo.
Figure 6: Analysis of Vps29p function in S. cerevisiae.

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Acknowledgements

The authors thank P. Evans and M. Noble for helpful scientific discussions, S. Höning for technical assistance, M. Harbour for mass spectrometry, D. Barford for the phage λ protein phosphatase expression construct, S.-H. Kim for the MJ0936 protein and the staff of Daresbury SRS beamline 9.6 for their assistance. This work was supported by a Wellcome Trust Senior Research Fellowship in Basic Biomedical Science to D.J.O.

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Authors and Affiliations

  1. Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge, CB2 2XY, UK

    Brett M Collins, Claire F Skinner, Peter J Watson, Matthew N J Seaman & David J Owen

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  1. Brett M Collins
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Correspondence to Matthew N J Seaman or David J Owen.

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Supplementary information

Supplementary Fig. 1

Structure of mVps29 and cross-species comparison. (PDF 2065 kb)

Supplementary Fig. 2

Comparison of apo mVps29 (magenta) and Mn2+-bound mVps29 (green). (PDF 1156 kb)

Supplementary Table 1

Active site residues of phosphoesterases of known structure. (PDF 56 kb)

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Collins, B., Skinner, C., Watson, P. et al. Vps29 has a phosphoesterase fold that acts as a protein interaction scaffold for retromer assembly. Nat Struct Mol Biol 12, 594–602 (2005). https://doi.org/10.1038/nsmb954

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