VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins

PLoS One. 2011;6(5):e20420. doi: 10.1371/journal.pone.0020420. Epub 2011 May 24.

Abstract

VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+) and Zn(2+) in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calorimetry
  • Crystallography, X-Ray
  • Humans
  • Immunoprecipitation
  • Magnetic Resonance Spectroscopy / methods*
  • Protein Binding
  • Sorting Nexins / genetics
  • Sorting Nexins / metabolism
  • Vesicular Transport Proteins / chemistry*
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / metabolism*

Substances

  • SNX1 protein, human
  • Sorting Nexins
  • VPS29 protein, human
  • Vesicular Transport Proteins