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Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells
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The human Vps29 retromer component is a metallo-phosphoesterase for a cation-independent mannose 6-phosphate receptor

Ester Damen1, Elmar Krieger, Jens E Nielsen

  • 1Department of Cell Biology, Faculty of Sciences, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.

The Biochemical Journal
|June 2, 2006
PubMed

Insights

The retromer complex, containing Vps29, Vps26, and Vps35, dephosphorylates proteins involved in endosomal transport. This Vps29-dependent phosphatase activity is crucial for the cation-independent mannose 6-phosphate receptor

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The retromer complex mediates retrograde transport from endosomes to the Golgi.
  • It is a heterotrimer of Vps26, Vps29, and Vps35 proteins.
  • Vps29 possesses a metallo-phosphoesterase fold.

Purpose of the Study:

  • To investigate the enzymatic activity of the retromer complex, specifically Vps29.
  • To determine the role of Vps29 in the dephosphorylation of the cation-independent mannose 6-phosphate receptor (CI-M6PR).

Main Methods:

  • Recombinant human Vps29, Vps26, and Vps35 were expressed and purified.
  • In vitro phosphatase activity assays were performed using a serine-phosphorylated peptide from CI-M6PR.
  • Inductively coupled plasma mass spectrometry (ICP-MS) was used to detect metal ion binding.
  • Site-directed mutagenesis and metal ion chelation studies were conducted.

Main Results:

  • Recombinant human Vps29 exhibited in vitro phosphatase activity on a CI-M6PR peptide.
  • This activity required the presence of Vps26 and Vps35.
  • Vps29 was found to bind zinc, and its phosphatase activity was dependent on Zn2+ ions.
  • Mutations in predicted active-site residues reduced phosphatase activity.

Conclusions:

  • The retromer complex possesses Vps29-dependent phosphatase activity.
  • This activity targets a serine-phosphorylated motif on the CI-M6PR.
  • The findings suggest a role for Vps29's phosphatase activity in regulating endosomal trafficking of CI-M6PR and other cycling proteins.

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