The retromer complex is involved in the retrograde transport of the CI-M6PR (cation-independent mannose 6-phosphate receptor) from endosomes to the Golgi. It is a hetero-trimeric complex composed of Vps26 (vacuolar sorting protein 26), Vps29 and Vps35 proteins, which are conserved in eukaryote evolution. Recently, elucidation of the crystal structure of Vps29 revealed that Vps29 contains a metallo-phosphoesterase fold [Wang, Guo, Liang, Fan, Zhu, Zang, Zhu, Li, Teng, Niu et al. (2005) J. Biol. Chem. 280, 22962–22967; Collins, Skinner, Watson, Seaman and Owen (2005) Nat. Struct. Mol. Biol. 12, 594–602]. We demonstrate that recombinant hVps29 (human Vps29) displays in vitro phosphatase activity towards a serine-phosphorylated peptide, containing the acidic-cluster dileucine motif of the cytoplasmatic tail of the CI-M6PR. Efficient dephosphorylation required the additional presence of recombinant hVps26 and hVps35 proteins, which interact with hVps29. Phosphatase activity of hVps29 was greatly decreased by alanine substitutions of active-site residues that are predicted to co-ordinate metal ions. Using inductively coupled plasma MS, we demonstrate that recombinant hVps29 binds zinc. Moreover, hVps29-dependent phosphatase activity is greatly reduced by non-specific and zinc-specific metal ion chelators, which can be completely restored by addition of excess ZnCl2. The binuclear Zn2+ centre and phosphate group were modelled into the hVps29 catalytic site and pKa calculations provided further insight into the molecular mechanisms of Vps29 phosphatase activity. We conclude that the retromer complex displays Vps29-dependent in vitro phosphatase activity towards a serinephosphorylated acidic-cluster dileucine motif that is involved in endosomal trafficking of the CI-M6PR. The potential significance of these findings with respect to regulation of transport of cycling trans-Golgi network proteins is discussed.
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Research Article|
August 29 2006
The human Vps29 retromer component is a metallo-phosphoesterase for a cation-independent mannose 6-phosphate receptor substrate peptide Available to Purchase
Ester Damen;
Ester Damen
*Department of Cell Biology, Faculty of Sciences, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
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Elmar Krieger;
Elmar Krieger
†Centre for Molecular and Biomolecular Informatics, Faculty of Sciences, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
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Jens E. Nielsen;
Jens E. Nielsen
‡Centre for Synthesis and Chemical Biology, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland
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Jelle Eygensteyn;
Jelle Eygensteyn
§Department of General Instrumentation, Faculty of Sciences, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
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Jeroen E. M. Van Leeuwen
Jeroen E. M. Van Leeuwen
1
*Department of Cell Biology, Faculty of Sciences, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
1To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
January 05 2006
Revision Received:
May 08 2006
Accepted:
June 01 2006
Accepted Manuscript online:
June 01 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 398 (3): 399–409.
Article history
Received:
January 05 2006
Revision Received:
May 08 2006
Accepted:
June 01 2006
Accepted Manuscript online:
June 01 2006
Citation
Ester Damen, Elmar Krieger, Jens E. Nielsen, Jelle Eygensteyn, Jeroen E. M. Van Leeuwen; The human Vps29 retromer component is a metallo-phosphoesterase for a cation-independent mannose 6-phosphate receptor substrate peptide. Biochem J 15 September 2006; 398 (3): 399–409. doi: https://doi.org/10.1042/BJ20060033
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