The human Vps29 retromer component is a metallo-phosphoesterase for a cation-independent mannose 6-phosphate receptor substrate peptide
The human Vps29 retromer component is a metallo-phosphoesterase for a cation-independent mannose 6-phosphate receptor substrate peptide
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DOI：
10.1042/bj20060033
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发表时间：
2006-09-15
影响因子：
4.1
通讯作者：
van Leeuwen, Jeroen E. M.
中科院分区：
文献类型：
--
作者：
Damen, Ester;Krieger, Elmar;van Leeuwen, Jeroen E. M.
逆转录复合物参与CI-M6 PR（阳离子非依赖性甘露糖6-磷酸受体）从内体到高尔基体的逆行转运。它是由Vps 26（vacuolar sorting protein 26）、Vps 29和Vps 35蛋白组成的异源三聚体复合物，这些蛋白在真核生物进化中是保守的。最近，Vps 29的晶体结构的阐明揭示了Vps 29含有金属-磷酸酯酶折叠[Wang，Guo，Liang，Fan，Zhu，Zang，Zhu，Li，Teng，Niu等人（2005）J.Biol.Chem.280，22962-22967;柯林斯，Skinner，沃森，Dahan和Owen（2005）Nat. Struct. Mol. 12，594-602]。我们证明，重组hVps 29（人Vps 29）显示在体外磷酸酶活性对丝氨酸磷酸化的肽，含有酸性簇双亮氨酸基序的细胞质尾的CI-M6 PR。有效的去磷酸化需要额外存在与hVps 29相互作用的重组hVps 26和hVps 35蛋白。磷酸酶活性的hVps 29大大降低了丙氨酸取代的活性位点残基，预计协调金属离子。使用电感耦合等离子体质谱，我们证明，重组hVps 29结合锌。此外，hVps 29依赖性磷酸酶活性大大降低了非特异性和锌特异性金属离子螯合剂，这可以通过添加过量的ZnCl 2完全恢复。将双核Zn ~（2+）中心和磷酸基团模拟到hVps 29的催化位点和pK（a）中，计算结果进一步揭示了Vps 29磷酸酶活性的分子机制。我们得出结论，retromer复合物显示Vps 29依赖性的体外磷酸酶活性对丝氨酸磷酸化的酸性簇双亮氨酸基序，参与内体运输的CI-M6 PR。这些研究结果的潜在意义与调节循环trans-Golgi网络蛋白的运输进行了讨论。
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 pK(a), 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.