Two different zinc transport complexes of cation diffusion facilitator proteins localized in the secretory pathway operate to activate alkaline phosphatases in vertebrate cells
Two different zinc transport complexes of cation diffusion facilitator proteins localized in the secretory pathway operate to activate alkaline phosphatases in vertebrate cells
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DOI：
10.1074/jbc.m506902200
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发表时间：
2005-09-02
影响因子：
4.8
通讯作者：
Kambe, T
中科院分区：
文献类型：
--
作者：
Suzuki, T;Ishihara, K;Kambe, T
锌是许多需要锌的酶的催化活性的必需组分。然而，直到最近，人们对这些酶所需的锌供应途径中所涉及的分子知之甚少。我们最近展示了（铃木，T.，石原，K.，Migaki，H.，Matsuura，W.，Kohda，A.，Okumura，K.，Nagao，M.，Yamaguchi-Iwai，Y.，Kambe，T.（2005）J.Biol.Chem.280，637 - 643），锌转运蛋白ZnT 5和ZnT 7是需要锌的酶碱性磷酸酶（ALP）的活化所需的，其通过将锌转运到高尔基体的内腔和ALP所在的囊泡隔室中并将apoALP转化为holoALP。ZnT 6也像ZnT 5和ZnT 7一样位于囊泡隔室中。然而，ZnT 6的功能和这三种转运蛋白之间的关系尚未被表征。在这里，我们通过使用DT 40细胞的基因靶向研究来表征ZnT 6与ZnT 5和ZnT 7一起的细胞功能。ZnT 6缺陷的DT 40细胞显示出低ALP活性，这表明ZnT 6是激活锌需要酶如ZnT 5和ZnT 7所必需的。三种转运蛋白基因的联合破坏和转基因的重新表达揭示了ZnT 5和ZnT 6在相同的途径中起作用，而ZnT 7单独起作用。此外，免疫共沉淀研究显示ZnT 5和ZnT 6形成异源寡聚体，而ZnT 7形成同源寡聚体.有趣的是，ZnT 6中的富含Ser的环，一个潜在的锌结合位点，被证明是ZnT 5/ZnT 6异源寡聚体的锌供应功能，这表明ZnT 5中的富含His的环可能对锌结合很重要，而ZnT 6中的环可能在异源寡聚体的形成中获得另一种功能.这些结果表明，两种不同的锌转运复合物激活ALP。
Zinc is an essential component for the catalytic activity of numerous zinc- requiring enzymes. However, until recently little has been known about the molecules involved in the pathways required for supplying zinc to these enzymes. We showed recently ( Suzuki, T., Ishihara, K., Migaki, H., Matsuura, W., Kohda, A., Okumura, K., Nagao, M., Yamaguchi- Iwai, Y., and Kambe, T. ( 2005) J. Biol. Chem. 280, 637 - 643) that zinc transporters, ZnT5 and ZnT7, are required for the activation of zinc- requiring enzymes, alkaline phosphatases ( ALPs), by transporting zinc into the lumens of the Golgi apparatus and the vesicular compartments where ALPs locate and converting apoALPs to holoALPs. ZnT6 is also located in the vesicular compartments like ZnT5 and ZnT7. However, the functions of ZnT6 and relationships among these three transporters have not been characterized yet. Here, we characterized the cellular function of ZnT6 together with ZnT5 and ZnT7 by gene- targeting studies using DT40 cells. ZnT6- deficient DT40 cells showed low ALP activity, suggesting that ZnT6 is required for the activation of zinc-requiring enzymes like ZnT5 and ZnT7. Combined disruptions of three transporter genes and re- expressions of transgenes revealed that ZnT5 and ZnT6 work in the same pathway, whereas ZnT7 acts alone. Furthermore, co- immunoprecipitation studies revealed that ZnT5 and ZnT6 formed hetero- oligomers, whereas ZnT7 formed homo- oligomers. Interestingly, the Ser- rich loop in ZnT6, a potential zinc- binding site, was dispensable for the zinc- supplying function of ZnT5/ ZnT6 hetero- oligomers, suggesting that the His- rich loop in ZnT5 may be important for zinc binding and that the loop in ZnT6 may acquire another function in the hetero- oligomer formation. These results suggest that two different zinc transport complexes operate to activate ALPs.