Two different zinc transport complexes of cation diffusion facilitator proteins localized in the secretory pathway operate to activate alkaline phosphatases in vertebrate cells

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.