Two Different Zinc Transport Complexes of Cation Diffusion Facilitator Proteins Localized in the Secretory Pathway Operate to Activate Alkaline Phosphatases in Vertebrate Cells

Abstract

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 zincrequiring 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.

Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway

“…In contrast, the characteristics of the hetero-oligomerization between ZnT5 and ZnT6 and their functional orthologues are poorly understood (19,24,25). This is because ZnT5 and ZnT6 differ from other CDF/ZnT proteins in two important respects.…”

“…Moreover, the functional yeast orthologues Zrg17p (Saccharomyces cerevisiae) and Zrg17 (S. pombe) show little homology to those of vertebrates. These discrepancies make it dif-ficult to understand the properties of these hetero-oligomers (19,24,25).…”

Cellular function and molecular structure of ecto-nucleotidases

“…Zn 2+ is loaded onto APs in the early secretory pathway that contains specific zinc transporters (ZnTs). Activation of human TNAP requires the presence of ZnT5/ZnT6 heterodimers and ZnT7 homooligomers [478,479]. It is thought that the activation takes place in two steps.…”

“…In the absence of ZnT5/ZnT6 heterodimers, the apo-form is degraded [481]. In contrast, the placental enzyme (PLAP) is constitutively expressed in the apo-form and trafficked to the plasma membrane in ZnT5/ZnT6-deficient cells [478,479], implicating that the activation processes of the two enzymes differ.…”

Hepatic ZIP14-mediated zinc transport is required for adaptation to endoplasmic reticulum stress

“…In the sense that ER function is dependent on zinc availability to provide zinc for incorporation into and folding of zinc metalloproteins (39), increased zinc may play a role in the restoration of ER homeostasis. In support of that notion, it has been shown that a supply of zinc to the early secretory pathway via ZnT5, ZnT6, and ZnT7 is essential for helping protein folding via zinc incorporation into alkaline phosphatases, which are zinc metalloproteins (40,41). The importance of an adequate zinc supply for protein folding was illustrated when TM-induced ER stress was exacerbated during zinc-deficient conditions in ZnT5 −/− /ZnT7 −/− cells (26).…”