title: Cooperative functions of ZnT1, metallothionein and ZnT4 in the cytoplasm are required for full activation of TNAP in the early secretory pathway.
abstract: The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1(-/-) MT(-/-) ZnT4(-/-) cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1(-/-) MT(-/-) ZnT4(-/-) cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1(-/-) MT(-/-) ZnT4(-/-) cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.
authors.affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Fujimoto
authors.forename: Naoya
authors.initials: N
authors.lastname: Itsumura
authors.forename: Tokuji
authors.initials: T
authors.lastname: Tsuji
authors.forename: Yasumi
authors.initials: Y
authors.lastname: Anan
authors.forename: Natsuko
authors.initials: N
authors.lastname: Tsuji
authors.forename: Yasumitsu
authors.initials: Y
authors.lastname: Ogra
authors.forename: Tomoki
authors.initials: T
authors.lastname: Kimura
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Miyamae
authors.forename: Seiji
authors.initials: S
authors.lastname: Masuda
authors.forename: Masaya
authors.initials: M
authors.lastname: Nagao
authors.forename: Taiho
authors.initials: T
authors.lastname: Kambe
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D030161:Avian Proteins / Q000235:genetics* / Q000378:metabolism; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism; D027682:Cation Transport Proteins / Q000172:deficiency / Q000235:genetics*; D002461:Cell Line, Transformed; D002645:Chickens / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D008668:Metallothionein / Q000172:deficiency / Q000235:genetics*; D020033:Protein Isoforms / Q000172:deficiency / Q000235:genetics; D021381:Protein Transport; D055571:Secretory Pathway / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D015032:Zinc / Q000378:metabolism*
chemical_list: D030161:Avian Proteins; D027682:Cation Transport Proteins; D020033:Protein Isoforms; D008668:Metallothionein; D000469:Alkaline Phosphatase; D015032:Zinc