title: Tissue nonspecific alkaline phosphatase is activated via a two-step mechanism by zinc transport complexes in the early secretory pathway.
abstract: A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.
authors.affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Fukunaka
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Kurokawa
authors.forename: Fumie
authors.initials: F
authors.lastname: Teranishi
authors.forename: Israel
authors.initials: I
authors.lastname: Sekler
authors.forename: Kimimitsu
authors.initials: K
authors.lastname: Oda
authors.forename: M Leigh
authors.initials: ML
authors.lastname: Ackland
authors.forename: Victor
authors.initials: V
authors.lastname: Faundez
authors.forename: Makoto
authors.initials: M
authors.lastname: Hiromura
authors.forename: Seiji
authors.initials: S
authors.lastname: Masuda
authors.forename: Masaya
authors.initials: M
authors.lastname: Nagao
authors.forename: Shuichi
authors.initials: S
authors.lastname: Enomoto
authors.forename: Taiho
authors.initials: T
authors.lastname: Kambe
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001051:Apoenzymes / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D002645:Chickens; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D015032:Zinc / Q000378:metabolism*
chemical_list: D001051:Apoenzymes; D027682:Cation Transport Proteins; C548267:ALPL protein, human; D000469:Alkaline Phosphatase; D015032:Zinc