Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

PLOS ONE, Dec 2019

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.

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Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway. PLoS ONE 8(10): e77445. doi:10.1371/journal.pone.0077445 Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway Shigeyuki Fujimoto Naoya Itsumura Tokuji Tsuji Yasumi Anan Natsuko Tsuji Yasumitsu Ogra Tomoki Kimura Yusaku Miyamae Seiji Masuda Masaya Nagao Taiho Kambe Stefano L Sensi, University G. D'Annunzio, Italy 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 membranebound 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 reexpression 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 (ZnT12/2MT2/2ZnT42/2 cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT12/2MT2/2ZnT42/2 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 ZnT12/2MT2/2 ZnT42/2 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. - . These authors contributed equally to this work. Of all transition metals, zinc is the most widely used catalytic and structural factor in proteins [1,2]. Zinc proteomics predicts that approximately 10% of proteins encoded in the human genome have a motif that potentially binds to zinc [3,4]. Among these proteins, approximately 1000 are enzymes, which are involved in diverse physiological functions and can be classified into six major classes [5]. Most zinc enzymes use zinc as a catalytic component [4], and therefore zinc coordination (metalation) following zinc transport, trafficking and transfer is fundamental for enzyme activity. The molecular mechanism for this activation process, however, remains unclear. Metallothionein (MT) has been suggested to control the activation of cytoplasmic zinc enzymes [6] and to play a zinc chaperoning role in in vitro studies [2,7]. All zinc transport proteins, including zinc transporters (ZnTs) and ZRT/IRT-related proteins (ZIPs), would potentially be involved in enzyme activation via zinc transport across the cell membrane [8,9]. However, at present there is little direct evidence. Secretory and membrane-bound zinc enzymes, such as matrix metalloproteinases, angiotensin-converting enzymes [10], A disintegrin and metalloproteinase (ADAM) family proteins [11], and alkaline phosphatase [12], are thought to become functional by incorporating zinc in the early secretory pathway (ESP) before reaching their final destination. Thus, zinc transport into the lumen of the ESP is one of the crucial steps for enzyme activation [9]. Compared with the well-known activation process of secretory cuproenzymes by Atox1-ATP7A/ATP7B pathways [1315], understanding of the activation process of secretory and membrane-bound zinc enzymes has been less clear. We have previously shown that the ZnT5-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes) are employed as zinc entry routes into the ESP [16,17]. We have also shown that the zinc transport complexes are indispensable for the activation of secretory and membrane-bound zinc enzymes by converting them from the apo to the holo form using tissue non-specific alkaline phosphatase (TNAP) as a reporter enzyme [18]. However, how other proteins involved in cellular zinc metabolism affect this activation process remains unknown [19]. Here, we examined the TNAP activation process by establishing a series of cells deficient in genes encoding molecules known to be involved in cytoplasmic zinc metabolism. Specifically, we disrupted the ZnT1, MT and ZnT4 genes in the cells, whose products play pivotal roles in the maintenance of cellular zinc homeostasis [8,15,20] via regulatory mechanisms called zinc buffering and muffling [21,22]. Using (...truncated)


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Shigeyuki Fujimoto, Naoya Itsumura, Tokuji Tsuji, Yasumi Anan, Natsuko Tsuji, Yasumitsu Ogra, Tomoki Kimura, Yusaku Miyamae, Seiji Masuda, Masaya Nagao, Taiho Kambe. Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway, PLOS ONE, 2013, Volume 8, Issue 10, DOI: 10.1371/journal.pone.0077445