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

<div><p>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 <i>ZnT1</i>, <i>Metallothionein</i> (<i>MT</i>) and <i>ZnT4</i> genes (<i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− 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 <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− 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.</p></div

Restoration of resistance of <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells by expression of the indicated genes against high zinc toxicity.

<p>The relative contribution of hZnT1, mMt-I or hZnT4 re-expression in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells against zinc toxicity was determined by counting the number of cells after 72 h exposure to the indicated concentrations of ZnSO₄. The relative contribution of exogenous expression of hZnT2 or mutant hZnT1 or hZnT4 is also indicated. Relative values presented are evaluations of the averages of three independent experiments. ++: growing to confluence; +: less growth compared with ++ (20–50% relative to ++); -: not growing.</p

Reduction of the cytosolic zinc levels in ZnT1^−/−MT^−/−ZnT4^−/− cells did not restore reduced TNAP activity.

<p>(<b>A</b>) Exogenous expression of hZnT2 reduced the cytosolic zinc levels in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells or <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells stably expressing hZnT2-HA were transiently transfected with MT-I luciferase as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077445#pone-0077445-g001" target="_blank">Fig. 1B</a>. Relative activity of luciferase is shown (the luciferase activity of <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells stably expressing hZnT2 cultured without ZnSO₄ is defined as 1). Each value is the mean ± SD of three independent experiments (*<i>P</i><0.01). (<b>B</b>) TNAP activity of the total cellular protein prepared from the indicated cells is expressed as the mean ± SD of three independent experiments (N.S., not significant, <i>upper panel</i>). Expression of hZnT2 was confirmed by immunoblot analysis using total cellular proteins. Tubulin is shown as a loading control (<i>lower panels</i>). (<b>C</b>) The change in cytosolic zinc levels in WT and <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells during zinc-deficient culture. Both cell lines were transiently transfected with MT-I luciferase, cultured in normal medium for 9 h, and then cultured in zinc-deficient medium for the indicated period of time prior to measuring luciferase activity. (<b>D</b>) The change in TNAP activity in WT and <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells during zinc-deficient culture. Both cells cultured in normal medium were washed once and then cultured in zinc-deficient medium for the indicated period. TNAP activity is expressed as the mean ± SD of three independent experiments. In the lower panels of <i>C</i> and <i>D</i>, relative rates of change are plotted with values of WT or <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells at time 0 h defined as 1. 1M4; <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells.</p

ZnT1, MT and ZnT4 are all involved in the activation process of TNAP.

<p>(<b>A</b>) TNAP activity decreased in DT40 cells deficient in genes coding for c<i>ZnT1</i>, c<i>MT</i> and/or c<i>ZnT4</i> (<i>upper panel</i>). TNAP activity of total cellular protein prepared from the indicated cells is expressed as the mean ± SD of three independent experiments (*<i>P</i><0.01 vs. WT cells). Disruption of each gene was confirmed by RT-PCR using the appropriate primers (<i>lower panel</i>). <i>cMT2</i> expression is shown in duplicate (first and nested) to show significant or moderate induction of <i>cMT2</i> mRNA expression in <i>ZnT1</i>^−/− or <i>ZnT4</i>^−/− cells. Confirmation of c<i>TNAP</i> expression is also shown. TKO; <i>ZnT5</i>⁻<i>ZnT6</i>^−/−<i>ZnT7</i>^−/− cells. (<b>B</b>) Re-expression of hZnT1, mMt-I and/or hZnT4 restored TNAP activity in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells (<i>upper panel</i>). Expression of FLAG-hZnT1, mMt-I and hZnT4-HA was confirmed by immunoblot analysis using total cellular or membrane proteins prepared from the indicated cells. Tubulin and calnexin (CNX) are shown as loading controls (<i>lower panels</i>). TNAP activity is expressed as the mean ± SD of three independent experiments (*<i>P</i><0.01 vs. <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells).</p

TNAP activity is restored by high zinc supplementation in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells.

<p>The indicated cells were cultured in medium supplemented with 0, 25 or 50 µM ZnSO₄ for 40 h. TNAP activity is the mean ± SD of three independent experiments (N.S., not significant). Note that TNAP activity in <i>ZnT5</i>⁻<i>ZnT6</i>^−/−<i>ZnT7</i>^−/− (TKO) cells was never restored.</p

Zinc transport activities of ZnT1 and ZnT4 are required for TNAP activation.

<p>(<b>A</b>) Expression of hZnT2 did not significantly affect TNAP activity restored by re-expression of all of hZnT1, mMt-I and hZnT4 in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells (<i>upper panel</i>). Expression of FLAG-hZnT1, mMt-I, hZnT4-HA and hZnT2-FLAG was confirmed by immunoblot analysis using total cellular or membrane proteins prepared from the indicated cells (<i>lower panels</i>). (<b>B</b>) Re-expression of zinc transport-incompetent mutants hZnT1 (FLAG-hZnT1 H43A) or/and hZnT4 (hZnT4-HA H146A) failed to significantly restore the reduced TNAP activity in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells (<i>upper panel</i>). Expression of FLAG-hZnT1 and hZnT4-HA mutants was confirmed by immunoblot analysis (<i>lower panels</i>). (<b>C</b>) Expression of hZnT1 and hZnT4 mutants failed to adequately restore reduced TNAP activity, even if mMt-I was expressed (<i>upper panel</i>). Expression of FLAG-hZnT1 H43A mutant, mMt-I and hZnT4-HA H146A mutant was confirmed by immunoblot analysis (<i>lower panels</i>). In A–C, TNAP activity is expressed as the mean ± SD of three independent experiments (*<i>P</i><0.01, N.S., not significant, <i>upper panels</i>), and tubulin and calnexin (CNX) are shown as loading controls (<i>lower panels</i>). (<b>D</b>) The subcellular localization of hZnT1, hZnT4 (<i>upper panels</i>) and their mutants (<i>lower panels</i>) expressed in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. Nomarski, FLAG-hZnT1 (green), hZnT4-HA (red) and the merged images are shown.</p

Homeostasis of the secretory pathway is not significantly impaired in ZnT1^−/−MT^−/−ZnT4^−/− cells.

<p>(<b>A</b>) Surface IgM expression was not impaired in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. WT and <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells were fixed and immunostained without permeabilization. (<b>B</b>) The cell surface proteins biotinylated with membrane-impermeable reagent were almost the same between WT and <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. Surface refers to the solubilized proteins captured using streptavidin beads, while input refers to the aliquot of the biotinylated proteins before avidin capture (that is total cell lysate). In the surface panel, lectin blotting using WGA detects cell surface glycosylated proteins, and CBB staining detects total cell surface proteins. 1M4; <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. (<b>C</b>) Secretory <i>Cypridina</i> luciferase expression was not significantly different between WT and <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. Both cells transiently transfected with secretory <i>Cypridina</i> luciferase reporter were cultured for 4 h after the medium change. Relative activity of <i>Cypridina</i> luciferase in the spent medium is shown (the luciferase activity/total cellular proteins in WT cells is defined as 1). Each value is the mean ± SD of three independent experiments (N.S., not significant).</p