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

The majority of CDF/ZnT zinc transporters form homo-oligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.     

Zinc transporters, ZnT5 and ZnT7, are required for the activation of alkaline phosphatases, zinc-requiring enzymes that are glycosylphosphatidylinositol-anchored to the cytoplasmic membrane

Numerous proteins are properly folded by binding with zinc during their itinerary in the biosynthetic-secretory pathway. Several transporters have been implicated in the zinc entry into secretory compartments from cytosol, but their precise roles are poorly understood. We report here that two zinc transporters (ZnT5 and ZnT7) localized in the secretory apparatus are responsible for loading zinc to alkaline phosphatases (ALPs) that are glycosylphosphatidylinositol-anchored membrane proteins exposed to the extracellular site. Disruption of the ZnT5 gene in DT40 cells decreased the ALP activity to 45% of that in the wild-type cells. Disruption of the ZnT7 gene lowered the ALP activity only by 20%. Disruption of both genes markedly decreased the ALP activity to <5%. Overexpression of human ZnT5 or ZnT7 in DT40 cells deficient in both ZnT5 and ZnT7 genes recovered the ALP activity to the level comparable to that in the wild-type cells. The inactive ALP protein in DT40 cells deficient in both ZnT5 and ZnT7 genes was transported to cytoplasmic membrane like the active ALP protein in the wild-type cells. Thus both ZnT5 and ZnT7 contribute to the conversion of apo-ALP to holo-ALP.     

Tissue nonspecific alkaline phosphatase is activated via a two-step mechanism by zinc transport complexes in the early secretory pathway

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.     

Two different zinc transport complexes of cation diffusion facilitator proteins localized in the secretory pathway operate to activate alkaline phosphatases in vertebrate cells

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 zinc-requiring 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.     

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

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.     

Analysis of zinc transporter,<Emphasis Type="Italic"> hZnT4</Emphasis> (<Emphasis Type="Italic">Slc30A4</Emphasis>), gene expression in a mammary gland disorder leading to reduced zinc secretion into milk

Zinc deficiency, causing impaired growth and development, may have a nutritional or genetic basis. We investigated two cases of inherited zinc deficiency found in breast-fed neonates, caused by low levels of zinc in the maternal milk. This condition is different from acrodermatitis enteropathica but has similarities to the "lethal milk" mouse, where low levels of zinc in the milk of lactating dams leads to zinc deficiency in pups. The mouse disorder has been attributed to a defect in the ZnT4 gene. Little is known about the expression of the human orthologue, hZnT4 (Slc30A4). Sequence analysis of cDNA, real-time PCR and Western blot analysis of hZnT4, carried out on control cells and cells from unrelated mothers of two infants with zinc deficiency, showed no differences. The hZnT4 gene was highly expressed in mouthwash buccal cells compared with lymphoblasts and fibroblasts. The hZnT4 protein did not co-localise with intracellular free zinc pools, suggesting that hZnT4 is not involved in transport of zinc into vesicles destined for secretion into milk. This observation, combined with phenotypic differences between the "lethal milk" mouse and the human disorder, suggests that the "lethal milk" mouse is not the corresponding model for the human zinc deficiency condition.     

Deficiency of ATP2C1, a Golgi ion pump, induces secretory pathway defects in endoplasmic reticulum (ER)-associated degradation and sensitivity to ER stress

Relatively few clues have been uncovered to elucidate the cell biological role(s) of mammalian ATP2C1 encoding an inwardly directed secretory pathway Ca2+/Mn2+ pump that is ubiquitously expressed. Deficiency of ATP2C1 results in a human disease (Hailey-Hailey), which primarily affects keratinocytes. ATP2C1-encoded protein is detected in the Golgi complex in a calcium-dependent manner. A small interfering RNA causes knockdown of ATP2C1 expression, resulting in defects in both post-translational processing of wild-type thyroglobulin (a secretory glycoprotein) as well as endoplasmic reticulum-associated protein degradation of mutant thyroglobulin, whereas degradation of a nonglycosylated misfolded secretory protein substrate appears unaffected. Knockdown of ATP2C1 is not associated with elevated steady state levels of ER chaperone proteins, nor does it block cellular activation of either the PERK, ATF6, or Ire1/XBP1 portions of the ER stress response. However, deficiency of ATP2C1 renders cells hypersensitive to ER stress. These data point to the important contributions of the Golgi-localized ATP2C1 protein in homeostatic maintenance throughout the secretory pathway.     

Effects of Zinc Transporter on Differentiation of Bone Marrow Mesenchymal Stem Cells to Osteoblasts

The differentiation of bone marrow mesenchymal stem cells (MSCs) into osteoblasts is a crucial step during bone formation. However, the exact mechanisms regulating the early stages of osteogenic differentiation remain unknown. In the present study, we found that ZnT7, a member of the zinc transporter family SLC30A(ZnTs), was downregulated during dexamethasone-induced differentiation of rat MSCs into osteoblasts. Dexamethasone treatment resulted in significantly lower levels of ZnT7 compared with cocultured cells without dexamethasone. Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity and staining for ALP, von Kossa, collagen type I, and osteocalcin. Overexpression of ZnT7 decreased the expression of the osteoblast alkaline phosphatase, type I collagen, as well as calcium deposition in mesenchymal cells. In contrast, knockdown of ZnT7 using siRNA promoted gene expression associated with osteoblast differentiation and matrix mineralization in vitro. Moreover, according to the ZnT7 inhibition or activation experiments, Wnt and ERK signaling pathways were found to be important signal transduction pathways in mediating the osteogenic effect of MSCs, and this effect is intensified by a decrease in the level of ZnT7 induced by dexamethasone. These findings suggest that ZnT7 is involved in the switch from the undifferentiated state of MSC to an osteogenic program, and marking the expression level of ZnT7 may be useful in the detection of early osteogenic differentiation.     

Cutting edge: differential self-peptide/MHC requirement for maintaining CD8 T cell function versus homeostatic proliferation

Memory T cells do not require self-peptide/MHC (spMHC) complexes to survive long term in vivo. However, memory CD4 T cells lose the ability to reject skin grafts when transiently placed in an environment in which these low-level TCR stimulations are absent. Whether or not spMHC alters the ability of CD8 T cells to respond to stimulation in vivo remains unknown. Here, we show that memory CD8 T cells retain the ability to respond to dendritic cell-mediated stimulation after adoptive transfer into either TAP(-/-) (MHC class I-deficient) or wild-type mice. Surprisingly, naive CD8 T cells, which fail to undergo homeostatic proliferation and erode in number in the absence of MHC class I, also retain the ability to respond to dendritic cell-mediated antigenic stimulation for at least 1 wk after transfer into TAP(-/-) mice. These findings suggest a differential requirement for spMHC signals for maintenance of CD8 T cell function and homeostatic proliferation.     

A role for the immunoglobulin-like domain of the human IL-6 receptor. Intracellular protein transport and shedding.

Interleukin (IL)-6, IL-11 and cililary neurotrophic factor (CNTF) belong to the same family of hematopoietic and neurotrophic cytokines. Their receptor complexes contain a cytokine-binding alpha receptor and the common glycoprotein (gp)130 subunit for signal transduction. The extracellular parts of the alpha-receptor subunits consist of a membrane-proximal cytokine-binding domain and an N-terminal immunoglobulin (Ig)-like domain with unknown function. We examined the role of the Ig-like domain of IL-6R by constructing deletion mutants lacking the Ig domain (IL-6RDeltaIg and soluble IL-6RDeltaIg). IL-6RDeltaIg was shed as effectively as wild-type IL-6R from transfected COS-7 cells upon 4beta-phorbol 12-myristate 13-acetate (PMA) treatment, whereas nonstimulated shedding of IL-6RDeltaIg was not observed. The shed sIL-6RDeltaIg from PMA-treated cells, as well as the transmembrane IL-6RDeltaIg, had the same biological activity as wild-type sIL-6R, as measured by the induction of haptoglobin secretion in HepG2-IL-6 cells and IL-6-dependent proliferation of IL-6RDeltaIg transfected BAF/gp130 cells. In COS-7 cells transfected with IL-6RDeltaIg or soluble IL-6RDeltaIg cDNA, transport of the deletion mutants through the secretory pathway appeared to be delayed because a sizeable proportion of the mutants was detected as an endo-beta-N-acetylglucosaminidase-sensitive intermediate, suggesting that transport and processing of the DeltaIg mutants on the secretory pathway were impaired. These experiments suggest that the Ig-like domain of the IL-6R is important for intracellular transport of IL-6R through the secretory pathway. Furthermore, the Ig-like domain is necessary for noninduced shedding of the IL-6R, whereas it has no function in PKC-dependent shedding of the IL-6R.     

Characterization of the mutant (A115V) tissue-nonspecific alkaline phosphatase gene from adult-type hypophosphatasia

Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. To date, various mutations in the TNSALP gene have been identified. Especially, A115V located in exon 5 has been detected in a Japanese patient with severe periodontitis and adult-type HOPS. In this study, we have characterized the protein translated from the mutant A115V gene. Wild-type and A115V mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h transfection, the cells were subjected to assay ALP activity. In order to identify possible dominant effect of the mutation, we performed co-transfections of wild-type and mutated cDNA, and evaluated the residual activities of each mutation. Detection of TNSALP synthesized by COS-1 cells transfected with the wild- or the mutated-type was also performed by using an immunofluorescent method. ALP activity of cell transfected with the mutant cDNA (A115V) plasmid after 48-h transfection exhibited 0.399+/-0.021 U/mg. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 16.9%. When co-transfected this mutant showed no inhibition of the wild-type enzyme. TNSALP in COS-1 cells with transfected with the mutant exhibited strong fluorescence at the surface of cells as wild-type. This study indicated that the mutant (A115V) TNSALP gene produced the defective ALP enzyme and it could be recessively transmitted and be a disease-causing mutation of the adult-type hypophosphatasia.     

Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation

The zinc transporter ZnT2 (SLC30A2) imports zinc into vesicles in secreting mammary epithelial cells (MECs) and is critical for zinc efflux into milk during lactation. Recent studies show that ZnT2 also imports zinc into mitochondria and is expressed in the non-lactating mammary gland and non-secreting MECs, highlighting the importance of ZnT2 in general mammary gland biology. In this study we used nulliparous and lactating ZnT2-null mice and characterized the consequences on mammary gland development, function during lactation, and milk composition. We found that ZnT2 was primarily expressed in MECs and to a limited extent in macrophages in the nulliparous mammary gland and loss of ZnT2 impaired mammary expansion during development. Secondly, we found that lactating ZnT2-null mice had substantial defects in mammary gland architecture and MEC function during secretion, including fewer, condensed and disorganized alveoli, impaired Stat5 activation, and unpolarized MECs. Loss of ZnT2 led to reduced milk volume and milk containing less protein, fat, and lactose compared with wild-type littermates, implicating ZnT2 in the regulation of mammary differentiation and optimal milk production during lactation. Together, these results demonstrate that ZnT2-mediated zinc transport is critical for mammary gland function, suggesting that defects in ZnT2 not only reduce milk zinc concentration but may compromise breast health and increase the risk for lactation insufficiency in lactating women.     

Widespread expression of zinc transporter ZnT (SLC30) family members in mouse endocrine cells

Zinc is abundant in most endocrine cell types, and plays a pivotal role in the synthesis and secretion of many hormones. Recent studies have demonstrated the expression of numerous zinc transporter (ZnT) family members in the pancreas, thyroid, and adrenal glands, suggesting a role for ZnTs in regulating cellular zinc homeostasis in endocrine cells. However, the cellular distribution of ZnTs in the endocrine organs has not been well established. In the present study, the mRNA expression level, cellular distribution of ZnTs as well as liable zinc ions were examined in the mouse pituitary, adrenal glands, thyroid, and pancreas. In general, ZnT1-10 mRNA was expressed to various degrees in the detected endocrine organs, with no detectable ZnT10 mRNA in the pancreas. In the anterior pituitary, both the acidophilic and basophilic cells were immunopositive to ZnT1-5, 7, 8, except for ZnT10. In the adrenal cortex, the immunoreactivity of all the tested ZnTs, including ZnT1-5, 7, 8, 10, was observed in the zona fasciculata, and some ZnTs were detected in the zona glomerulosa, zona reticularis, and the adrenal medulla. Both the follicle epithelial cells and parafollicular cells in the thyroid gland were immunostained with ZnT1-5, 7, 8, but not ZnT10. In the endocrine pancreas, the immunoreactivity of tested ZnTs was observed to various degrees except for ZnT10 in the cytoplasm of islet cells. Furthermore, autometallographic staining showed that liable zinc was observed in the endocrine cells, such as the adrenal cortical cells, thyroid follicle epithelial cells, and the pancreatic islet cells. All together, the wide distribution of liable zinc and the phenomenon that numerous ZnT family members are partially overlapped in a subset of endocrine cells suggest an important role for the ZnT family in controlling cellular zinc levels and subsequently regulating the synthesis and secretion of hormones in the endocrine system.     

Zinc Transporter 7 Induced by High Glucose Attenuates Epithelial-to-Mesenchymal Transition of Peritoneal Mesothelial Cells

Zinc (Zn) is an essential micronutrient and cytoprotectant involved in preventing many types of epithelial-to-mesenchymal transition (EMT)-driven fibrosis in vivo. The zinc-transporter family SLC30A (ZnT) is a pivotal factor in the regulation of Zn homeostasis. However, its function in EMT in peritoneal mesothelial cells (PMCs) remains unknown. This study explored the regulation of zinc transporters and the role they play in cell EMT, particularly in rat peritoneal mesothelial cells (RPMCs), surrounding glucose concentrations and the molecular mechanism involved. The effects of high glucose (HG) on zinc transporter gene expression were measured in RPMCs by real-time PCR. We explored ZnT7 (Slc30A7): the effect of ZnT7 over-expression and siRNA-mediated knock-down on HG-induced EMT was investigated as well as the underlying molecular mechanisms. Over-expression of ZnT7 resulted in significantly inhibited HG-induced EMT in RPMCs, while inhibition of ZnT7 expression using a considerable siRNA-mediated knock-down of RPMCs increased the levels of EMT. Furthermore, over-expression of ZnT7 is accompanied by down-regulation of TGF-β/Smad pathway, phospho-Smad3,4 expression levels. The finding suggests that the zinc-transporting system in RPMCs is influenced by the exposure to HG. The ZnT7 may account for the inhibition of HG-induced EMT in RPMCs, likely through targeting TGF-β/Smad signaling.     

Differential Targeting of SLC30A10/ZnT10 Heterodimers to Endolysosomal Compartments Modulates EGF‐Induced MEK/ERK1/2 Activity

The solute carrier 30A (SLC30A) family of zinc exporters transports zinc into the lumen of intracellular organelles in order to prevent zinc toxicity. We reported that formation of tyrosine dimers is required for ZnT3 (zinc transporter 3) zinc transport activity and targeting to synaptic‐like microvesicles (SLMVs) in PC12 cells and the formation of ZnT3/ZnT10 heterodimers. Here, we focused on ZnT10 to determine the role of heterodimerization in the sorting of ZnTs in the endolysosomal pathway. Using cell fractionation, immunoprecipitation and immunofluorescence approaches, we found that ZnT10 resides in transferrin receptor and Rab5‐positive endosomes and forms covalent heterodimers and oligomers with ZnT2, ZnT3 and ZnT4. The interaction of ZnT10 with ZnT3, mediated by dityrosine bonds, was unable to target ZnT10 into SLMVs in vitro or into synaptic vesicles isolated from mouse brain in vivo. However, ZnT3/ZnT10 heterodimers regulate epidermal growth factor receptor (EGF‐R) signaling by increasing the phosphorylation of mitogen‐activated protein kinase kinase (MEK) and extracellular signal‐regulated kinase (ERK1/2), but not EGF‐R, C‐Raf or Akt phosphorylation in response to EGF. Further, mutation of tyrosine 4 in ZnT10 reduced ZnT3/ZnT10 dityrosine‐mediated heterodimerization and zinc transport, as well as MEK and ERK1/2 phosphorylation, which were also reduced by the zinc chelator TPEN. Phosphorylation of these kinases is likely to occur in the cytosol as no differences in phosphorylation were observed in membrane fractions of control and ZnT3/ZnT10‐expressing cells. We propose that ZnT10 plays a role in signal transduction, which is mediated by homo and heterodimerization with other ZnTs.      

Complementation of a bovine papilloma virus low-copy-number mutant: evidence for a temporal requirement of the complementing gene.

We identified a bovine papilloma virus function encoded by the E6/E7 gene, which is required for both BPV high-copy-number replication and maintenance of transformation of cultured cells. A cDNA copy of this gene was isolated and expressed from a retrovirus vector. We found that complete complementation of a BPV low-copy-number mutant (dl576) by the cDNA encoding the E6/E7 gene was temporally dependent. When both the E6/E7 cDNA and dl576 were introduced together into cells, wild-type replication and stable transformation resulted. In contrast, introduction of the complementing cDNA into cells already carrying dl576 led to only partial amplification of the resident mutant DNA accompanied by a restoration of the transformed phenotype. These results, along with other findings, suggest that the establishment of BPV plasmid replication occurs in two stages: an initial amplification of the incoming DNA followed by stable homeostatic replication which maintains the existing copy number.