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.     

ZnT5 variant B is a bidirectional zinc transporter and mediates zinc uptake in human intestinal Caco-2 cells

Zinc is an essential micronutrient, so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the functional properties of zinc transporters. Mammalian zinc transporters are classified in two major families: the SLC30 (ZnT) family and the SLC39 family. The prevailing view is that SLC30 family transporters function to reduce cytosolic zinc concentration, either through efflux across the plasma membrane or through sequestration in intracellular compartments, and that SLC39 family transporters function in the opposite direction to increase cytosolic zinc concentration. We demonstrated that human ZnT5 variant B (ZnT5B (hZTL1)), an isoform expressed at the plasma membrane, operates in both the uptake and the efflux directions when expressed in Xenopus laevis oocytes. We measured increased activity of the zinc-responsive metallothionein 2a (MT2a) promoter when ZnT5b was co-expressed with an MT2a promoter-reporter plasmid construct in human intestinal Caco-2 cells, indicating increased total intracellular zinc concentration. Increased cytoplasmic zinc concentration mediated by ZnT5B, in the absence of effects on intracellular zinc sequestration by the Golgi apparatus or endoplasmic reticulum, was also confirmed by a dramatically enhanced signal from the zinc fluorophore Rhodzin-3 throughout the cytoplasm of Caco-2 cells overexpressing ZnT5B at the plasma membrane when compared with control cells. Our findings demonstrate clearly that, in addition to mediating zinc efflux, ZnT5B at the plasma membrane can function to increase cytoplasmic zinc concentration, thus indicating a need to reevaluate the current paradigm that SLC30 family zinc transporters operate exclusively to decrease cytosolic zinc concentration.     

Heterodimerization,Altered Subcellular Localization,and Function of Multiple Zinc Transporters in Viable Cells Using Bimolecular Fluorescence Complementation

Zinc plays a crucial role in numerous key physiological functions. Zinc transporters (ZnTs) mediate zinc efflux and compartmentalization in intracellular organelles; thus, ZnTs play a central role in zinc homeostasis. We have recently shown the in situ dimerization and function of multiple normal and mutant ZnTs using bimolecular fluorescence complementation (BiFC). Prompted by these findings, we here uncovered the heterodimerization, altered subcellular localization, and function of multiple ZnTs in live cells using this sensitive BiFC technique. We show that ZnT1, -2, -3, and -4 form stable heterodimers at distinct intracellular compartments, some of which are completely different from their homodimer localization. Specifically, unlike the plasma membrane (PM) localization of ZnT1 homodimers, ZnT1-ZnT3 heterodimers localized at intracellular vesicles. Furthermore, upon heterodimerization with ZnT1, the zinc transporters ZnT2 and ZnT4 surprisingly localized at the PM, as opposed to their vesicular homodimer localization. We further demonstrate the deleterious effect that the G87R-ZnT2 mutation, associated with transient neonatal zinc deficiency, has on ZnT1, ZnT3, and ZnT4 upon heterodimerization. The functionality of the various ZnTs was assessed by the dual BiFC-Zinquin assay. We also undertook a novel transfection competition assay with ZnT cDNAs to confirm that the driving force for heterodimer formation is the core structure of ZnTs and not the BiFC tags. These findings uncover a novel network of homo- and heterodimers of ZnTs with distinct subcellular localizations and function, hence highlighting their possible role in zinc homeostasis under physiological and pathological conditions.     

Functional characterization of a novel mammalian zinc transporter, ZnT6

We describe ZnT6, a new member of the CDF (cation diffusion facilitator) family of heavy metal transporters. The human ZNT6 gene was mapped at 2p21-22, while the mouse Znt6 was localized to chromosome 17. Overexpression of ZnT6 in both wild-type yeast and mutants that are deficient in cytoplasmic zinc causes growth inhibition, but this inhibition is abolished in mutant cells with high cytoplasmic zinc. ZnT6 may function in transporting the cytoplasmic zinc into the Golgi apparatus as well as the vesicular compartment, as evidenced by its overlapping intracellular localization with TGN38 and transferrin receptor in the normal rat kidney cells. We also demonstrate that the intracellular distributions of ZnT6 as well as ZnT4 are regulated by zinc in the normal rat kidney cells. The results from this report, combined with those from other studies, suggest that the intracellular zinc homeostasis is mediated by many ZnT proteins, which act in tissue-, cell-, and organelle-specific manners.     

In Situ Dimerization of Multiple Wild Type and Mutant Zinc Transporters in Live Cells Using Bimolecular Fluorescence Complementation

Zinc transporters (ZnTs) facilitate zinc efflux and zinc compartmentalization, thereby playing a key role in multiple physiological processes and pathological disorders, presumed to be modulated by transporter dimerization. We recently proposed that ZnT2 homodimerization is the underlying basis for the dominant negative effect of a novel heterozygous G87R mutation identified in women producing zinc-deficient milk. To provide direct visual evidence for the in situ dimerization and function of multiple normal and mutant ZnTs, we applied here the bimolecular fluorescence complementation (BiFC) technique, which enables direct visualization of specific protein-protein interactions. BiFC is based upon reconstitution of an intact fluorescent protein including YFP when its two complementary, non-fluorescent N- and C-terminal fragments (termed YN and YC) are brought together by a pair of specifically interacting proteins. Homodimerization of ZnT1, -2, -3, -4, and -7 was revealed by high subcellular fluorescence observed upon co-transfection of non-fluorescent ZnT-YC and ZnT-YN; this homodimer fluorescence localized in the characteristic compartments of each ZnT. The validity of the BiFC assay in ZnT dimerization was further corroborated when high fluorescence was obtained upon co-transfection of ZnT5-YC and ZnT6-YN, which are known to form heterodimers. We further show that BiFC recapitulated the pathogenic role that ZnT mutations play in transient neonatal zinc deficiency. Zinquin, a fluorescent zinc probe applied along with BiFC, revealed the in situ functionality of ZnT dimers. Hence, the current BiFC-Zinquin technique provides the first in situ evidence for the dimerization and function of wild type and mutant ZnTs in live cells.     

Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter

Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn²⁺ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn²⁺ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on ZnT5. Consistent with this model, replacement of these residues, Asp⁵⁹⁹ and His⁴⁵¹, with alanine was sufficient to block Zn²⁺ transport. These findings indicate, for the first time, that Zn²⁺ transport mediated by a mammalian ZnT is catalyzed by H⁺/Zn²⁺ exchange and identify the zinc binding site of ZnT proteins essential for zinc transport.     

Differential expression of zinc transporters in functionally contrasting tissues involved in zinc homeostasis

Abstract

Zinc homeostasis is maintained by 24 tissue-specific zinc transporters which include ZnTs (ZnT1-10), ZIPs (ZIP1-14), in addition to metallothionein (MT). Current study aimed the role of zinc transporters in maintaining the basal levels of zinc in functionally contrasting tissue specific THP-1 (monocyte), RD (muscle), and Saos-2 (bone) cells. Zinc transporters expression was assessed by qRT-PCR. The mRNA levels of ZnTs (ZnT5-7 & ZnT9), ZIPs (ZIP6-10, ZIP13-14), and MT were significantly (p?<?0.05) higher in Saos-2 compared to THP-1 and RD. The present study suggests that distinct expression pattern of zinc transporters and metallothionein might be responsible for the differential zinc assimilation.     

Immunohistochemical analysis of ZnT1, 4, 5, 6, and 7 in the mouse gastrointestinal tract.

Expression of five zinc transporters (ZnT1, 4, 5, 6, and 7) of the Slc30 family in the mouse gastrointestinal tract was studied by immunohistochemical analysis. Results demonstrated unique expression patterns, levels, and cellular localization among ZnT proteins in the mouse gastrointestinal tract with some overlapping. ZnT1 was abundantly expressed in the epithelium of the esophagus, duodenum of the small intestine, and cecum of the large intestine. ZnT4 was predominantly detected in the large intestine. ZnT5 was mainly expressed in the parietal cell of the stomach and in the absorptive epithelium of the duodenum and jejunum. ZnT6 was predominantly detected in the chief cell of the stomach, columnar epithelial cells of the jejunum, cecum, colon, and rectum. Lastly, ZnT7 was observed in all epithelia of the mouse gastrointestinal tract with the highest expression in the small intestine. Expression of ZnT proteins in the absorptive epithelial cell of the gastrointestinal tract suggests that ZnT proteins may play important roles in zinc absorption and endogenous zinc secretion.     

AP-3-dependent mechanisms control the targeting of a chloride channel (ClC-3) in neuronal and non-neuronal cells

Adaptor protein (AP)-2 and AP-3-dependent mechanisms control the sorting of membrane proteins into synaptic vesicles. Mouse models deficient in AP-3, mocha, develop a neurological phenotype of which the central feature is an alteration of the luminal synaptic vesicle composition. This is caused by a severe reduction of vesicular levels of the zinc transporter 3 (ZnT3). It is presently unknown whether this mocha defect is restricted to ZnT3 or encompasses other synaptic vesicle proteins capable of modifying synaptic vesicle contents, such as transporters or channels. In this study, we identified a chloride channel, ClC-3, whose level in synaptic vesicles and hippocampal mossy fiber terminals was reduced in the context of the mocha AP-3 deficiency. In PC-12 cells, ClC-3 was present in transferrin receptor-positive endosomes, where it was targeted to synaptic-like microvesicles (SLMV) by a mechanism sensitive to brefeldin A, a signature of the AP-3-dependent route of SLMV biogenesis. ClC-3 was packed in SLMV along with the AP-3-targeted synaptic vesicle protein ZnT3. Co-segregation of ClC-3 and ZnT3 to common intracellular compartments was functionally significant as revealed by increased vesicular zinc transport with increased ClC3 expression. Our work has identified a synaptic vesicle protein in which trafficking to synaptic vesicles is regulated by AP-3. In addition, our findings indicate that ClC-3 and ZnT3 reside in a common vesicle population where they functionally interact to determine vesicle luminal composition.     

Zinc fluxes during acute and chronic exposure of INS-1E cells to increasing glucose levels.

Zinc in beta-cell secretory vesicles is essential for insulin hexamerization, and tight vesicular zinc regulation is mandatory. Little is known about zinc ion fluxes across the secretory vesicle membrane and the influence of changes in the extracellular environment on vesicular zinc. Our study aim was to investigate the effect of acute and chronic exposure to various glucose concentrations on zinc in secretory vesicles, the relation between zinc and insulin, and the presence of two zinc transporters, ZnT1 and ZnT4, in INS-1E cells. Zinc ions were demonstrated and semi-quantified using zinc-sulfide autometallography. Insulin content and secreted insulin were measured. Measurements were made on INS-1E cells after exposure to 2.0, 6.6, 16.7, and 24.6 mmol/l glucose for 1, 24, and 96 hours. 1h: Increasing glucose resulted in no changes in intravesicular zinc ions at 2, and 24.6 mmol/l glucose, but a slight increase at 16.7 mmol/l glucose. 24 and 96 h: Increasing glucose led to decreased vesicular zinc ion content accompanied by a decrease in insulin content. ZnT1 and ZnT4 were present in the cytoplasm. Our results demonstrate that intra-vesicular zinc ions respond to changes in the extra-cellolar glucose concentration, especially during chronic high glucose concentrations, where the content of vesicular zinc ions decreases.     

SLC30A3 (ZnT3) Oligomerization by Dityrosine Bonds Regulates Its Subcellular Localization and Metal Transport Capacity

Non-covalent and covalent homo-oligomerization of membrane proteins regulates their subcellular localization and function. Here, we described a novel oligomerization mechanism affecting solute carrier family 30 member 3/zinc transporter 3 (SLC30A3/ZnT3). Oligomerization was mediated by intermolecular covalent dityrosine bonds. Using mutagenized ZnT3 expressed in PC12 cells, we identified two critical tyrosine residues necessary for dityrosine-mediated ZnT3 oligomerization. ZnT3 carrying the Y372F mutation prevented ZnT3 oligomerization, decreased ZnT3 targeting to synaptic-like microvesicles (SLMVs), and decreased resistance to zinc toxicity. Strikingly, ZnT3 harboring the Y357F mutation behaved as a “gain-of-function” mutant as it displayed increased ZnT3 oligomerization, targeting to SLMVs, and increased resistance to zinc toxicity. Single and double tyrosine ZnT3 mutants indicate that the predominant dimeric species is formed between tyrosine 357 and 372. ZnT3 tyrosine dimerization was detected under normal conditions and it was enhanced by oxidative stress. Covalent species were also detected in other SLC30A zinc transporters localized in different subcellular compartments. These results indicate that covalent tyrosine dimerization of a SLC30A family member modulates its subcellular localization and zinc transport capacity. We propose that dityrosine-dependent membrane protein oligomerization may regulate the function of diverse membrane protein in normal and disease states.     

Zinc transporter 2 (SLC30A2) can suppress the vesicular zinc defect of adaptor protein 3-depleted fibroblasts by promoting zinc accumulation in lysosomes

Zinc accumulation in the lumen of cytoplasmic vesicles is one of the mechanisms by which cells can store significant amounts of this essential but potentially toxic biometal. Previous studies had demonstrated reduced vesicular zinc levels in fibroblasts from mutant mice deficient in adaptor protein 3 (AP-3), a complex involved in protein trafficking to late endosomes and lysosomes. We have observed a similar phenotype in the human fibroblastoid cell line, M1, upon small interference RNA-mediated AP-3 knockdown. A survey of the expression and localization of zinc transporter (ZnT) family members identified ZnT2, ZnT3, and ZnT4 as likely mediators of vesicular zinc accumulation in M1 cells. Expression of green fluorescence protein (GFP)-tagged ZnT2 and ZnT3 promoted accumulation of vesicular zinc as visualized using the indicator zinquin. Moreover, GFP-ZnT2 overexpression elicited a significant accumulation of zinc within mature lysosomes, which in untransfected M1 cells contained little or no chelatable zinc, and restored the zinc storage capability of AP-3-deficient cells. These results suggest that ZnT2 can facilitate vesicular zinc accumulation independently of AP-3 function, and validate the M1 fibroblastoid line as a human cell culture system amenable to the study of vesicular zinc regulation using techniques compatible with functional genomic approaches.     

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.