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ZnT-3, a putative transporter of zinc into synaptic vesicles.

ABSTRACT: The murine ZnT3 gene was cloned by virtue of its homology to the ZnT2 gene, which encodes a membrane protein that facilitates sequestration of zinc in endosomal vesicles. ZnT-3 protein is predicted to have six transmembrane domains and shares 52% amino acid identity with ZnT-2, with the homology extending throughout the two sequences. Human ZnT-3 cDNAs were also cloned; the amino acid sequence is 86% identical to murine ZnT-3. The mouse ZnT3 gene has 8 exons and maps to chromosome 5. Northern blot and reverse transcriptase-PCR analyses demonstrate that murine ZnT-3 expression is restricted to the brain and testis. In situ hybridization reveals that within the brain, ZnT-3 mRNA is most abundant in the hippocampus and cerebral cortex. Antibodies raised against the C-terminal tail of mouse ZnT-3 react with the projections from these neurons and produce a pattern similar to that obtained with Timm's reaction, which reveals histochemically reactive zinc within synaptic vesicles. We propose that ZnT-3 facilitates the accumulation of zinc in synaptic vesicles.

SUBMITTER: Palmiter RD

PROVIDER: S-EPMC26240 | biostudies-literature | 1996 Dec

REPOSITORIES: biostudies-literature

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ZnT-3, a putative transporter of zinc into synaptic vesicles.

Palmiter R D RD Cole T B TB Quaife C J CJ Findley S D SD

Proceedings of the National Academy of Sciences of the United States of America 19961201 25

The murine ZnT3 gene was cloned by virtue of its homology to the ZnT2 gene, which encodes a membrane protein that facilitates sequestration of zinc in endosomal vesicles. ZnT-3 protein is predicted to have six transmembrane domains and shares 52% amino acid identity with ZnT-2, with the homology extending throughout the two sequences. Human ZnT-3 cDNAs were also cloned; the amino acid sequence is 86% identical to murine ZnT-3. The mouse ZnT3 gene has 8 exons and maps to chromosome 5. Northern bl ...[more]

PMID: 8962159

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Project description:BackgroundWe examined the effects of the R325W mutation on the three-dimensional (3D) structure of the beta-cell-specific Zn2+ (zinc) transporter ZnT-8.MethodsA model of the C-terminal domain of the human ZnT-8 protein was generated by homology modeling based on the known crystal structure of the Escherichia coli (E. coli) zinc transporter YiiP at 3.8 A resolution.ResultsThe homodimer ZnT-8 protein structure exists as a Y-shaped architecture with Arg325 located at the ultimate bottom of this motif at approximately 13.5 A from the transmembrane domain juncture. The C-terminal domain sequences of the human ZnT-8 protein and the E. coli zinc transporter YiiP share 12.3% identical and 39.5% homologous residues resulting in an overall homology of 51.8%. Validation statistics of the homology model showed a reasonable quality of the model. The C-terminal domain exhibited an alphabetabetaalphabeta fold with Arg325 as the penultimate N-terminal residue of the alpha2-helix. The side chains of both Arg325 and Trp325 point away from the interface with the other monomer, whereas the epsilon-NH3+ group of Arg325 is predicted to form an ionic interaction with the beta-COO- group of Asp326 as well as Asp295. An amino acid alignment of the beta2-alpha2 C-terminal loop domain revealed a variety of neutral amino acids at position 325 of different ZnT-8 proteins.ConclusionsOur validated homology models predict that both Arg325 and Trp325, amino acids with a helix-forming behavior, and penultimate N-terminal residues in the alpha2-helix of the C-terminal domain, are shielded by the planar surface of the three cytoplasmic beta-strands and hence unable to affect the sensing capacity of the C-terminal domain. Moreover, the amino acid residue at position 325 is too far removed from the docking and transporter parts of ZnT-8 to affect their local protein conformations. These data indicate that the inherited R325W abnormality in SLC30A8 may be tolerated and results in adequate zinc transfer to the correct sites in the pancreatic islet cells and are consistent with the observation that the SLC30A8 gene variant R325W has a low predicted value for future type 2 diabetes at population-based level.

Project description:Corynebacterium diphtheriae, a Gram-positive, aerobic bacterium, is the causative agent of diphtheria and cutaneous infections. While mechanisms required for heme iron acquisition are well known in C. diphtheriae, systems involved in the acquisition of other metals such as zinc and manganese remain poorly characterized. In this study, we identified a genetic region that encodes an ABC-type transporter (iutBCD) and that is flanked by two genes (iutA and iutE) encoding putative substrate binding proteins of the cluster 9 family, a related group of transporters associated primarily with the import of Mn and Zn. We showed that IutA and IutE are both membrane proteins with comparable Mn and Zn binding abilities. We demonstrated that the iutABCD genes are cotranscribed and repressed in response to iron by the iron-responsive repressor DtxR. Transcription of iutE was positively regulated in response to iron availability in a DtxR-dependent manner and was repressed in response to Zn by the Zn-dependent repressor Zur. Electrophoretic mobility shift assays showed that DtxR does not bind to the iutE upstream region, which indicates that DtxR regulation of iutE is indirect and that other regulatory factors controlled by DtxR are likely responsible for the iron-responsive regulation. Analysis of the iutE promoter region identified a 50-bp sequence at the 3' end of the iutD gene that is required for the DtxR-dependent and iron-responsive activation of the iutE gene. These findings indicate that transcription of iutE is controlled by a complex mechanism that involves multiple regulatory factors whose activity is impacted by both Zn and Fe.IMPORTANCE Vaccination against diphtheria prevents toxin-related symptoms but does not inhibit bacterial colonization of the human host by the bacterium. Thus, Corynebacterium diphtheriae remains an important human pathogen that poses a significant health risk to unvaccinated individuals. The ability to acquire iron, zinc, and manganese is critical to the pathogenesis of many disease-causing organisms. Here, we describe a gene cluster in C. diphtheriae that encodes a metal importer that is homologous to broadly distributed metal transport systems, some with important roles in virulence in other bacterial pathogens. Two metal binding components of the gene cluster encode surface exposed proteins, and studies of such proteins may guide the development of second-generation vaccines for C. diphtheriae.

Project description:BackgroundHuntington's disease (HD) is a neurodegenerative disease that involves a complex combination of psychiatric, cognitive and motor impairments. Synaptic dysfunction has been implicated in HD pathogenesis. However, the mechanisms have not been clearly delineated. Synaptic vesicular zinc is closely linked to modulating synaptic transmission and maintaining cognitive ability. It is significant to assess zinc homeostasis for further revealing the pathogenesis of synaptic dysfunction and cognitive impairment in HD.ResultsHistochemical staining by autometallography indicated that synaptic vesicular zinc was decreased in the hippocampus, cortex and striatum of N171-82Q HD transgenic mice. Analyses by immunohistochemistry, Western blot and RT-PCR found that the expression of zinc transporter 3 (ZnT3) required for transport of zinc into synaptic vesicles was obviously reduced in these three brain regions of the HD mice aged from 14 to 20 weeks and BHK cells expressing mutant huntingtin. Significantly, dual-luciferase reporter gene and chromatin immunoprecipitation assays demonstrated that transcription factor Sp1 could activate ZnT3 transcription via its binding to the GC boxes in ZnT3 promoter. Moreover, mutant huntingtin was found to inhibit the binding of Sp1 to the promoter of ZnT3 and down-regulate ZnT3 expression, and the decline in ZnT3 expression could be ameliorated through overexpression of Sp1.ConclusionsThis is first study to reveal a significant loss of synaptic vesicular zinc and a decline in ZnT3 transcriptional activity in the HD transgenic mice. Our work sheds a novel mechanistic insight into pathogenesis of HD that mutant huntingtin down-regulates expression of ZnT3 through inhibiting binding of Sp1 to the promoter of ZnT3 gene, causing disruption of synaptic vesicular zinc homeostasis. Disrupted vesicular zinc ultimately leads to early synaptic dysfunction and cognitive deficits in HD. It is also suggested that maintaining normal synaptic vesicular zinc concentration is a potential therapeutic strategy for HD.

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ZnT-3, a putative transporter of zinc into synaptic vesicles.

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ZnT-3, a putative transporter of zinc into synaptic vesicles.

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