Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc.
A cDNA encoding a zinc transporter (ZnT-1) was isolated from a rat kidney cDNA expression library by complementation of a mutated, zinc-sensitive BHK cell line. This cDNA was used to isolate the homologous mouse ZnT-1 gene. The proteins predicted for these transporters contain six membrane-spanning domains, a large intracellular loop and a C-terminal tail. ZnT-1 is homologous to zinc and cobalt resistance genes of yeast. Immunocytochemistry with an antibody to a myc epitope added to the C-terminus of ZnT-1 revealed localization to the plasma membrane. Transformation of normal cells with a mutant ZnT-1 lacking the first membrane-spanning domain conferred zinc sensitivity on wild-type cells, suggesting that ZnT-1 functions as a multimer. Deletion of the first two membrane-spanning domains resulted in a non-functional molecule, whereas deletion of the C-terminal tail produced a toxic phenotype. Mutant cells have a slightly higher steady-state level of intracellular zinc and high basal expression of a zinc-dependent reporter gene compared with normal cells. Mutant cells have a lower turnover of 65Zn compared with normal cells or mutant cells transformed with ZnT-1. We propose that ZnT-1 transports zinc out of cells and that its absence accounts for the increased sensitivity of mutant cells to zinc toxicity.
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Howard Hughes Medical Institute, University of Washington, Seattle 98195.
Findley
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D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D027682:Cation Transport Proteins; D002451:Cell Compartmentation; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D015870:Gene Expression; D005796:Genes; D007668:Kidney; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D014162:Transfection; D015032:Zinc / Q000378:metabolism*
D027682:Cation Transport Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; C485803:Slc30a1 protein, mouse; C092392:Slc30a1 protein, rat; D015032:Zinc