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 kid ney cDNA expression library by complementation of a mutated, zinc-sens itive BHK cell line. This cDNA was used to isolate the homologous mous e ZnT-1 gene. The proteins predicted for these transporters contain si x membrane-spanning domains, a large intracellular loop and a C-termin al tail. ZnT-1 is homologous to zinc and cobalt resistance genes of ye ast. Immunocytochemistry with an antibody to a myc epitope added to th e C-terminus of ZnT-1 revealed localization to the plasma membrane. Tr ansformation of normal cells with a mutant ZnT-1 lacking the first mem brane-spanning domain conferred zinc sensitivity on wild-type cells, s uggesting that ZnT-1 functions as a multimer. Deletion of the first tw o membrane-spanning domains resulted in a non-functional molecule, whe reas deletion of the C-terminal tail produced a toxic phenotype. Mutan t cells have a slightly higher steady-state level of intracellular zin c and high basal expression of a zinc-dependent reporter gene compared with normal cells. Mutant cells have a lower turnover of Zn-65 compar ed with normal cells or mutant cells transformed with ZnT-1, We propos e that ZnT-1 transports zinc out of cells and that its absence account s for the increased sensitivity of mutant cells to zinc toxicity.