Hundreds of proteins with crucial cellular functions contain catalytic and/or structural zinc atoms. The multiple roles of zinc require spatiotemporal regulation of its uptake, transport, and distribution. This proposal addresses the virtually unknown (bio)chemical mechanisms of cellular zinc distribution. The experiments proposed in this application will investigate the roles of metallothionein (MT) and its apoprotein thionein (T) in this process and the role of the MT/T ratio as a regulated zinc donor/acceptor pair that controls the availability of cellular zinc, directs cellular zinc fluxes to and from cellular compartments, and controls zinc-dependent cellular events. The experiments are based on recent discoveries of new biological activities of these proteins. First, MT is a redox-active zinc protein, in which biological oxidants react with the thiolate ligands of its clusters and mobilize zinc. This remarkable feature is typical for biological metal metabolism as contrasted to pure inorganic chemistry. It links the cellular redox state and zinc distribution from zinc/thiolate coordination sites. Second, T is an endogenous chelating agent that activates zinc-inhibited enzymes. Third, significant amounts of T are present in different tissues under normal physiological conditions. And fourth, MT is imported into the mitochondrial intermembrane space and releases zinc, which in turn inhibits respiration. Employing a new differential fluorescence labeling procedure, MT/T ratios will be determined in subcellular fractions. Then, the translocation of MT and T from the cytosol to the mitochondrion, the nucleus, and the extracellular space will be studied and the effect of these translocations on the MT/T ratio evaluated. The proposal will further examine how the redox state controls the MT/T ratio, and hence affects the meta-bolically active pool of cellular zinc that is available to zinc-chelating fluorophores. In cellular model systems of proliferation and differentiation, it will then be investigated how changing the availability of cellular zinc affects functions of zinc in regulatory and enzymatic sites of proteins. Finally, an enzymatic activity that releases zinc from MT in the mitochondrial intermembrane space will be identified and characterized and its effect on the mechanism of zinc release from MT studied. The proposed studies will establish how MT and T are involved in the cellular control of zinc and will provide basic insights into the cellular homeostatic system of this essential element. The experiments are of fundamental importance for an understanding of the role of zinc in cellular metabolism, gene expression, and signal transduction, and in the future will aid in devising new diagnostic, prognostic, and therapeutic means to address zinc-related disorders.
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