ABSTRACT
Metallothioneins (MTs) are low molecular weight, stress-activated proteins that protect cells against heavy metals, oxidants, and some electrophilic drugs. Both nuclear and cytoplasmic MT phenotypes have been observed in cells even though MTs (6 kDa) are well below the size exclusion limit for diffusion through the nuclear envelope. To study the factors controlling MT subcellular partitioning, we covalently linked MTII to a fluorescent label and examined its subcellular distribution in response both to pharmacologic and physical perturbations. Fluorescent MTII localized to the nucleus of digitonin-permeabilized human SCC25 carcinoma cells, consistent with its endogenous distribution in these cells. Nuclear sequestration of the fluorescent MTII was inhibited by a 100-fold molar excess of unlabeled MTII and by wheat germ agglutinin, indicating a saturable binding mechanism and the involvement of one or more glycoproteins, respectively. Depletion of adenosine triphosphate (ATP) inhibited MTII nuclear localization, implying energy-dependent nuclear translocation or retention of MT. Neither chilling nor the absence of cytosolic extracts inhibited nuclear sequestration of MTII, supporting diffusion-based entry mechanism. In situ biochemical extractions of the nuclear MTII revealed at least two distinct binding activities. Collectively, these data indicate that MTII diffuses into the nucleus of SCC25 cells, where it is selectively and actively retained by nuclear binding factors, imparting its localization phenotype.
Subject(s)
Cell Nucleus/metabolism , Energy Metabolism , Metallothionein/metabolism , Adenosine Triphosphate/metabolism , Animals , Biological Transport/drug effects , Cadmium/metabolism , Calmodulin/metabolism , Carbocyanines/metabolism , Cell Extracts/pharmacology , Cell Line , Cell Membrane Permeability , Cell Nucleus/drug effects , Cold Temperature , Cytosol/drug effects , Cytosol/metabolism , Diffusion , Energy Metabolism/drug effects , Fluorescent Dyes/metabolism , Glycosylation , Humans , Metallothionein/chemistry , Metallothionein/pharmacology , Mice , Protein Binding , Wheat Germ Agglutinins/pharmacologyABSTRACT
Embryonic cells from transgenic mice with targeted disruption of metallothionein I and II genes expressed no detectable metallothionein either constitutively or after treatment with cadmium, in contrast to cultured cells that were wild type or heterozygous for the loss of the metallothionein genes. Metallothionein null cells were most sensitive to the cytotoxic effects of cadmium, the membrane permeant oxidant tert-butylhydroperoxide, and the redox cycling toxin paraquat. No marked differences were seen among the wild type, heterozygous, or metallothionein null cells in glutathione levels or in the activity of CuZn-superoxide dismutase, glutathione peroxidase, or catalase. Nevertheless, metallothionein null cells were more sensitive to tert-butylhydroperoxide-induced oxidation as ascertained by confocal microscopic imaging of dichlorofluoroscein fluorescence. These results indicate basal metallothionein levels can function to regulate intracellular redox status in mammalian cells.
