Energy-dependent nuclear binding dictates metallothionein localization

Metallothioneins (MTs) are low molecular weight, stress-activated proteins that protect cells against heavy metals, oxidants, and some electrophilic d rugs. Both nuclear and cytoplasmic MT phenotypes have been observed in cell s even though MTs (6 kDa) are well below the size exclusion limit for diffu sion through the nuclear envelope. To study the factors controlling MT subc ellular 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 digit onin-permeabilized human SCC25 carcinoma cells, consistent with its endogen ous distribution in these cells. Nuclear sequestration of the fluorescent M TII was inhibited by a 100-fold molar excess of unlabeled MTII and by wheat germ agglutinin, indicating a saturable binding mechanism and the involvem ent of one or more glycoproteins, respectively. Depletion of adenosine trip hosphate (ATP) inhibited MTII nuclear localization, implying energy-depende nt nuclear translocation or retention of MT. Neither chilling nor the absen ce of cytosolic extracts inhibited nuclear sequestration of MTII, supportin g diffusion-based entry mechanism. In situ biochemical extractions of the n uclear MTII revealed at least two distinct binding activities. Collectively , these data indicate that MTII diffuses into the nucleus of SCC25 cells, w here it is selectively and actively retained by nuclear binding factors, im parting its localization phenotype. J. Cell. Physiol. 182:69-76, 2000. (C) 2000 Wiley-Liss, Inc.