The transport mechanism of metallothionein is different from that of classical NLS-bearing protein

A nuclear localization signal (NLS) has been detected in several nuclear pr oteins. Classical NLS-mediated nuclear pore targeting is performed by using the cytosolic factors, importin alpha and importin beta, whereas nuclear t ranslocation requires the small GTPase, Ran. In the present study, we demon strated that nuclear localization of metallothionein (MT) differs from that of classical NLS-mediated substrates. In digitonin-permeabilized BALB/c3T3 cells, biotinylated MT was localized in the nucleus in the presence of ATP and erythrocyte cytosol in the same manner as for SV40 large T NLS-conjuga ted allophycocyanin (APC-NLS). Under ATP-free conditions, nuclear rim-bindi ng was observed in both transport substrates. Rim-binding of labeled MT was competitively inhibited by the addition of an excess amount of unlabeled M T. Different elution profiles were observed for the localizalion-promoting activities of MT in the cytosol compared to those of APC-NLS. Furthermore, nuclear localization of MT was determined to be a wheat germ agglutinin-ins ensitive, GTP gammaS-sensitive, and anti-Ran antibody-sensitive process. Gr een fluorescent protein-metallothionein (GFP-MT) fusion protein was also lo calized in the nucleus in the stable transformant of CHL-IU cells. These re sults strongly suggest that the targeting by MT of the nuclear pore is medi ated by cytosolic factor(s) other than importins and that MT requires Ran f or its nuclear localization. J. Cell. Physiol. 185:440-446, 2000. (C) 2000 Wiley-Liss, Inc.