. 111: [807][808][809][810][811][812][813][814][815][816] 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30°C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.Large karyophilic proteins are imported into nuclei through the nuclear pore complex (NPC) in a process that can be divided into at least two steps (4,10,43,63). In the first step of the nuclear translocation process, proteins destined for import bind to the NPC via their association with soluble receptor proteins in an ATP-independent, N-ethylmaleimide (NEM)-sensitive reaction (44,45,55). The second step in this process is an ATP-dependent translocation through the pore. The translocation step requires both soluble factors (41) and components of the NPC (14, 66), which include a family of glycoproteins with 0-linked N-acetylglucosamine modifications (15,26).Karyophilic proteins generally contain nuclear localization signal sequences (NLSs) (30, 32), sequences of amino acids that serve as recognition sites for the soluble receptor proteins (2, 33, 72). In some cases, proteins destined for nuclear import which lack discernible NLSs can be cotransported into nuclei presumedly via stable interactions with NLS-containing proteins (22,65). Although the initial prototypic NLS identified within the simian virus 40 (SV40) large tumor antigen (TAg) is composed of a contiguous stretch of basic residues flanked by prolines (30, 32), most other NLSs appear to be more complex and bipartite in nature (11,56 61), and an unrelated 70-kDa phosphoprotein from S. cerevisiae (68). While the functions of the 54/56-kDa proteins in nuclear import are sensitive to NEM in vitro, it appears that they are not the only NEM-sensitive cytosolic components that ...