The nuclear accumulation of ␤-catenin plays an important role in the Wingless/Wnt signaling pathway. This study describes an examination of the nuclear import of ␤-catenin in living mammalian cells and in vitro semi-intact cells. When injected into the cell cytoplasm, ␤-catenin rapidly migrated into the nucleus in a temperature-dependent and wheat germ agglutinin-sensitive manner. In the cell-free import assay, ␤-catenin rapidly migrates into the nucleus without the exogenous addition of cytosol, Ran, or ATP/GTP. Cytoplasmic injection of mutant Ran defective in its GTP hydrolysis did not prevent ␤-catenin import. Studies using tsBN2, a temperature-sensitive mutant cell line that possesses a point mutation in the RCC1 gene, showed that the import of ␤-catenin is insensitive to nuclear Ran-GTP depletion. These results show that ␤-catenin possesses the ability to constitutively translocate through the nuclear pores in a manner similar to importin ␤ in a Ran-unassisted manner. We further showed that ␤-catenin also rapidly exits the nucleus in homokaryons, suggesting that the regulation of nuclear levels of ␤-catenin involves both nuclear import and export of this molecule. INTRODUCTIONThe trafficking of macromolecules across the nuclear envelope plays a key role in the coordination of cytoplasmic and nuclear events. The exchange of macromolecules occurs at the nuclear pore complex (NPC), which spans the double lipid bilayer of the nuclear envelope. The NPC is a large proteinaceous structure of ϳ125 MDa in size and mediates bidirectional transport via several different mechanisms (for reviews, see Davis, 1995;Fabre and Hurt, 1997). Small molecules, such as ions, low-molecular-weight metabolites, and proteins smaller than 20 -40 kDa cross 10-nm-diameter aqueous channels of the NPC by passive diffusion, whereas larger molecules are generally transported through the gated channels of the NPC via an active, receptor-mediated mechanism.A number of recent discoveries have led to the development of a model for receptor-mediated active nuclear import and export (for reviews, see Corbett and Silver, 1997;Gö rlich, 1997;Nakielny et al., 1997;Nigg, 1997;Ullman et al., 1997;Imamoto et al., 1998;Mattaj and Englmeier, 1998;Ohno et al., 1998). The model involves two essential elements, which are required for both the import and export pathways: 1) soluble transport factors, which recognize respective signals present in each protein, which is either imported into or exported out of the nucleus; and 2) a small GTPase Ran that affects the affinity between the transport factors and signals by binding directly to the transport factors. Import substrates form a complex with import factors in the cytoplasm, are transported through the NPC, and are then released from the import factors on the nucleoplasmic side of NPC when the GTP-bound form of Ran binds to the import factors. Export substrates form a complex with export factors and Ran-GTP inside the nucleus, are transported through the NPC, and are then released from the export factors w...

Cadmium-resistant Saccharomyces cerevisiae strain 301N exhibits high basal as well as cadmium-induced expression of the CUP1 metallothionein gene. Since regulation of CUP1 is usually restricted to copper ions, our goal was to identify the factor responsible for the high metallothionein levels in strain 301N. The gene responsible for the observed phenotype is a spontaneously mutated heat shock transcription factor gene (HSF1). A double, semidominant HSF1 mutant with substitutions at codons 206 and 256 within the DNA-binding domain of the heat shock factor (HSF) confers two phenotypes. The first phenotype is elevated transcriptional activity of the HSF mutant (HSF301), which results in constitutive thermotolerance. A second HSF301 phenotype is enhanced binding affinity for the heat shock element (HSE) within the CUP1 5-sequences, resulting in high basal transcription of metallothionein. The CUP1 HSE is a minimal heat shock element containing only two perfectly spaced inverted repeats of the basic nGAAn block. Cells containing HSF301 are resistant to cadmium salts. The single R206S mutation is responsible for the high affinity binding to the CUP1 HSE. In addition, the R206S HSF substitution exhibits constitutive transcriptional activation from a consensus HSE (HSE2). The F256Y substitution in HSF attenuates the effects of R206S on the consensus HSE2, but not on the CUP1 HSE.All cells are capable of coping with changes in their environment, such as exposure to elevated temperatures, toxins, and oxidants. In response to certain stress conditions, activation of stress gene expression occurs, resulting in an elevated synthesis of stress proteins, commonly called heat shock proteins (hsp) 1 (1, 2). That these hsp genes are induced by a variety of stress conditions implies that they have broadly protective functions.The induction of heat shock protein(s) occurs at the level of transcription (3-5). Genes encoding the various hsp molecules contain a conserved promoter element, designated a heat shock element (HSE) (1, 6). The induction of hsp70 in animal cells by heat or metal ions requires only the HSE in the promoter (7,8). HSEs contain multiple 5-bp inverted repeats of the sequence nGAAn (9 -12). The number of 5-bp boxes may range from three to six (9 -11). A perfect consensus array of three boxes would be the sequence 5Ј-nGAAnnTTCnnGAAn-3Ј. Not all HSEs have perfect inverted repeats, but it appears that they have at least two perfect nGAAn boxes (9 -12). A compilation of 40 naturally occurring HSEs from different organisms revealed that seven contained only three nGAAn blocks, and in each case, these three nGAAn boxes were in combination with additional HSE units, permitting cooperative interactions (12).Transcriptional activation of genes containing heat shock promoter elements is mediated by the heat shock factor (HSF). Saccharomyces cerevisiae has one HSF encoded by the HSF1 locus (13,14). Yeast HSF is a trimeric protein reported to bind HSE sequences constitutively at low temperature (5,(15)(16)(17). Within the N-t...

␤-Catenin is an example of a typical molecule that can be translocated bidirectionally through nuclear pore complexes (NPCs) on its own in a facilitated manner. In this work the nuclear import and export of ␤-catenin were examined to compare the sequence requirement of this molecule and to determine whether molecular interactions required for its bidirectional NPC passage are distinct or not. Deletion analysis of ␤-catenin revealed that armadillo repeats 10 -12 and the C terminus comprise the minimum region necessary for nuclear migration activity. Further dissection of this fragment showed that the C terminus tail plays an essential role in nuclear migration. The region of ␤-catenin required for export substantially overlapped the region required for import. Therefore, the NPC translocation of ␤-catenin is apparently reversible, which is consistent with findings reported previously. However, different translocating molecules blocked nuclear import and export of ␤-catenin differentially. The data herein indicate that ␤-catenin shows an overlapping sequence requirement for its import and export but that bidirectional movement through the NPC proceeds through distinct molecular interactions.