The yeast AP-1-like transcription factor, Yap1p, activates genes required for the response to oxidative stress. Yap1p is normally cytoplasmic and inactive, but will activate by nuclear translocation if cells are placed in an oxidative environment. Here we show that Yap1p is a target of the β-karyopherin-like nuclear exporter, Crm1p. Yap1p is constitutively nuclear in a crm1 mutant, and Crm1p binds to a nuclear export sequence (NES)-like sequence in Yap1p in the presence of RanGTP. Recognition of Yap1p by Crm1p is inhibited by oxidation, and this inhibition requires at least one of the three cysteine residues flanking the NES. These results suggest that Yap1p localization is largely regulated at the level of nuclear export, and that the oxidation state affects the accessibility of the Yap1p NES to Crm1p directly. We also show that a mutation in RanGAP (rna1-1) is synthetically lethal with crm1 mutants. Yap1p export is inhibited in both rna1-1 and prp20 (RanGNRF) mutant strains, but Yap1p rapidly accumulates at the nuclear periphery after shifting rna1-1, but not other mutant cells to the non-permissive temperature. Thus, disassembly of export complexes in response to RanGTP hydrolysis may be required for release of substrate from a terminal binding site at the nuclear pore complex (NPC).

Rel and IB protein families form a complex cellular regulatory network. A major regulatory function of IB proteins is to retain Rel proteins in the cell cytoplasm. In addition, IB proteins have also been postulated to serve nuclear functions. These include the maintenance of inducible NF-B-dependent gene transcription, as well as termination of inducible transcription. We show that IB␣ shuttles between the nucleus and the cytoplasm, utilizing the nuclear export receptor CRM1. A CRM1-binding export sequence was identified in the N-terminal domain of IB␣ but not in that of IB␤ or IB. By reconstituting major aspects of NF-B-IB sequestration in yeast, we demonstrate that cytoplasmic retention of p65 (also called RelA) by IB␣ requires Crm1p-dependent nuclear export. In mammalian cells, inhibition of CRM1 by leptomycin B resulted in nuclear localization of cotransfected p65 and IB␣ in COS cells and enhanced nuclear relocation of endogenous p65 in T cells. These observations suggest that the main function of IB␣ is that of a nuclear export chaperone rather than a cytoplasmic tether. We propose that the nucleus is the major site of p65-IB␣ association, from where these complexes must be exported in order to create the cytoplasmic pool.The NF-B family of transcription factors consists of proteins that share a domain of approximately 300 amino acids known as the Rel homology domain (RHD) (10, 18). The RHD is required for sequence-specific DNA binding and also mediates protein-protein interactions. Homotypic interactions between RHDs generates a complex array of homo-and heterodimeric NF-B-related proteins in cells, with the term NF-B usually referring to the p50-p65 heterodimer. The RHDs also interact with other structural motifs, including ankyrin domains found in the family of IB proteins (29,31). Interactions between RHD and IB proteins results in inhibition of DNA binding and retention of Rel complexes in the cytoplasm. Signals that induce NF-B lead to the phosphorylation of IB proteins, which are then targeted for ubiquitination and proteasome-mediated degradation. Rel proteins are thereby released to translocate to the nucleus, bind DNA, and activate gene expression. IB proteins are therefore central regulators of NF-B function.The IB proteins are a family of functionally diverse molecules. IB␣, IB␤, and IBε are the most similar, to the extent that they all interact with p65 (also known as RelA) or c-Rel to inhibit DNA binding and are targeted by signal induced phosphorylation for degradation (29, 31). p100 and p110, which are the precursors of RHD-containing p50 and p52 proteins, also contain at their C termini multiple ankyrin repeats that serve IB-like functions by intramolecularly inhibiting DNA binding by the respective N-terminal RHDs. However, it is unclear whether these IB proteins are targeted for signal induced degradation. Finally, the protooncogene bcl-3 contains multiple ankyrin domains and looks IB-like, yet it does not inhibit DNA binding by Rel proteins and has been proposed to be a transcriptional a...