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We conclude that Crm1p interacts with the Rev NES and nuclear pore proteins during delivery of cargo to the nuclear pore complex. Our findings also agree well with current experiments on Crm1p orthologs in Schizosaccharomyces pombe and in vertebrate systems.

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).

No abstract

Using a monoclonal antibody (mAb 414), we previously identified a protein of 62 kDa (p62) that was localized to the nuclear pore complex by immunoelectron microscopy. We also showed that p62 binds specifically to wheat germ agglutinin. Therefore, we proposed that this nuclear pore complex protein might be a member of a recently characterized family of glycoproteins that are labeled by in vitro galactosylation of rat liver nuclei and contain O-linked monosaccharidic GlcNAc residues. In support of this, we now show that incubation with N-acetylglucosaminidase reduces the molecular mass of p62 by =3 kDa because of the removal of terminal GlcNAc residues. Moreover, p62 can be galactosylated in vitro by using UDP-[3HJgalactose and galactosyltransferase. We also show that most of the GlcNAc residues are added within 5 min of synthesis, when p62 is soluble and cytosolic. Thus, the addition of GlcNAc is carried out in the cytoplasm and is clearly distinct from the N-and O-linked glycosylation pathways of the endoplasmic reticulum and Golgi complex. Using another mAb with a broad specificity for nuclear GlcNAc-containing proteins, we show by immunofluorescence and protein blotting of subnuclear fractions that some of these proteins are in the interior of the nucleus, and others are most likely located in the pore complex.

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...

The nuclear pore complex (NPC) creates an aqueous channel across the nuclear envelope through which macromolecular transport between nucleus and cytoplasm occurs. Nucleocytoplasmic traffic is bidirectional and involves diverse substrates, including protein and RNA. It is unclear whether import and export are mechanistically similar, but evidence suggests that numerous pathways may be involved. The discovery of filaments that extend out from each side of the NPC suggests that the NPC may also have a structural role, perhaps providing a connection between cytoskeletal elements of the nucleus and cytoplasm. If this suggestion is valid, it remains to be determined whether this aspect of NPC function is related to its role in nuclear transport. This review discusses recent developments regarding the structure of the NPC, characterization of its constituent proteins (nucleoporins), the mechanism by which transport occurs, the function of individual nucleoporins, and the pathway of NPC assembly and disassembly.

We have isolated a new gene, NUP2, that encodes a constituent of the yeast-nuclear pore complex (NPC). The NUP2 protein sequence shares a central repetitive domain with NSP1 and NUP1, the two previously characterized yeast nucleoporins. Like NUP1 and NSP1, NUP2 localizes to discrete spots in the nuclear envelope, as determined by indirect immunofluorescence. Although the sequence similarity among these three nucleoporins suggests that they have a similar role in the nuclear pore complex, NUP2, in contrast to NSP1 and NUP1, is not required for growth. Some combinations of mutant alleles of NUP1, NSP1, and NUP2 display "synthetic lethal" relationships that provide evidence for functional interaction between these NPC components. This genetic evidence of overlapping function suggests that the nucleoporins act in concert, perhaps participating in the same step of the recognition or transit of macromolecules through the NPC.