No abstract

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.