Importin/karyopherin protein family members required for mRNA export from the nucleus

Abstract

The yeast Saccharomyces cerevisiae contains three proteins (Kap104p, Pse1p, and Kap123p) that share similarity to the 95-kDa ␤ subunit of the nuclear transport factor importin (also termed karyopherin and encoded by KAP95͞RSL1 in yeast). Proteins that contain nuclear localization sequences are recognized in the cytoplasm and delivered to the nucleus by the heterodimeric importin complex. A second importin-related protein, transportin, delivers a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) to the nucleoplasm. We now show that in contrast to loss of importin ␤ (Kap95p͞Rsl1p) and transportin (Kap104p), conditional loss of Pse1p in a strain lacking Kap123p results in a specific block of mRNA export from the nucleus. Overexpression of Sxm1p, a protein related to Cse1p in yeast and to the human cellular apoptosis susceptibility protein, relieves the defects of cells lacking Pse1p and Kap123p. Thus, a major role of Pse1p, Kap123p, and Sxm1p may be nuclear export rather than import, suggesting a symmetrical relationship between these processes.Movement of macromolecules in and out of the nucleus is a highly regulated process essential for proper progression though the cell cycle, response to extracellular signals, and viral maturation. Our knowledge of the mechanism by which proteins are imported into and RNAs exported from the nucleus has grown significantly in recent years. Proteins have been identified that mediate both inward and outward macromolecular movement and both processes appear to be regulated by some of the same factors (reviewed in refs. 1 and 2).Import of proteins into the nucleus is a highly conserved, multistep process initiated by recognition of a nuclear localization sequence (NLS) by a cognate receptor, followed by docking of the complex at the nuclear pore, and GTPdependent translocation into the nucleoplasm. The receptor for the canonical NLS is a heterodimeric protein complex variously called importin or karyopherin, composed of an ␣ (NLS-binding) and a ␤ (docking) subunit (3-6). Translocation through the nuclear pore is driven by GTP hydrolysis, catalyzed by the small ras-related GTP-binding protein Ran and its regulators (7-10). Once inside the nucleus, the importin͞ karyopherin complex is probably dissociated and the transport factors recycled to the cytoplasm (5). This relatively simple model fails to explain the nuclear import of proteins that lack an NLS conforming to the canonical sequences. The recent identification of a role for the importin ␤ homolog transportin, in the nuclear import of heterogeneous nuclear ribonucleoprotein (hnRNP) A1 raised the possibility that different classes of nuclear proteins would be transported by different pathways, defined by the identity of the ␤ subunit involved (11, 12).In contrast to protein import, how RNAs are exported from the nucleus to the cytoplasm is less well understood. Nascent RNA is subject to a number of posttranscriptional modifications, and it seems likely that RNA moves through the nucleoplasm and the nuclear pore...

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

“…Data represent the average values of six independent assays. Note: N795 K513-515A localization was unaffected in an ⌬sxm1 strain constructed in the Blobel lab (Rosenblum et al, 1997) in a different strain background than PSY1200 (Seedorf and Silver, 1997). These results suggest the presence of a genetic modifier, implying the existence of multiple import receptors for NL2 in some yeast strains.…”

“…Fragments encoding residues 540 -795 of rat-GR (540C) and the lacZ gene were amplified as described above and ligated into the SalI-BglII and BglII sites of pJW248, respectively. The pL2G3z GR lacZ reporter plasmid (Bohen and Yamamoto, 1993) EB0347 expressing Pho4-GFP (Kaffman et al, 1998b), pyxLhp1gfp expressing a fusion between Lhp1p-GFP (Rosenblum et al, 1997), pPS1574 expressing Sxm1p, and pPS1117 expressing Sxm1p-GFP (Seedorf and Silver, 1997) have been described.…”

“…The localization of GR-GFP was monitored in 14 yeast strains each deficient in a different nuclear transport receptor: CRMI (Stade et al, 1997), CSEI (Xiao et al, 1993), Kap95 (Iovine et al, 1995), Kap104 (Aitchison et al, 1996), Kap123 (Seedorf and Silver, 1997), LOS1 (Hopper et al, 1980), MSN5 (Kaffman et al, 1998a), MTR10 (Kadowaki et al, 1994), NMD5 (He and Jacobson, 1995), PSEI (Seedorf and Silver, 1997), SRP1 (Yano et al, 1992), SXM1 (Seedorf and Silver, 1997), Kap114 (Morehouse et al, 1999;Pemberton et al, 1999), and PDR6 (Titov and Blobel, 1999). Yeast strains PSY580, pse1-1, PSY1200, and PSY902 have been described (Seedorf and Silver, 1997).…”

“…Yeast strains PSY580, pse1-1, PSY1200, and PSY902 have been described (Seedorf and Silver, 1997). For temperature-sensitive strains, suspension cultures were grown overnight at permissive temperature, diluted to an OD595 of 0.1, and placed at the restrictive temperature.…”

Nuclear Export and Plasma Membrane Recruitment of the Ste5 Scaffold Are Coordinated with Oligomerization and Association with Signal Transduction Components

“…Wang and E.A. Elion nsp1 ts mutations (Nehrbass et al, 1993;Seedorf and Silver, 1997) blocked the recruitment of Ste5-GST during vegetative growth at both permissive (25°C) and nonpermissive (37°C) temperatures, and greatly reduced recruitment at nonpermissive temperature in the presence of ␣ factor ( Figure 6D; shown is rsl1-4; see MATERIALS AND METHODS for details on nsp1 ts ). Consistent with this, Ste5-GST was also unable to induce morphological changes at either temperature in either mutant.…”