The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88

Abstract

The oncogenic nucleoporin CAN/Nup214 is essential in vertebrate cells. Its depletion results in defective nuclear protein import, inhibition of messenger RNA export and cell cycle arrest. We recently found that CAN associates with proteins of 88 and 112 kDa, which we have now cloned and characterized. The 88 kDa protein is a novel nuclear pore complex (NPC) component, which we have named Nup88. Depletion of CAN from the NPC results in concomitant loss of Nup88, indicating that the localization of Nup88 to the NPC is dependent on CAN binding. The 112 kDa protein is the human homologue of yeast CRM1, a protein known to be required for maintenance of correct chromosome structure. This human CRM1 (hCRM1) localized to the NPC as well as to the nucleoplasm. Nuclear overexpression of the FG-repeat region of CAN, containing its hCRM1-interaction domain, resulted in depletion of hCRM1 from the NPC. In CAN-/- mouse embryos lacking CAN, hCRM1 remained in the nuclear envelope, suggesting that this protein can also bind to other repeat-containing nucleoporins. Lastly, hCRM1 shares a domain of significant homology with importin-beta, a cytoplasmic transport factor that interacts with nucleoporin repeat regions. We propose that hCRM1 is a soluble nuclear transport factor that interacts with the NPC.

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates

“…A direct interaction between TAP and the C-terminal domain of CAN was reported by Katahira et al+ (1999)+ In this study we analyzed the interaction between TAP and the nucleoporin Nup98 and further characterized its interaction with CAN+ Previously, the C-terminal domain of CAN (residues 1690-2090), containing the FGrepeats, was shown to interact with CRM1 (Fornerod et al+, 1997b)+ Figure 6A shows a comparison of TAP and CRM1 binding to C-terminal fragments of CAN+ In this assay, CAN fragments were expressed in E. coli as GST fusions and immobilized on glutathione agarose beads, and TAP and CRM1 were translated in vitro+ Both TAP and CRM1 bound to a fragment of CAN encompassing residues 1690-2090 (Fig+ 6A, lanes 4 and 10) and to a shorter fragment encompassing residues 1690-1894 (Fig+ 6A, lanes 3 and 8)+ A fragment of CAN comprising residues 1983-2090 no longer interacted with TAP (data not shown)+ Furthermore, TAP binding was not regulated by Ran, whereas binding of CRM1 was dramatically stimulated by RanQ69L-GTP (Fig+ 6A, lanes 8 and 10 vs+ 7 and 9), as previously reported (Askjaer et al+, 1999;Kehlenbach et al+, 1999)+ Similarly, TAP/Nup98 interaction was mediated by the N-terminal FG-repeat-containing domain of the nucleoporin (residues 66-515; data not shown)+ This domain also interacts with CRM1, but its binding is not dramatically stimulated by RanQ69L-GTP (data not shown)+…”

“…Nuclear transport occurs through the nuclear pore complexes (NPC) and depends on the presence of nuclear localization signals (NLSs) or nuclear export signals (NESs) in the transported molecules+ NLSs or NESs are recognized and bound by saturable import or export receptors that shuttle between the nucleus and cytoplasm+ Upon binding, the transport receptors dock their cargoes to the NPC and facilitate their translocation+ After delivering their cargoes, the receptors are recycled to initiate additional rounds of transport (reviewed by Görlich, 1997;Nakielny & Dreyfuss, 1997;Mattaj & Englmeier, 1998)+ A key regulator of nucleocytoplasmic transport is the small GTPase Ran (reviewed by Görlich, 1997;Dahlberg & Lund, 1998;Mattaj & Englmeier, 1998)+ Ran exists in a GTP-or GDPbound form+ The location of RanGAP1 primarily in the cytoplasm and RanGEF in the nucleus is believed to generate a nucleocytoplasmic gradient of RanGTP across the NPC that imparts directionality to the transport process (Görlich et al+, 1996;Izaurralde et al+, 1997;Richards et al+, 1997)+ Additional Ran cofactors are the Ran-binding protein 1 (RanBP1), which costimulates the activity of RanGAP1 (Bischoff et al+, 1995a(Bischoff et al+, , 1995b, and the Ran-binding protein 2 (RanBP2/Nup358) (Wu et al+, 1995;Yokoyoma et al+, 1995), which may have, in part, a similar function to RanBP1+ RanBP2/Nup358 is localized to the cytoplasmic fibrils of the NPC (Wu et al+, 1995;Yokoyoma et al+, 1995), whereas RanBP1 is predominantly cytoplasmic (reviewed by Görlich, 1997;Mattaj & Englmeier, 1998)+ A fraction of RanGAP1 conjugated with the ubiquitin-like molecule SUMO-1, binds to RanBP2 and localizes to the cytoplasmic fibrils of the NPC (Matunis et al+, 1996;Mahajan et al+, 1997;Saitoh et al+, 1997)+ Transport receptors identified to date are members of a large family of RanGTP-binding proteins exhibiting limited sequence similarity with the Ran-binding domain of importin-b (Görlich et al+, 1997;Fornerod et al+, 1997b), and have been termed importins/exportins or karyopherins+ The interaction of these b-related receptors with their cargoes or with nucleoporins is regulated by the binding of Ran to the receptor (reviewed by Mattaj & Englmeier, 1998)+ During export, the binding of RanGTP to the receptor is required for interaction of the receptor with its cargo and probably for the binding of the receptor to the pore+ The opposite situation exists d...…”

Structural and functional analysis of the avian leukemia virus constitutive transport element

“…As noted above, the MPMV CTE directly recruits the Tap nuclear export factor whereas the HIV-1 RRE:Rev complex instead recruits Crm1+ Previously, we and others have shown that a truncated form of the Nup214/ CAN nucleoporin, termed ⌬CAN, can directly bind to Crm1 and block its ability to target the Rev:RRE complex to the nuclear pore (Fornerod et al+, 1997b;Bogerd et al+, 1998;Zolotukhin & Felber, 1999)+ In contrast, ⌬CAN exerts no effect on Tap-dependent RNA export or on cellular mRNA export+ To examine whether Crm1 plays a role in mediating ALV CTE function, we tested the effect of ⌬CAN expression on ALV CTE or Rev:RRE function in transfected QCl-3 cells+ As shown in Figure 4, ⌬CAN effectively blocked Rev:RRE function, as expected, yet had no effect on the activity of the ALV CTE+ Similar data were obtained using the drug leptomycin B, which is also a selective inhibitor of Crm1 function (data not shown)+ We therefore conclude that the ALV CTE, like the MPMV CTE, functions independently of Crm1+…”

Crm1 (XpoI) dependent nuclear export of the budding yeast transcription factor yAP‐1 is sensitive to oxidative stress

“…The importin ␣ subunit acts as a receptor by binding directly to the NLS of proteins bound for transport to the nucleus (for a review Nigg 1997). In the case of protein export, recent evidence suggests that one of the receptors that binds to the NES is the Crm1 protein (Fornerod et al 1997a;Fukuda et al 1997;OssarehNazari et al 1997;Stade et al 1997), which also associates with the nuclear pore complex (Fornerod et al 1997b). Overexpression of Crm1 enhances the nuclear export of NES-containing proteins (Fornerod et al 1997a) whereas leptomycin B, which binds and inactivates Crm1, represses this export (Fornerod et al 1997a;Fukuda et al 1997).…”

“…Overexpression of Crm1 enhances the nuclear export of NES-containing proteins (Fornerod et al 1997a) whereas leptomycin B, which binds and inactivates Crm1, represses this export (Fornerod et al 1997a;Fukuda et al 1997). Crm1 (also known as Xpo1) is a highly conserved protein (Toda et al 1992;Fornerod et al 1997b;Kudo et al 1997) and its role in protein export has now been described in mammalian (Fornerod et al 1997a;Fukuda et al 1997;Ossareh-Nazari et al 1997), amphibian (Fornerod et al 1997a;Fukuda et al 1997), fission yeast (Fukuda et al 1997;Kudo et al 1997) and budding yeast systems (Neville et al 1997;Stade et al 1997).…”