NXT1 is necessary for the terminal step of Crm1-mediated nuclear export.
Soluble factors are required to mediate nuclear export of protein and RNA through the nuclear pore complex (NPC). These soluble factors include receptors that bind directly to the transport substrate and regulators that determine the assembly state of receptor-substrate complexes. We recently reported the identification of NXT1, an NTF2-related export factor that stimulates nuclear protein export in permeabilized cells and undergoes nucleocytoplasmic shuttling in vivo (Black, B.E., L. Lévesque, J.M. Holaska, T.C. Wood, and B.M. Paschal. 1999. Mol. Cell. Biol. 19:8616-8624). Here, we describe the molecular characterization of NXT1 in the context of the Crm1-dependent export pathway. We find that NXT1 binds directly to Crm1, and that the interaction is sensitive to the presence of Ran-GTP. Moreover, mutations in NXT1 that reduce binding to Crm1 inhibit the activity of NXT1 in nuclear export assays. We show that recombinant Crm1 and Ran are sufficient to reconstitute nuclear translocation of a Rev reporter protein from the nucleolus to an antibody accessible site on the cytoplasmic side of the NPC. Further progress on the export pathway, including the terminal step of Crm1 and Rev reporter protein release, requires NXT1. We propose that NXT1 engages with the export complex in the nucleoplasm, and that it facilitates delivery of the export complex to a site on the cytoplasmic side of NPC where the receptor and substrate are released into the cytoplasm.
Black
B E
BE
Center for Cell Signaling, Department of Biochemistry and Molecular Genetics, Cell and Molecular Biology Program, University of Virginia, Charlottesville, Virginia 22908, USA.
Holaska
J M
JM
Lévesque
L
L
Ossareh-Nazari
B
B
Gwizdek
C
C
Dargemont
C
C
Paschal
B M
BM
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D028884:Karyopherins; D016296:Mutagenesis; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D000097606:Exportin 1 Protein
D002352:Carrier Proteins; D015694:Gene Products, rev; D028884:Karyopherins; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D012343:RNA, Transfer; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D000097606:Exportin 1 Protein; C402484:Nxt1 protein, mouse; C405483:U1 small nuclear RNA