The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP.
Nuclear export of intron-containing human immunodeficiency virus type 1 (HIV-1) RNA is mediated by the viral Rev protein that contains both an RNA binding domain specific for the viral Rev response element (RRE) and a nuclear export signal (NES). The cellular CRM1 (Exportin1) protein functions as a nuclear export receptor for proteins carrying a Rev-like NES in a process that also requires the GTP bound form of the Ran GTPase. Using purified recombinant factors, we show by co-precipitation, gel mobility shift and protein footprinting assays that full-length Rev protein interacts directly with CRM1 in vitro independently of both the integrity of the characteristic leucine residues of the NES and the presence of the cytotoxin leptomycin B (LMB). Addition of RanGTP induces the formation of an RRE-Rev-CRM1-RanGTP complex that is sensitive to LMB, NES mutations, and Ran being charged with GTP. Within this complex, CRM1 is readily cross-linked to Cys89 near the NES of Rev. By protein footprinting, we demonstrate that the NES of Rev and two regions in CRM1 become inaccessible to endoproteinases upon binding suggesting that these regions are involved in protein-protein interactions. Our data are consistent with a model in which CRM1 is the nuclear export receptor for the Rev-RRE ribonucleoprotein complex and that RanGTP binds to a preformed Rev-CRM1 complex and specifies a functional interaction with the NES.
Askjaer
P
P
Department of Molecular and Structural Biology, University of Aarhus, C. F. Mollers Allé, Building 130, DK-8000 Aarhus C, Denmark.
Jensen
T H
TH
Nilsson
J
J
Englmeier
L
L
Kjems
J
J
D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism* / Q000528:radiation effects; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers; D015694:Gene Products, rev / Q000378:metabolism* / Q000528:radiation effects; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D028884:Karyopherins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014466:Ultraviolet Rays; D000097606:Exportin 1 Protein
D002352:Carrier Proteins; D017931:DNA Primers; D015694:Gene Products, rev; D006160:Guanosine Triphosphate; D028884:Karyopherins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D000097606:Exportin 1 Protein