Kinetic and molecular analysis of nuclear export factor CRM1 association with its cargo in vivo.
The nucleocytoplasmic transport receptor CRM1 mediates the export of macromolecules from the nucleus to the cytoplasm by forming a ternary complex with a cargo molecule and RanGTP. The in vivo mechanism of CRM1 export complex formation and its mobility throughout the nucleus have not been fully elucidated. More information is required to fully understand complex formation and the dynamics of CRM1-cargo-RanGTP complexes in space and time. We demonstrate true molecular interaction of CRM1 with its Rev cargo in living cells by using fluorescence resonance energy transfer (FRET). Interestingly, we found that the inhibitory effect of leptomycin B on this CRM1-cargo interaction is Ran dependent. Using fluorescence recovery after photobleaching (FRAP), we show that CRM1 moves at rates similar to that of free green fluorescent protein in the nucleoplasm. A slower mobility was detected on the nuclear membrane, consistent with known CRM1 interactions with nuclear pores. Based on these data, we propose an in vivo model in which CRM1 roams through the nucleus in search of high-affinity binding sites. CRM1 is able to bind Rev cargo in the nucleolus, and upon RanGTP binding a functional export complex is produced that is exported to the cytoplasm.
Daelemans
Dirk
D
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium. dirk.daelemans@uz.kuleuven.ac.be
Costes
Sylvain V
SV
Lockett
Stephen
S
Pavlakis
George N
GN
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D002466:Cell Nucleolus / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D015694:Gene Products, rev / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D046912:Multiprotein Complexes; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism*; D000097606:Exportin 1 Protein
D015694:Gene Products, rev; D028884:Karyopherins; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein