The C-terminal extension of the small GTPase Ran is essential for defining the GDP-bound form.
The small GTPase Ran controls cellular processes by interacting with members of the importin beta family that bind specifically to the GTP-bound form of Ran, and this regulates the interaction between importin beta-like proteins and cellular factors. The structures of RanGDP and RanGTP are markedly different, and major structural changes are found in the switch I and switch II regions and in the C-terminal extension of Ran. Here, we show that a deletion mutant of Ran, lacking the entire C-terminal extension, termed Ran Core, can bind to importin beta in its GDP-bound form with high affinity. The ability of Ran CoreGDP to dissociate cargo from importin beta results in an import block in digitonin-permeabilized cells and leads to microtubule aster formation in mitotic Xenopus egg extract. As for importin beta, also transportin, importin 7 and exportin-t can no longer discriminate efficiently between the two nucleotide-bound forms of Ran Core. In contrast, a significant reduction in affinity of the RanGDP-binding protein NTF2 for Ran CoreGDP is observed, indicating that the switch regions have changed conformation in the Ran Core mutant. Our results demonstrate that the C terminus of Ran is a major determinant of the state of Ran, and that removal of this allows the GDP-bound form to adopt a GTP-like conformation, thereby creating a constitutively active protein.
Nilsson
Jakob
J
Department of Molecular and Structural Biology, University of Aarhus, C.F. Møllers Alle, Building 130, DK-8000 Arhus C, Denmark.
Weis
Karsten
K
Kjems
Jørgen
J
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005260:Female; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D028884:Karyopherins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008958:Models, Molecular; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014981:Xenopus; D028961:beta Karyopherins / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D028884:Karyopherins; D011994:Recombinant Proteins; D028961:beta Karyopherins; D006153:Guanosine Diphosphate; D020931:ran GTP-Binding Protein