Fluorescence resonance energy transfer biosensors that detect Ran conformational changes and a Ran x GDP-importin-beta -RanBP1 complex in vitro and in intact cells.
The Ran GTPase plays a central role in nucleocytoplasmic transport. Association of Ran x GTP with transport carriers (karyopherins) triggers the loading/unloading of export or import cargo, respectively. The C-terminal tail of Ran x GTP is deployed in an extended conformation when associated with a Ran binding domain or importins. To monitor tail orientation, a Ran-GFP fusion was labeled with the fluorophore Alexa546. Fluorescence resonance energy transfer (FRET) occurs efficiently between the green fluorescent protein (GFP) and Alexa546 for Ran x GDP and Ran x GTP, suggesting that the tail is tethered in both states. However, Ran x GTP complexes with importin-beta, RanBP1, and Crm1 all show reduced FRET consistent with tail extension. Displacement of the C-terminal tail of Ran by karyopherins may be a general mechanism to facilitate RanBP1 binding. A Ran x GDP-RanBP1-importin-beta complex also displayed a low FRET signal. To detect this complex in vivo, a bipartite biosensor consisting of Ran-Alexa546 plus GST-GFP-RanBP1, was co-injected into the cytoplasm of cells. The Ran redistributed predominantly to the nucleus, and RanBP1 remained cytoplasmic. Nonetheless, a robust cytoplasmic FRET signal was detectable, which suggests that a significant fraction of cytoplasmic Ran.GDP may exist in a ternary complex with RanBP1 and importins.
Plafker
Kendra
K
Center for Cell Signaling and the Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0577, USA.
Macara
Ian G
IG
D000818:Animals; D015374:Biosensing Techniques; D002460:Cell Line; D006224:Cricetinae; D004735:Energy Transfer; D006153:Guanosine Diphosphate / Q000737:chemistry*; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D028961:beta Karyopherins / Q000737:chemistry*; D020931:ran GTP-Binding Protein / Q000737:chemistry*
D009687:Nuclear Proteins; D028961:beta Karyopherins; C091775:ran-binding protein 1; D006153:Guanosine Diphosphate; D020931:ran GTP-Binding Protein