Different structural and kinetic requirements for the interaction of Ran with the Ran-binding domains from RanBP2 and importin-beta.
The cytoplasmic disassembly of Ran.GTP.importin and Ran.GTP.exportin. cargo complexes is an essential step in the corresponding nuclear import and export cycles. It has previously been shown that such disassembly can be mediated by RanBP1 in the presence of RanGAP. The nuclear pore complex protein RanBP2 (Nup358) contains four Ran-binding domains (RanBDi) that might function like RanBP1. We used biophysical assays based on fluorescence-labeled probes and on surface plasmon resonance to investigate the dynamic interplay of Ran in its GDP- and GTP-complexed states with RanBDis and with importin-beta. We show that RanBP1 and the four RanBDis from RanBP2 have comparable affinities for Ran.GTP (10(8)-10(9) M(-1)). Deletion of Ran's C-terminal (211)DEDDDL(216) sequence weakens the interaction of Ran.GTP with RanBPis approximately 2000-fold, but accelerates the association of Ran.GTP with importin-beta 10-fold. Importin-beta binds Ran.GTP with a moderate rate, but attains a high affinity for Ran (K(D) = 140 pM) via an extremely low dissociation rate of 10(-5) s(-)(1). Association with Ran is accelerated 3-fold in the presence of RanBP1, which presumably prevents steric hindrance caused by the Ran C-terminus. In addition, we show that the RanBDis of RanBP2 are full equivalents of RanBP1 in that they also costimulate RanGAP-catalyzed GTP hydrolysis in Ran and relieve the GTPase block in a Ran.GTP.transportin complex. Our data suggest that the C-terminus of Ran functions like a loose tether in Ran.GTP complexes of importins or exportins that exit the nucleus. This flag is then recognized by the multiple RanBDis at or near the nuclear pore complex, allowing efficient disassembly of these Ran.GTP complexes.
Villa Braslavsky
C I
CI
Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.
Nowak
C
C
Görlich
D
D
Wittinghofer
A
A
Kuhlmann
J
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002384:Catalysis; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D028884:Karyopherins; D007700:Kinetics; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D020931:ran GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism
D004268:DNA-Binding Proteins; D028884:Karyopherins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; C094493:ran-binding protein 2; D006160:Guanosine Triphosphate; D020931:ran GTP-Binding Protein