Different Structural and Kinetic Requirements for the Interaction of Ran with the Ran-Binding Domains from RanBP2 and Importin-
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- 作者：Carolina I. Villa Braslavsky ; Christine Nowak ; Dirk Gö ; rlich ; Alfred Wittinghofer ; and Jü ; rgen Kuhlmann
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：September 26, 2000
- 年：2000
- 卷：39
- 期：38
- 页码：11629 - 11639
- 全文大小：265K
- 年卷期：v.39,no.38(September 26, 2000)
- ISSN：1520-4995
文摘
The cytoplasmic disassembly of Ran·GTP·importin and Ran·GTP·exportin·cargo complexesis an essential step in the corresponding nuclear import and export cycles. It has previously been shownthat such disassembly can be mediated by RanBP1 in the presence of RanGAP. The nuclear pore complexprotein 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 toinvestigate the dynamic interplay of Ran in its GDP- and GTP-complexed states with RanBDis and withimportin-
. We show that RanBP1 and the four RanBDis from RanBP2 have comparable affinities forRan·GTP (108-109 M-1). Deletion of Ran's C-terminal 211DEDDDL216 sequence weakens the interactionof Ran·GTP with RanBPis approximately 2000-fold, but accelerates the association of Ran·GTP withimportin- 10-fold. Importin- binds Ran·GTP with a moderate rate, but attains a high affinity for Ran(KD = 140 pM) via an extremely low dissociation rate of 10-5 s-1. Association with Ran is accelerated3-fold in the presence of RanBP1, which presumably prevents steric hindrance caused by the RanC-terminus. In addition, we show that the RanBDis of RanBP2 are full equivalents of RanBP1 in thatthey 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 inRan·GTP complexes of importins or exportins that exit the nucleus. This flag is then recognized by themultiple RanBDis at or near the nuclear pore complex, allowing efficient disassembly of these Ran·GTPcomplexes.
. We show that RanBP1 and the four RanBDis from RanBP2 have comparable affinities forRan·GTP (108-109 M-1). Deletion of Ran's C-terminal 211DEDDDL216 sequence weakens the interactionof Ran·GTP with RanBPis approximately 2000-fold, but accelerates the association of Ran·GTP withimportin- 10-fold. Importin- binds Ran·GTP with a moderate rate, but attains a high affinity for Ran(KD = 140 pM) via an extremely low dissociation rate of 10-5 s-1. Association with Ran is accelerated3-fold in the presence of RanBP1, which presumably prevents steric hindrance caused by the RanC-terminus. In addition, we show that the RanBDis of RanBP2 are full equivalents of RanBP1 in thatthey 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 inRan·GTP complexes of importins or exportins that exit the nucleus. This flag is then recognized by themultiple RanBDis at or near the nuclear pore complex, allowing efficient disassembly of these Ran·GTPcomplexes.