Interaction of the Nuclear GTP-Binding Protein Ran with Its Regulatory Proteins RCC1 and RanGAP1

Klebe, Christian; Bischoff, F. Ralf; Ponstingl, Herwig; Wittinghofer, Alfred

doi:10.1021/bi00002a031

Cited by 329 publications

(341 citation statements)

References 66 publications

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“…(22), we examined the effect of Kap95p on the binding of guanine nucleotides to Gsp1p. First, we examined the effect of Kap95p on the Gsp1p-GDP dissociation by monitoring the loss of Gsp1p-bound ]GDP was stable over 60 min, whereas it was strongly destabilized in the presence of EDTA, consistent with previous observations (3,23 . Ran has a 10-fold higher affinity for GDP compared with GTP (23); therefore, this result suggested that upon formation of the Kap95p⅐Gsp1p complex, Gsp1p adopted a conformational state that favored the binding of GTP over GDP.…”

Section: Methodssupporting

confidence: 75%

“…1). However, although RCC1 has almost no preference for the nucleotide state of Ran and catalyzes the nucleotide exchange in both directions (23), nucleotide exchange by Kap95p (importin-␤) is unidirectional because of the stable association of Kap95p (importin-␤) with Gsp1p-GTP (Ran-GTP). The stability of the nucleotide-free Ran⅐Kap95p complex, demonstrated by the inability of Kap60p to dissociate it (Fig.…”

Section: Kap95p (Importin-␤) As a Nucleotide Exchange Factor; Comparimentioning

confidence: 99%

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Importin-β Is a GDP-to-GTP Exchange Factor of Ran

Lonhienne

Forwood

Marfori

et al. 2009

Journal of Biological Chemistry

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Ran-GTP interacts strongly with importin-␤, and this interaction promotes the release of the importin-␣-nuclear localization signal cargo from importin-␤. Ran-GDP also interacts with importin-␤, but this interaction is 4 orders of magnitude weaker than the Ran-GTP⅐importin-␤ interaction. Here we use the yeast complement of nuclear import proteins to show that the interaction between Ran-GDP and importin-␤ promotes the dissociation of GDP from Ran. The release of GDP from the Ran-GDP-importin-␤ complex stabilizes the complex, which cannot be dissociated by importin-␣. Although Ran has a higher affinity for GDP compared with GTP, Ran in complex with importin-␤ has a higher affinity for GTP. This feature is responsible for the generation of Ran-GTP from Ran-GDP by importin-␤. Ran-binding protein-1 (RanBP1) activates this reaction by forming a trimeric complex with Ran-GDP and importin-␤. Importin-␣ inhibits the GDP exchange reaction by sequestering importin-␤, whereas RanBP1 restores the GDP nucleotide exchange by importin-␤ by forming a tetrameric complex with importin-␤, Ran, and importin-␣. The exchange is also inhibited by nuclear-transport factor-2 (NTF2). We suggest a mechanism for nuclear import, additional to the established RCC1 (Ran-guanine exchange factor)-dependent pathway that incorporates these results.Ran (Gsp1p in yeast) is a Ras-like GTPase that regulates diverse cellular processes, including nuclear transport, mitotic spindle assembly, and post-mitotic nuclear assembly (1, 2). Like other GTPases, Ran can bind GTP and GDP. Ran-GTP is generated in the nucleus by the guanine exchange factor RCC1 (regulator of chromosome condensation 1), which is associated with the chromatin (3). Ran-GDP is produced in the cytoplasm by the activation of the intrinsic GTPase activity of Ran by Ran-GAP1 (GTPase-activating protein) (4) and RanBP1 (Ran-binding protein-1, Yrb1p in yeast). The compartmentalization of RanGAP1 (cytoplasm) and RCC1 (nucleus) gives rise to the asymmetric distribution of Ran-GDP (cytoplasm) and Ran-GTP (nucleus) across the nuclear envelope. This asymmetric distribution of Ran-GDP and Ran-GTP plays a central role in nucleocytoplasmic transport by mediating assembly and disassembly of import and export complexes through interaction with the nuclear import machinery (for reviews, see Refs. 5-9).The passage of molecules into the nucleus occurs through the nuclear pore complexes (NPCs) 6 (10). Nucleocytoplasmic transport is driven by a series of protein-protein interactions and involves several soluble carriers named ␤-karyopherins. Import carriers are called importins and export carriers are called exportins. The classical nuclear import pathway involves importin-␤ (Kap95p in yeast) and the adaptor protein importin-␣ (Kap60p in yeast). In the cytoplasm importin-␤ binds to importin-␣. Their interaction triggers a conformational change of importin-␣ that increases its affinity for cargo proteins containing a nuclear localization signal (NLS) (11,12). The translocation of the resulting complex (import...

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Section: Methodssupporting

confidence: 75%

Section: Kap95p (Importin-␤) As a Nucleotide Exchange Factor; Comparimentioning

confidence: 99%

Importin-β Is a GDP-to-GTP Exchange Factor of Ran

Lonhienne

Forwood

Marfori

et al. 2009

Journal of Biological Chemistry

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“…Ran acetylation only marginally affects the intrinsic nucleotide hydrolysis rate. The intrinsic GTP hydrolysis rate of Ran is very slow (5.4 × 10 −5 s −1 at 37°C) and would not be of biological significance if not 10 5 -fold (2.1 s −1 at 25°C) accelerated by RanGAP (31). We speculated that acetylation of lysines in the switch regions (K37 R and K71 R ) modulates intrinsic and/or RanGAP-mediated GTP hydrolysis.…”

Section: Ran Acetylation Impairs the Rcc1-catalyzed Nucleotide Exchangementioning

confidence: 98%

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Boor

Knyphausen

Kuhlmann

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular Ran•GTP/Ran•GDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed.Ran | lysine acetylation | genetic code expansion concept | nucleus | nuclear cytosolic transport T he small GTP-binding protein Ran (Ras-related nuclear) is involved in nucleo-cytoplasmic transport processes, nuclear envelope formation, and the formation of the mitotic spindle (1). Ran, furthermore, has a variety of cytosolic functions and is involved in the cross-talk with the actin cytoskeleton. As a member of the Ras superfamily, Ran is structurally composed of a fold known as the G-domain (GTP-binding domain), a central sixstranded β-sheet that is surrounded by α-helices. Ras-family members bind to GTP and GDP nucleotides with high picomolar affinity. However, only in the GTP-bound form and the switch Iand switch II-loops adopt a stable conformation. Ran has been structurally characterized in great detail, including different nucleotide states and various protein complexes (2-4).In interphase cells, about 90% of cellular Ran is nuclear, and only a minor proportion is cytosolic (5). The localization of the guanine-nucleotide exchange factor (GEF) RCC1 (Regulator of chromosome condensation 1) at the nuclear chromatin and the RanGAP (RanGTPase-activating protein) at the cytosolic site of the nuclear pore creates a gradient of Ran•GTP in the nucleus and Ran•GDP in the cytosol (6-8).In the nucleus, Ran•GTP binds to exportins such as CRM1 (Chromosome region maintenance 1) to transport cargo proteins containing a nuclear export signal (NES) into the cytosol (3, 9, 10). Ran•GTP, furthermore, binds to Importin-β•cargo complexes to release the cargo in the nucleus (11-15). In the cytosol, the Importin•Ran•GTP complexes, as well as the ternary exportin•Ran•GTP-cargo complexes, dissociate on binding of RanBP1 and subsequent GTP hydrolysis catalyzed by RanGAP (16,17). The Ran transport cycle closes by translocation of Ran•GDP to the nucleus by the nuclear transport factor 2 (NTF2) (4,(17)(18)(19)(20). Many of these Ran interactions also ...

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“…Moreover, asymmetric distribution of Ran, RCC1, RanGAP1 and RanBP1 [46][47][48] results in a steep RanGTP gradient across the nuclear envelope; High RanGTP concentration in the nucleus, whereas high RanGDP concentration in the cytoplasm [30]. Furthermore, Ran is required directly for nuclear export rather than for re-import of potential export mediators [30].…”

Section: Failure Of Exp6 Binding To Rangtp Both In the Replicative Anmentioning

confidence: 99%

Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

Park¹,

Park²,

Lim³

2011

Experimental Cell Research

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Interaction of the Nuclear GTP-Binding Protein Ran with Its Regulatory Proteins RCC1 and RanGAP1

Cited by 329 publications

References 66 publications

Importin-β Is a GDP-to-GTP Exchange Factor of Ran

Importin-β Is a GDP-to-GTP Exchange Factor of Ran

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

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