Ran-binding Protein 1 (RanBP1) Forms a Ternary Complex with Ran and Karyopherin β and Reduces Ran GTPase-activating Protein (RanGAP) Inhibition by Karyopherin β

Lounsbury, Karen M.; Macara, Ian G.

doi:10.1074/jbc.272.1.551

Cited by 134 publications

(133 citation statements)

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“…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).…”

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confidence: 99%

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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confidence: 99%

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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“…In mammalian cells, two proteins, RanBP1 (24) and Nup358 (also called RanBP2) (25,26), can perform this function. RanBP1 contains one copy and Nup358 contains four copies of a highly conserved, 135-residue domain that can bind Ran⅐GTP with high affinity, form a ternary complex with Ran⅐GTP and importins, and co-activate RanGAP (27)(28)(29)(30)(31). This domain is referred to as the Ran-binding domain or RanBD.…”

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confidence: 99%

Random Mutagenesis and Functional Analysis of the Ran-binding Protein, RanBP1

Petersen

Orem

Trueheart

et al. 2000

Journal of Biological Chemistry

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Ran GTPase is required for nucleocytoplasmic transport of many types of cargo. Several proteins that recognize Ran in its GTP-bound state (Ran⅐GTP) possess a conserved Ran-binding domain (RanBD). Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins). In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells. We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51 ts ) in vivo. In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs. However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1. These mutants bound to Ran⅐GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51 ts yeast cells. Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1 ts mutant and did not bind detectably to Ran in vitro. However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to coactivate RanGAP. A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1 ts mutation. When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1 ts cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin-␤ and Ran-GTP. The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran⅐GTP⅐importin-5 complex. Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran⅐GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate Ran-GAP activity toward GTP-bound Ran in these complexes.

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“…Formation of these exportin-Ran⅐GTP-export substrate complexes requires Ran⅐GTP, so assembly would be favored in the nucleus (high [Ran⅐GTP]). After the complexes traverse the nuclear pore, RanGAP-stimulated conversion of Ran⅐GTP to Ran⅐GDP in the cytosol could drive the complexes to disassemble (Gör-lich, 1997;Lounsbury and Macara, 1997). The same mechanism could explain the efficient recycling to the cytosol of karyopherin ␤ importins as Ran⅐GTP complexes (Floer et al, 1997).…”

Section: Introductionmentioning

confidence: 93%

“…The docked complex is then transported through the nuclear pore. Once in the nucleus, the complex could be disrupted by Ran⅐GTP, which in vitro can bind to karyopherin ␤ and displace the import substrate (Rexach and Blobel, 1995;Chi et al, 1996Chi et al, , 1997Gö rlich et al, 1996;Izaurralde et al, 1997;Lounsbury and Macara, 1997;Siomi et al, 1997). Complex assembly would thus be favored in the cytosol (low [Ran⅐GTP]), and disassembly would be favored in the nucleus (high [Ran⅐GTP]).…”

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confidence: 99%

Isolated Mammalian andSchizosaccharomyces pombeRan-binding Domains RescueS. pombe sbp1(RanBP1) Genomic Mutants

Novoa¹,

Rush

D'Eustachio

1999

MBoC

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Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein-mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1 Ϫ yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1 Ϫ yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells. INTRODUCTIONProteins to be transported across the eukaryotic nuclear membrane often contain sequence motifs that function as nuclear import or export signals (Nigg, 1997). Transport requires both insoluble components, associated with the nuclear pore, and soluble components that interact with the transport cargo and/or pore. Among the soluble factors are members of the karyopherin ␤ family, the small GTPase Ran, Ranbinding protein-1 (RanBP1), and nuclear transport factor 2 (Mattaj and Englmeier, 1998). Components of the nuclear pore complex, termed nucleoporins, have been identified by biochemical analysis of partially purified pore complexes and by genetic analysis of nuclear transport in budding yeast (Fabre and Hurt, 1997;Ohno et al., 1998). A pore component that appears to play a central role in interactions with the soluble factors is RanBP2 (also known as Nup358) (Wu et al., 1995;Yokoyama et al., 1995).Many models of nuclear transport have been proposed, and central to them all is the control exerted by Ran in regulating the association and dissociation of transport complexes, linked to the hydrolysis and turnover of Ran-bound guanine nucleotides (Koepp and Silver, 1996;Panté and Aebi, 1996;Rush et al., 1996;Gö rlich, 1997;Nakielny and Dreyfuss, 1997;Nigg, 1997;Yoneda, 1997;Izaurralde and Adam, 1998;Mattaj and Englmeier, 1998;Melchior and Gerace, 1998;Moore, 1998). The intrinsic rates of GTP hydrolysis and guanine nucleotide release by Ran are slow ‡ Corresponding author. E-mail address: deustp01@mcrcr0.med. nyu.edu. Abbreviations used: GFP, green fluorescent protein; HA, hemagglutinin; NES, nuclear export signal; NLS, nuclear localizatio...

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Ran-binding Protein 1 (RanBP1) Forms a Ternary Complex with Ran and Karyopherin β and Reduces Ran GTPase-activating Protein (RanGAP) Inhibition by Karyopherin β

Cited by 134 publications

References 34 publications

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

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

Random Mutagenesis and Functional Analysis of the Ran-binding Protein, RanBP1

Isolated Mammalian andSchizosaccharomyces pombeRan-binding Domains RescueS. pombe sbp1(RanBP1) Genomic Mutants

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