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Novel G Proteins, Rag C and Rag D, Interact with GTP-binding Proteins, Rag A and Rag B*

  • Takeshi Sekiguchi
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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  • Eiji Hirose
    Affiliations
    Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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  • Nobutaka Nakashima
    Affiliations
    Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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  • Miki Ii
    Affiliations
    Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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  • Takeharu Nishimoto
    Affiliations
    Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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  • Author Footnotes
    * This work was supported by Grants-in-aid for C-2 (10680673) (to T. S.) and Specially Promoted Research from the Ministry of Education, Science and Culture of Japan (to T. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) and .
Open AccessPublished:March 09, 2001DOI:https://doi.org/10.1074/jbc.M004389200
      Rag A/Gtr1p are G proteins and are known to be involved in the RCC1-Ran pathway. We employed the two-hybrid method using Rag A as the bait to identify proteins binding to Rag A, and we isolated two novel human G proteins, Rag C and Rag D. Rag C demonstrates homology with Rag D (81.1% identity) and with Gtr2p ofSaccharomyces cerevisiae (46.1% identity), and it belongs to the Rag A subfamily of the Ras family. Rag C and Rag D contain conserved GTP-binding motifs (PM-1, -2, and -3) in their N-terminal regions. Recombinant glutathione S-transferase fusion protein of Rag C efficiently bound to both [3H]GTP and [3H]GDP. Rag A was associated with both Rag C and Rag D in their C-terminal regions where a potential leucine zipper motif and a coiled-coil structure were found. Rag C and D were associated with both the GDP and GTP forms of Rag A. Both Rag C and Rag D changed their subcellular localization, depending on the nucleotide-bound state of Rag A. In a similar way, the disruption of S. cerevisiae GTR1 resulted in a change in the localization of Gtr2p.AF272035AF272036
      PCR
      polymerase chain reaction
      GST
      glutathione S-transferase
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      GTPγS
      guanosine 5′-3-O-(thio)triphosphate
      DTT
      dithiothreitol
      PBS
      phosphate-buffered saline
      HA
      hemagglutinin
      a.a.
      amino acids
      FITC
      fluorescein isothiocyanate
      EGFP
      enhanced green fluorescent protein
      G proteins are a superfamily of regulatory GTP hydrolases and are composed of a large number of proteins. These include Ras family proteins, hetrotrimeric G protein α subunits, and elongation factors TU and G, among others (
      • Sprang S.R.
      ). Ras-like small G proteins such as Ras, Rab, Rho, ARF, and Ran are monomeric and bind to the guanine nucleotides, GTP or GDP, to function as molecular switches while also playing crucial roles in cell growth, differentiation, and protein traffic between different compartments within the cells (
      • Exton J.H.
      ,
      • Milburn M.V.
      • Tong L.
      • DeVos A.M.
      • Brunger A.
      • Yamaizumi Z.
      • Nishimura S.
      • Kim S.-H.
      ). Ras is a key regulator of cell growth and is an essential component of the signal transduction pathways initiated by receptor tyrosine kinase (
      • Wittinghofer A.
      ). The Rho family members consist of Rho, Rac, and Cdc42 subtypes that control the actin cytoskeleton and that play a role in the regulation of transcription (
      • Deborah J.
      • Mackay G.
      • Hall A.
      ). ADP-ribosylation factors play a role in the vesicular trafficking pathway (
      • Moss J.
      • Vaughan M.
      ). The Rab subfamily plays a role in secretory and endocytic pathways and is located within a distinct cellular compartment (
      • Schimmoller F.
      • Simon I.
      • Pfeffer S.R.
      ).
      Ran is a well characterized nuclear Ras-like small G protein that plays an essential role in the import and export of proteins and RNAs across the nuclear membrane through the nuclear pore complex (
      • Moore M.S.
      ) and also plays a role in the induction of microtubule self-organization inXenopus egg extracts (
      • Carazo S.R.
      • Guarguaglini G.
      • Gruss O.J.
      • Segref A.
      • Karsenti E.
      • Mattaj I.W.
      ,
      • Kalab P.
      • Pu R.T.
      • Dasso M.
      ,
      • Ohba T.
      • Nakamura M.
      • Nishitani H.
      • Nishimoto T.
      ,
      • Wilde A.
      • Zheng Y.
      ,
      • Zhang C.
      • Hughes M.
      • Clarke P.R.
      ,
      • Nishimoto T.
      ). There are a large number of factors that interact with either the GDP-bound form or the GTP-bound form of Ran (Gsp1p), these being nucleoporin, RanBP2/NUP358 (
      • Yokoyama N.
      • Hayashi N.
      • Seki T.
      • Pante N.
      • Ohba T.
      • Nishii K.
      • Kuma K.
      • Hayashida T.
      • Miyata T.
      • Aebi U.
      • Fukui M.
      • Nishimoto T.
      ,
      • Wu J.
      • Matunis M.J.
      • Kraemer D.
      • Blobel G.
      • Coutavas E.
      ), Prp20p interacting protein, RanBP3(Yrb2p) (
      • Noguchi E.
      • Hayashi N.
      • Nakashima N.
      • Nishimoto T.
      ,
      • Taura T.
      • Schlenstedt G.
      • Silver P.A.
      ,
      • Mueller L.
      • Cordes V.C.
      • Bischoff F.R.
      • Ponstingl H.
      ), the exosome involved in ribosomal RNA processing, Dis3p (
      • Noguchi E.
      • Hayashi N.
      • Azuma Y.
      • Seki T.
      • Nakamura M.
      • Nakashima N.
      • Yanagida M.
      • He X.
      • Mueller U.
      • Sazer S.
      • Nishimoto T.
      ,
      • Shiomi T.
      • Fukushima K.
      • Suzuki N.
      • Nakashima N.
      • Noguchi E.
      • Nishimoto T.
      ), microtubule nucleation, RanBPM (
      • Nakamura M.
      • Masuda H.
      • Horii J.
      • Kuma K.I.
      • Yokoyama N.
      • Ohba T.
      • Nishitani H.
      • Miyata T.
      • Tanaka M.
      • Nishimoto T.
      ), and regulators of Ran, RanGAP1 (
      • Matunis M.J.
      • Coutavas E.
      • Blobel G.
      ,
      • Bischoff F.R.
      • Klebe C.
      • Kretschmer J.
      • Wittinghofer A.
      • Ponstingl H.
      ,
      • Bischoff F.R.
      • Krebber H.
      • Kempf T.
      • Hermes I.
      • Ponstingl H.
      ), RanBP1(Yrb1p) (
      • Bischoff F.R.
      • Krebber H.
      • Smirnova E.
      • Dong W.
      • Ponstingl H.
      ,
      • Hayashi N.
      • Yokoyama N.
      • Seki T.
      • Azuma Y.
      • Ohba T.
      • Nishimoto T.
      ), RCC1/RanGEF (
      • Seki T.
      • Hayashi N.
      • Nishimoto T.
      ), and Mog1p (
      • Oki M.
      • Nishimoto T.
      ).
      RCC1 catalyzes guanine nucleotide exchange on Ran (
      • Bischoff F.R.
      • Ponstingl H.
      ) and is located inside the nucleus, bound to chromatin (
      • Ohtsubo M.
      • Okazaki H.
      • Nishimoto T.
      ). The concentration of GTP within the cell is ∼30 times higher than the concentration of GDP, thus resulting in the preferential production of the GTP form of Ran by RCC1 within the nucleus (
      • Moore M.S.
      ). In the cytoplasm, the GTP of Ran is hydrolyzed to GDP through the aid of RanGAP1, which is located within the cytoplasm, thus producing a difference in the concentration of the GTP form of Ran between the nucleus and the cytoplasm. The loss of RCC1 resulted in hamster (tsBN2) and yeast pleiotropic phenotypes, such as premature chromosome condensation, lack of chromosome condensation, the suppression of the receptorless mating process, chromosome instability, an abnormal mRNA metabolism, and mRNA export defects (reviewed in Ref.
      • Seki T.
      • Hayashi N.
      • Nishimoto T.
      ).
      Mutation of GTR1 suppressed the prp20 mutation ofSaccharomyces cerevisiae RCC1, thus suggesting that the function of GTR1 is related to that of the RCC1-Ran (PRP20/MTR1/SRM1-GSP1) system (
      • Nakashima N.
      • Hayashi N.
      • Noguchi E.
      • Nishimoto T.
      ). Rag A is a human homologue of GTR1 as shown by a high sequence similarity and by the fact that the mutation of Rag A suppressed theprp20/mtr1 mutation of S. cerevisiae (
      • Hirose E.
      • Nakashima N.
      • Sekiguchi T.
      • Nishimoto T.
      ). Among the RCC1-Ran system proteins, Gtr1p/Rag A is another subfamily of Ras-like small G proteins (
      • Nakashima N.
      • Noguchi E.
      • Nishimoto T.
      ). Gtr1p is located within both the cytoplasm and the nucleus and has been reported to play a role in cell growth (
      • Bun Y.M.
      • Harashima S.
      • Oshima Y.
      ,
      • Yompakdee C.
      • Bun Y.M.
      • Shikata K.
      • Ogawa N.
      • Harashima S.
      • Oshima Y.
      ). Rag A was originally isolated by polymerase chain reaction (PCR)1 methods during the search for novel G proteins (
      • Schurmann A.
      • Brauers A.
      • Massmann S.
      • Becker W.
      • Joost H.G.
      ) and also independently by two-hybrid screening using adenovirus E3 14.7 kDa as the bait and thus was shown to be involved in apoptosis (
      • Li Y.
      • Kang J.
      • Horwitz M.S.
      ). Rag A and Gtr1p were shown to belong to the sixth subfamily of the Ras-like small GTPase superfamily (
      • Nakashima N.
      • Noguchi E.
      • Nishimoto T.
      ,
      • Schurmann A.
      • Brauers A.
      • Massmann S.
      • Becker W.
      • Joost H.G.
      ). Rag A and Rag B differ by seven conservative amino acid substitutions (98% identity) and by 33 additional residues at the N terminus of Rag B (
      • Schurmann A.
      • Brauers A.
      • Massmann S.
      • Becker W.
      • Joost H.G.
      ). There are three alternative splice variants in Rag B, such as Rag Bs, Rag Bl, and Rag Bn (
      • Hirose E.
      • Nakashima N.
      • Sekiguchi T.
      • Nishimoto T.
      ,
      • Schurmann A.
      • Brauers A.
      • Massmann S.
      • Becker W.
      • Joost H.G.
      ).
      We herein identify two novel Rag A-interacting proteins, Rag C and Rag D. Rag C had GTP-binding motifs in its N-terminal region and was also shown to bind significantly to [3H]GTP and [3H]GDP in vitro. Both Rag A and Rag C had a mutual binding region in their C-terminal regions downstream of the GTP-binding region. These structural features led us to propose that Rag C and Rag D belong to the Rag A subfamily of the Ras-like small G protein superfamily. Rag A and Rag B bound to both Rag C and Rag D. Rag A was also colocalized with Rag C and Rag D in BHK21 cells, thus suggesting that Rag A may form a heterodimer with Rag C or with Rag D.

      DISCUSSION

      We herein demonstrated that the G protein, Rag A, is associated with the novel G proteins, Rag C and Rag D. Human Rag C bound to guanine nucleotides efficiently and specifically, demonstrating that Rag C is in fact a G protein. Ras-like small G proteins are present as monomeric proteins by nature, although Ras-like small G proteins transiently interact with various effectors. Rag A-Rag C association is the first example of a Ras-like small G protein forming a stable heterodimer. It is well known that stable heterodimers and trimers are present in other members of the G protein family, such as trimeric G proteins and α- and β-tubulin, which form trimers and dimers, respectively. Trimeric G proteins act as switches that regulate information-processing circuits (reviewed in Ref.
      • Simon M.I.
      • Strathmann M.P.
      • Gautam N.
      ). Gα is a GTP-binding protein in trimeric G protein and is associated with the non-G proteins, Gβ and Gγ, whereas α- and β-tubulin are both G proteins. It appears that α- and β-tubulin GTPases have separate functions; the α-tubulin GTPase is important for heterodimer formation, whereas the β-tubulin GTPase is important for microtubule assembly. Although the β-tubulin-bound GTP is rapidly hydrolyzed to GDP after microtubule assembly, the α-tubulin-bound GTP is nonexchangeable and is never hydrolyzed (reviewed in Ref.
      • Desai A.
      • Mitchison T.J.
      ), due to strong association of the effector region of α-tubulin with β-tubulin. The association between Rag A and Rag C occurred in the C-terminal regions that were downstream of the effector region.
      In S. cerevisiae, Gtr1p and Gtr2p are associated with each other (
      • Nakashima N.
      • Noguchi E.
      • Nishimoto T.
      ) and are homologous to Rag A and Rag B and to Rag C and Rag D, respectively. To date, homologous genes in Caenorhabditis elegans are reported in each of GTR1(GenBankTM accession number CET24F1-1) and GTR2(CEY24A-a, probably incomplete due to the lack of a C-terminal region). As a result, GTR1 and GTR2 DNA may become duplicated in higher eukaryotes during evolution. Schurmannet al. (
      • Schurmann A.
      • Brauers A.
      • Massmann S.
      • Becker W.
      • Joost H.G.
      ) suggested that Rag A was a member of the superfamily of Ras-like G proteins, which did not belong to any of the previously known subfamilies, but which did represent a sixth subfamily conferring a unique specialized function. Moreover, Rag A/Gtr1p and Gtr2p were shown to belong to a novel subfamily of small G proteins by phylogenetic tree analysis (
      • Nakashima N.
      • Noguchi E.
      • Nishimoto T.
      ). It appears very likely that Rag C and Rag D are also members of the Rag A/Gtr1p subfamily, because G2 and G3 motifs that are different from other G proteins are highly conserved between Rag A/Gtr1p and Rag C/Gtr2p, as shown in Fig.1 b.
      In all G proteins, GTP is bound as a complex with Mg2+. Since many G proteins are unstable in the absence of bound nucleotides, nucleotide affinities are estimated from the rates of nucleotide dissociation (
      • Goody R.S.
      • Frech M.
      • Wittinghofer A.
      ). The off-rates of Ras are in the order of 104 and 105s1 for GDP and GTP, respectively, in the presence of Mg2+ (
      • Feuerstein J.
      • Kalbitzer H.R.
      • John J.
      • Goody R.S.
      • Wittinghofer A.
      ). Mg2+ was also required for GTPγS binding to Rag C. On the other hand, Mg2+ was not required for GDP binding to Rag C. The off-rate of GDP from Rag C was in the order of 102s1, which was different from Ras but which was similar to those of the trimeric G proteins Giα, Goα, and Gsα. Moreover, the GDP complexes of these trimeric G proteins have little affinity for Mg2+. As a result, Rag C seems to have a similar biochemical character to these trimeric G proteins. Rag C had intrinsic GTPase activity, although the GTPase activity was low. We were not able to measure the intrinsic rate of GTP hydrolysis correctly, because Rag C rapidly released GDP.
      We have herein shown that Rag C and Rag A have a substantially higher sequence similarity to spGtr2p (63.3% identity) and spGtr1p (66.4% identity), respectively. As a result, the C-terminal region of spGtr2p can also be expected to be involved in the binding to spGtr1p. The Y190 strain harboring the spGTR2 fragment ranging from 161 to 314 amino acids in pACT2 and the spGTR1 fragment in pAS1 was constructed. As expected, the strain was able to grow on a selection plate (SD, −Leu, −Trp, −His, +3-aminotriazole) (data not shown), thus demonstrating that the C-terminal region of spGtr2p is responsible for binding to spGtr1p.
      Although Gtr1p also interacted with both Gtr2p and Gtr1p (
      • Nakashima N.
      • Noguchi E.
      • Nishimoto T.
      ), we were not able to detect any Rag A interaction with itself (data not shown). This is probably due to the weak homology between Rag A and Gtr1p in the C-terminal region (40% identity). Furthermore, the potential leucine zipper sequences of Rag A (VX 6LX 6IX 6LX 6L) are different from those of Gtr1p (MX 6LX 6MX 6LX 6L). Although Rag A seems to contain two coiled-coil structures in its C-terminal region, Gtr1p contains one coiled-coil structure. It is possible that these structural differences are the reason why Rag A does not form a homodimer.
      Rag A changed its subcellular localization depending on the nucleotide-bound forms, T21L (GDP) and Q66L (GTP), and it seems probable that it shuttles between the nucleus and the cytoplasm (
      • Hirose E.
      • Nakashima N.
      • Sekiguchi T.
      • Nishimoto T.
      ). Whereas wild-type Rag A was soluble in the presence of 0.1% Nonidet P-40 in vivo, the T21L form of Rag A (GDP) was largely insoluble (data not shown), thus suggesting that a conformational change of Rag A resulted in a change in its biochemical character, which then influences its subcellular localization. As shown in Fig. 7,a and b, Rag C and Rag D changed their localization depending on the changes in Rag A localization. Moreover, the removal of Gtr1p resulted in the nuclear localization of Gtr2p (Fig. 8). Rag A/Gtr1p may influence Rag C and Rag D/Gtr2p regarding their subcellular localization, thus suggesting that Rag A/Gtr2p is associated with Rag C and Rag D/Gtr2p. The GTP concentration in the nucleus is about 30 times higher than the GDP concentration, which may result in the preferential production of the GTP form of Rag A within the nucleus. The GTP form of Rag A/Gtr1p is thought to have an ability to bind to and transport Rag C/Gtr2p from the nucleus to the cytoplasm through heterodimer formation.
      We previously showed that the T21L mutant form of Rag A was partially colocalized with the splicing factor SC35 in the nuclear speckle (
      • Hirose E.
      • Nakashima N.
      • Sekiguchi T.
      • Nishimoto T.
      ). Since Rag C was colocalized with the T21L mutation of Rag A when coexpressed, the function of Rag C may thus be related with either transcription or splicing. The prp20/mtr1 mutation of RCC1 was suppressed by the gtr1-11 mutation (GDP form) of Rag A/Gtr1p. It should be noted that the RCC1-Ran system is involved in transcription/splicing (
      • Aebi M.
      • Clark M.W.
      • Vijayraghavan U.
      • Abelson J.
      ). At present, we do not know the molecular mechanism behind the suppression of the prp20/mtr1 mutation of RCC1 by the gtr1-11 mutation of Rag A/Gtr1p. Disruption of GTR2 was able to rescue the prp20/mtr1mutation as well as the gtr1-11 mutation (
      • Nakashima N.
      • Noguchi E.
      • Nishimoto T.
      ). It is possible that an overexpression of the GDP form of Rag A/Gtr1p results in the nuclear accumulation of Rag C/Gtr2p. The inactivation ofGTR2 either by a mutation of GTR1/Rag A or by gene disruption of GTR2 is necessary for the suppression of RCC1 mutation.

      Acknowledgments

      We thank the members of the Nishimoto laboratory for their help and valuable discussions. We also thank J. Fukumura for technical assistance. The English used in this manuscript was revised by K. Miller (Royal English Language Center, Fukuoka, Japan).

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