Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1749-1753, February 1995 Biochemistry

Human RanGTPase-activating RanGAPi is a homologue of yeast Rnalp involved in mRNA processing and transport (TC4/RCCl/fi*gl/nucleocytoplasmic transport) F. RALF BISCHOFF, HEIKE KREBBER, TORE KEMPF, INGRID HERMES, AND HERWIG PONSTINGL Division for Molecular Biology of Mitosis, German Cancer Research Center, POB 101949, D-69009 Heidelberg, Germany Communicated by Hans Neurath, University of Washington, Seattle, WA, November 22, 1994

ABSTRACT RanGAPI is the GTPase activator for the parallel to our work on human RanGAP1 we have purified the nuclear Ras-related regulatory protein , converting it to major RanGAP activity from Sc. pombe cells and identified it the putatively inactive GDP-bound state. Here, we report the to be Rnalp. amino acid sequence of RanGAPI, derived from cDNA and peptide sequences. We found it to be homologous to murine Fugl, implicated in early embryonic development, and to MATERIALS AND METHODS Rnalp from Saccharomyces cerevisiae and Schizosaccharomyces Purification of Rnalp from Sc. pombe. The Sc. pombe strain pombe. Mutations of budding yeast RNA] are known to result 972h-, provided by Jorg D. Hoheisel (German Cancer Re- in defects in RNA processing and nucleocytoplasmic mRNA search Center, Heidelberg), was grown to a density of OD6wi transport. Concurrently, we have isolated Rnalp as the major = 1 in M9 minimal medium (18). The cells were collected by RanGAP activity from Sc. pombe. Both this protein and re- 10-min centrifugation at 5000 x g, and spheroblasts were combinant Rnalp were found to stimulate RanGTPase activ- prepared as described by Melchior et al. (19). Spheroblasts ity to an extent almost identical to that of human RanGAP1, from 10 ml of packed cells were lysed by Dounce homogeni- indicating the functional significance of the sequence homol- zation in 50 ml of lysis buffer (20 mM Tris HCl, pH 7.5/1 mM ogy. The Ran-specific guanine nucleotide exchange factor EDTA/1 mM 2-mercaptoethanol) containing protease inhib- RCC1 and its yeast homologues are restricted to the nucleus, itors (1). After centrifugation for 1 hr at 70,000 x g, ammo- while Rnalp is reported to be localized to the cytoplasm. We nium sulfate to 60% saturation was added to the supernatant. suggest a model in which both activities, nuclear GDP-to-GTP The precipitate was removed by centrifugation for 20 min at exchange on Ran and cytoplasmic hydrolysis of Ran-bound 70,000 x g, and the supernatant was directly chromatographed GTP, are essential for shuttling of Ran between the two on a Fractogel EMD AFTA 650/S column (10 x 50 mm; cellular compartments. Thus, a defect in either of the two Merck) at a flow rate of 2 ml/min, applying a linear gradient antagonistic regulators of Ran would result in a shutdown of from 60% saturated ammonium sulfate in lysis buffer to lysis Ran-dependent transport processes, in agreement with the buffer containing 100 mM NaCl. The buffer of fractions con- almost identical phenotypes described for such defects in taining RanGAP activity was changed on a Nap 25 column budding yeast. (Pharmacia) to 100 mM NaCl in lysis buffer, and the sample was applied to a Mono Q column (HR 5/5; Pharmacia) equil- Ran is a Ras-related, mainly nuclear protein (1, 2) which in its ibrated in the same buffer. Pure RanGAP/Rnalp was eluted GTP-bound form is thought to represent the ON state of a at "400 mM NaCl, using a linear gradient to 1 M salt in lysis regulatory pathway involved in the onset of mitosis (3, 4), buffer. RanGAP assays using Ran [y-32P]GTP have been done initiation of S-phase (4), exit from mitosis (5), import of as described (16). with nuclear localization signals into the nucleus (6, Generation of Fragments and Amino Acid Sequence Deter- 7), maintenance of nuclear structure, and pre-mRNA process- mination. Purified human RanGAP1 was cleaved with trypsin ing and export into the cytoplasm (8, 9). Homologues of Ran (sequencing grade; Boehringer Mannheim) in 100 mM am- have been identified in many different species (for review, see monium bicarbonate, pH 8.3/1 M guanidinium chloride for 16 ref. 10). Activation of Ran requires the exchange of bound hr at 37°C. Rnalp isolated from Sc. pombe was incubated for GDP for GTP, which is stimulated by its specific nucleotide 16 hr at 37°C with endoproteinase Lys-C (sequencing grade; exchange factor (11), designated RCC1 in mammals (12), Boehringer Mannheim) in 50 mM Tris-HCl, pH 8.5/10% SRM1/PRP20/MTR1 in Saccharomyces cerevisiae (9, 13, 14), (vol/vol) acetonitrile. Cleavage with CNBr, separation of pep- and piml in Schizosaccharomyces pombe (5, 15). Suppression tides by reversed-phase chromatography, and sequence deter- of the phenotypes that result from temperature-sensitive mu- mination was done as described (16). tations in the RCC1 homologues of Sc. pombe and Sa. cerevi- Cloning of Human RanGAP1. Based on the peptide se- siae, by overexpressing the corresponding Ran homologues (5, quences of purified RanGAP1 indicated in Fig. 1, degenerate 8, 9, 15), indicates that RCC1 and Ran participate in the same oligonucleotides were synthesized containing inosine in the regulatory pathway. wobble position at a degeneracy of three or four. PCR ampli- Ran has a very low intrinsic GTPase activity. It is stimulated fication with a HeLa Agtll cDNA library (Clontech) resulted 105-fold by the Ran GTPase activator RanGAP1 (16, 17), in a 800-bp fragment that was cloned into pBluescript KS' which we have purified from HeLa cells. This protein is an after digestion with EcoRI and BamHI. The 800-bp EcoRI- antagonistic regulator to RCC1, converting Ran into its GDP- BamHI fragment that was found to code for RanGAP1, as bound putative OFF form. Here we report the determination of verified by the sequences of additional RanGAP1 peptides, its amino acid sequence, based on data from peptides and was used to generate a hybridization probe labeled with cDNA.t The completed sequence revealed homologies to mu- [a-32P]ATP using the DECAprimeII-DNA-labeling kit (Am- rine Fugl and to Sa. cerevisiae and Sc. pombe Rnalp. In bion, Austin, TX). A HeLa Agtll cDNA library was screened as in ref. 20. Cross-hybridizing clones were purified and used The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in tThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. EMBL/GenBank data base (accession no. X82260). 1749 Downloaded by guest on September 25, 2021 1750 Biochemistry: Bischoff et aL Proc. Natl. Acad Sci. USA 92 (1995) for DNA isolation. Inserts cut with EcoRI were isolated from shaker. After the time intervals indicated in Fig. 5, 10 ,lI of agarose gels using the JetSorb-DNA-elution kit (Genomed, tsBN2 lysate and 10 ,ul of a 1:4 dilution of BHK21 lysate in lysis Bad Oeynhausen, Germany) and subcloned into pBluescript buffer, respectively, were added to 70 ,ul of 1 ,uM Ran- KS'. Both strands of the inserts were sequenced by using the [3H]GDP in incubation buffer containing 250 ,tM unlabeled T7Sequencing kit (Pharmacia). GDP. Ran-bound radioactivity was determined after 10-min Cloning and Expression of Sc. pombe RNA1. Oligonucleo- incubation at 25°C on a rotation shaker by filtering the sample tides representing the N terminus (5'-CGGAATTCATAT- through nitrocellulose as described (1). GTCGCGTTTTTCAATA-3') and C terminus of Rnalp (5'- CGGGATCCCTAAATATGAGCTTTTGA-3') were used to RESULTS AND DISCUSSION amplify the coding sequence from Sc. pombe genomic DNA, which was provided by Jorg D. Hoheisel. The resulting 1161-bp Human RanGAPi Is Structurally Homologous to Murine Nde I-BamHI fragment was ligated into the pET3a expression Fugl and to Yeast Rnalp. RanGAP1 was purified from HeLa vector. After transformation into Escherichia coli BL21(DE3), cells as a homodimeric 65-kDa protein (16) and cleaved into protein expression was induced for 2 hr with 0.5 mM isopropyl peptides that were sequenced. The partial amino acid se- /3-D-thiogalactoside after bacteria were grown at 37°C to an quences (Fig. 1) were used to design degenerate primers, and OD6wo of 0.4 in a 4-liter culture. Cells were harvested and lysed a 800-bp DNA fragment was obtained from a HeLa kgtll as described (16). Recombinant Rnalp was purified from the cDNA library by PCR. By screening the same library with the soluble fraction as described above for endogenous Rnalp. amplification product, a cDNA of 2.9 kb could be isolated. The However, a larger proportion of recombinant Rnalp already nucleotide sequence comprises an open reading frame coding precipitated at <60% ammonium sulfate saturation. for a protein of 587 amino acid residues and a calculated Mr Determination of Ran-Specific Guanine Nucleotide Ex- of 63,577. The primary structure suggests an N-terminal do- change Activity in tsBN2 and BHK21 Cells. Ran-[3H]GDP was main of 358 residues, separated by an extremely acidic string prepared by incubating 2 ,tM bacterially expressed Ran-GDP of some 40 residues from a C-terminal region of 190 residues. (16) with 10 ,tM [3H]GDP (10 Ci/mmol; NEN; 1 Ci = 37 GBq) The sequence appears to be unrelated to that of GTPase in 25 mM Mes, pH 6.5/500 mM NaCl/1 mM 2-mercaptoetha- activators for other Ras-related proteins, but it is highly ho- nol/10% (vol/vol) glycerol/10 mM EDTA for 30 min on ice. mologous (88% identical residues) to that of Fugl (21), the The buffer was changed to 20 mM Hepes-NaOH, pH 7.4/5 mM murine homologue ofyeast Rnalp (Fig. 1). Fugl was identified MgCl2/0.05% hydrolyzed gelatin on a NAP-5 column (Phar- by -disruption experiments, with homozygous mutant macia). About i07 adherent BHK21 and tsBN2 cells grown at mouse embryos arresting at the egg cylinder stage at about day 30°C as described (12) were collected in the logarithmic- 6 of embryonic development. This arrest concurs with the growth phase with a rubber policeman. After centrifugation at normal onset of fugl expression, as determined by Northern 1000 x g, cells were sonicated three times for 5 sec (Branson blot analysis (21). Because the corresponding RNA1 in sonifier B15, microtip, 30% cycle, output control 3) in 1 ml of the yeasts Sa. cerevisiae and Sc. pombe (see below) were shown lysis buffer, resulting in complete fragmentation of nuclei. to be essential for growth (19, 23), requirement for a functional Aliquots of the lysates were preincubated at 39°C on a rotation RNA1 homologue in early mammalian embryonic develop-

0 0 0 0 0 0 0 hum. 1 MASEDIAKLAETLAK,YAESFKGKSLKLN--TAEDAKDVIKEIEDFDSL-EALRLESNTVGVEA------ARVIAKALEKKSELKRCHWSDMFTGRLRTEIPPALISLGEGLITAGAQ I1111I mur. 1 MASEDIAKLAETLAKTQVAGGQLSFKGKGLKLN- -TAEDAKDVIKEIEEFDGL-EALRLEGNTVGVEA------ARVIAKALEKSELKRCHWSDMFTGRLRSEIPPALISLGEGLITAGAQ 11 III 11 III 11 11 1 1111 III 11 S.p. 1 MSRFSIEGKSLKLDAITTEDEKSVFAVLLEDDSVKE-IVLSGNTIGTEA----ARWLSENIASKKDLEIAEFSDIFTGRVKDEIPEALRLLLQALLK-CPK 11 11 III 11 III11111 11 I 11 11 III 11 S.c. 1 MATLHFVPQHttEEEVYSISGKALKL--- -TTSDDIKYLEirELAALKTCTKLDLSGNTIGTEASEALAKCIAENTQVRESLvEvWFADLYTSRLVDEVVDSLRFLLPVLLK-CPH

0 0 0 0 0 0000 hum. 114 LVELDLSDNAFGPDGVQGFEALLKSSACFT-LQELKLNNCGMGI-GGGKILAAALTECHRKSSAQGKP-LALKVFVAGR RYI-P G IT A L A mur. 114 LVELDLSDNAFGPDGVRGFEALLKSPACFT-LQELKLNNCGMGI -GGGKILAAALTECHRKSSAQGKP-LRLKVFVAGRNRLENDGATALAEAGI I-GTLEEVHMPQNGINHPGVTALA 11111111 11 11 11 1111111 1111 S.p. 96 LHTVRLSDNAFGP---TAQEPLIDFLS = N :PAGAKI-_ L RPLRS--IICGRNRLENGSNWAKTEQS-BLHTVKMVQNGIRPEGIEHLL 1111111 11 III 11 11 11 11 MI1 11111111111 11 11 111111 S.C. 110 LEIVNLSDNAFGL--- -RTIELLEDYIAHAVNIKHLILSNNGMGPFAGERI -GKALFHLAQNKKAASKPFLET- -FICGRNRLENGSAVYLAILGLKSHSEGLKVVKLYQNGIRPKGVAT,LI

'D 0 ~~0 0 0 0 0 0 hum. 230 -, NTFTEKGETLKTLRQ-VEVINFGDCLVRSKGAVAIADAI-RGGLPKLKELNLSFCEIKRDAALAVAEAMADK---AELEKLDLNGNTLGEEGCEQLQE

mur. 230 -QAFAINPLLRVI:NLNNTFMKGGVAMAETLKTLRQ-VEVINGIDCLVRSKGAVAIADAV-RGGLPKLKELNi1LSFCEIKRDAALVVAEAVADK---AELEKLDLNGNAIGEEGCEQLQE 11111 11 III 11 11 1I 1111 11 S.p. 209 LEGLAYCQELKVLDLQ IFTHLGSSALATAII I-LELG 1IDCLLSARGAAAVVDAFSKLENIGLQTLRLQYNEIELDAVRTLKTVI--- -MPDLLFLELNGNRFSEEDDVVDEI 111 111111111 11 11 11 111111 III 11 11 MI1 S.C. 224 HYGLQYLKNLEILDLQDNTFTKHASLILAKALPTWKDSLFEININDCLLKTAGSDEVFKi,vF,iTEvKFPNLHVLKFEYNEKAQETIEVSFLPAMEKGNLPELEKLEINGNRLDEDSDALplLI

hum. 344 VLEGFNMAKVALSDDE--- -DEEEEEEGE-EEEEPPAEEEEEEDEEEEEEEEEEEEEEPQQRGQGEKSATPSRKILDPNTGEPAPVLSSPPPA.DVSTFILAFPSPEK.LLGPKSSVLIAQ I11II1111 11111111 11 1 111 11111 11111111111 11111111111 Ill1111111 111111 11111111111 1111 1 mur. 344 VMDSFN4MAVLASLSDDE-GEDEDEEEBGE -EDDEEE:EDEEEEDDDE H:l;R QE'PEPPPQRGSGEEPATPSRKILDPNSGEPAPVLSSPTPTDLSTFLSFPSPEKLLRLGPKVSVLIVQ 11 11111 S.p. 326 REVFSTRGRGELDELDDM-=EED:-QSE SE TSt;n;DKEL ADELSKHI. 386 11 111 111 1 11 S.c. 344 QSKFDDLEVDDFEEVDSEDEEGEDEEDEDEDEKLEEIETERLEKELLEVQVDDLAERLAETEIK 407

hum. 460 QTDTSDPEKVVSAF RKAFNSSSFNSNTFLTRLLVHIGLLKSED1VaIANLYGPLMALNHMVQQDYFPKALAPLLLAFVTKPNSALESCSFARHS mur. 462 QTSDPEKVVSAFLVVFRDDASVKTAVLDAIDAAFSCSSFNSNTFLTRLLIHMGLLKSEDKIKAIPSLHGP]VINHVVQDYFPKALAPLLLAFVTKPNGALETCSF

hum. 580 LLQTLYKV 587 111111 nur. 582 LLQTLYNI 589 FIG. 1. Sequence alignments of human RanGAPl (hum.), murine Fugl (mur.) (21), Sc. pombe Rnalp (S.p.) (19), and Sa. cerevisiae Rnalp (S.c.) (22) in single-letter amino acid notation. Residues identified for human RanGAPi and Sc. pombe Rnalp by peptide sequencing are underlined; vertical lines indicate identical residues. Asterisks indicate peptide-derived sequences used to construct degenerate primers for PCR. 0, Leucine residues conserved in all four species. Downloaded by guest on September 25, 2021 Biochemistry: Bischoff et aL Proc. NatL Acad Sci USA 92 (1995) 1751 M 1 2 3 4 5 -0.8 , kDa

-0.4 Z 67 _ -0.0 43 1 -w MOWWO 36 f

29 m 20 - 80 ° 14 40O -40 FIG. 3. Purification of RanGAP activity from Sc. pombe cells. --7 -O * Coomassie-stained proteins of fractions obtained during purification Ift c I A 1 9 OM U J iU 1J LU after separation on a 12% SDS gel (the lane numbers refer to fractions in Table 1). In lane 4, most of the 44-kDa band consists of comigrating Volume, ml phosphoglycerate kinase as determined by sequencing. FIG. 2. Final purification of Sc. pombe RanGAP. After hydropho- bic chromatography, RanGAP-containing fractions were subjected to 1). Using PCR primers based on the published DNA sequence ion-exchange chromatography on a Mono Q column. RanGAP activ- (19), we amplified the coding region of RNA1 and expressed ity (hatched bars) is expressed as the percentage of Ran-bound Rnalp in E. coli. The specific RanGAP activity of the purified [-y-32P]GTP hydrolyzed within 5 min after addition of the fraction. recombinant protein was comparable to that determined for mnent would be assumed. It could be provided by maternal Rnalp purified from Sc. pombe and for the HeLa homologue contribution of Fugl protein or by a functionally redundant RanGAP1 (Fig. 4b). Thus, posttranslational modification does Fugl homologue preferentially expressed up to this stage of not seem to be required for RanGAP activity. As both yeasts development. are devoid of the C-terminal domain present in mammals, the The high degree of sequence homology of RanGAP1 and interaction with Ran should occur through the N-terminal Fugl led us to assume that RanGAP1 represents the HeLa cell domain. It is rich in leucines (19), and they appear to play Fugl homologue, but the limited sequence homology to Rnalp important structural roles, as 25 leucine positions are con- in Sa. cerevisiae (26% homology) and Sc. pombe (35% homol- served in the RanGAP homologues of all four species. A motif ogy in the overlapping sequence) and the lack of a sequence LxxxXLxDNxF is found twice in all four proteins in homolo- in the yeast homologues corresponding to the C-terminal 185 gous positions (in human RanGAP1 at positions 119-129 and residues in RanGAP1 and Fugl did not warrant the conclusion 243-253). Two additional conserved residues are altered in the that they were the GTPase-activating proteins for the corre- mal-1 mutant of the budding yeast (22), which results in block sponding Ran homologues. of RNA processing and export at the nonpermissive temper- Rnalp Represents the Major RanGAP Activity in Sc. pombe. ature. They are Ser-17 -- Phe (in human RanGAP1 correspond- In parallel to our work on human RanGAP we decided to ing to Ser-24) and Ala-194 -- Val (in human RanGAP corre- purify the corresponding RanGAP from Sc. pombe. The high sponding to Ala-201). degree of homology among Ran sequences from widely dif- Implications of Intracellular Localization of RanGAP for a ferent species indicated functional conservation throughout Model of the Ran Pathway. Immunofluorescence and cell- evolution, and using human Ran-GTP as a substrate, we could fractionation studies in Sa. cerevisiae (24) and Sc. pombe (19) a in crude lysates ofSc. indeed detect specific RanGAP activity revealed Rnalp to be located exclusively in the cytoplasm, pombe cells. This activity was enriched >300-fold from the soluble fraction of logarithmically growing Sc. pombe cells, whereas the Ran-specific guanine nucleotide exchange factor and its are considered to be restricted to yielding a 44-kDa protein on final purification on Mono Q RCC1 homologues columns (Figs. 2 and 3, Table 1). The 44-kDa protein specif- the nucleus (1, 25-27). Cell-fractionation studies with HeLa ically stimulates hydrolysis of Ran-bound GTP, whereas it does cells revealed that in addition to soluble cytoplasmic RanGAP not activate Ras GTPase, even if added in 100-fold higher a significant amount of RanGAP activity is associated with the concentration (Fig. 4a). Sequences of peptides generated by nuclear fraction (16). An antiserum that specifically recog- CNBr cleavage or by digestion with Lys-C protease were nizes RanGAP1 is still not available for use in immunofluo- identical with the deduced amino acid sequence of Rnalp (Fig. rescence analysis, and it therefore remains a possibility that Table 1. Enrichment of RanGAP/Rnalp from Sc. pombe cells Fraction Total Activity of protein, RanGAP, Purification, Yield, No. Description mg units x 10-4 -fold % 1 Complete lysate 580 ND 2 70,000 x g supernatant 490 30.8 1.0 100 3 (NH4)2SO4 supernatant 60 16.7 4.4 54 4 Hydrophobic chromatography 1.5 7.1 75.3 23 5 Mono Q 0.056 1.1 312.5 3.6 Values refer to 10 ml of packed Sc. pombe cells as starting material. RanGAP/Rnalp was quantitated densitometrically on 12% SDS gels stained with Coomassie blue. One unit is the amount of RanGAP that stimulates hydrolysis of50% of 100 nM Ran-bound [y-32P]GTP in 100 lI after 5 min of incubation at 25°C. ND, not determined. Downloaded by guest on September 25, 2021 1752 Biochemistry: Bischoff et aL Proc. NatL Acad Sci USA 92 (1995) nuclear membrane, after its translocation through the , clearing the pore for another transport event. Loss of RanGAP function, or the presence of nonhydrolyzable GTP inhibit this hydrolysis-mediated dissociation '4 75 - A 75 analogues, should step, thereby sealing the nuclear pore against further nuclear export and probably also against import of karyophiles. This ~~~50~ ~~0 mechanism agrees with the observed mRNA export defect in the rnal-1 budding yeast mutant and the inhibition of protein 50-44 -4== import after addition of guanosine [y-thio]triphosphate to in vitro transport assays in higher (6, 7). The putative |25 I)0 1 0Tine, 30 ability of Ran to shuttle between nucleus and cytoplasm ap- pears to be linked to Ran activation by GDP/GTP exchange in the nucleus and inactivation by GTP hydrolysis in the 0 cytoplasm. This model would predict the same phenotype to 0 10 20 30 result from inhibition of either of these steps, as illustrated by Time, mL the rnal-l and the prp20-1 mutants of Sa. cerevisiae (30). Search for a Ran-Specific Nucleotide Exchange Factor 100 Other Than RCC1. If Ran is converted into its GTP-bound ON state only by nuclear RCC1, Rnalp would convert all b Ran-GTP reaching the cytoplasm into the OFF state. Yet recently, cytosolic Ran has been claimed to be necessary for 775 0 the import of proteins containing a nuclear localization signal 4-4 into the nuclei of digitonin-permeabilized mammalian cells (6, 0 7). Although the nucleotide-binding state of this cytoplasmic pool of Ran is not definitively established, persistence of nuclear import in the presence of excess GTP but not excess GDP leads to the conclusion that the added Ran was required 25- in its GTP-bound form. Persistence of Ran-GTP in the cyto- plasm would require either a mechanism by which Ran*GTP is protected against RanGAPl-induced hydrolysis or a second, cytoplasmic, guanine nucleotide-exchange factor might be pos- tulated, which counteracts the effect of RanGAPl and regen- 0.01 0.1 1 10 erates active GTP-bound Ran. However, we were unable to RanGAPl/Rnalp, nM detect such an activity in the of interphase HeLa cells. FIG. 4. Rnalp induces GTPase activity on Ran. (a) GTPase re- To clarify whether a Ran-specific nucleotide-exchange factor action was started by adding 5 ,Ll of 0.4 ,uM (0) and 40 l,M (0) Rnalp other than RCC1 exists, perhaps associated with insoluble or 5 ,lI ofbuffer as control (A, O) to 1.6 ml of 100 nM Ran.[y-32P]GTP cytoplasmic structures, we analyzed the total nucleotide- (i, A) or Ras-[y-32P]GTP (0, O). After incubation at 25°C for the time exchange activity in tsBN2 cells, which express temperature- intervals indicated, 100 ,ul of the reaction mixture was filtered through sensitive mutated RCC1 (12). We prepared total lysates of nitrocellulose, and filter-bound radioactivity was determined. (Inset) tsBN2 cells and of wild-type golden hamster kidney cells a and A, Liberated [32P]phosphate was determined in parallel in 100 (BHK21), both grown at 30°C. Ran-specific guanine nucleo- ,lI of the reaction mixture. (b) To 90 ,ul of 111 nM Ran.[y32P]GTP 10 tide-exchange activity was determined after preincubating the ,A of RanGAP1 purified from HeLa cells (o), Rnalp purified from Sc. different time intervals at the restrictive pombe (0), or recombinant Rnalp (AL) was added, resulting in the lysates for tempera- concentrations indicated. After 5 min of incubation at 25°C liberated ture of 39°C (Fig. 5). Within 20 min, nucleotide-exchange [32P]phosphate (Pi) was determined. Values are corrected for protein- activity was completely abolished in tsBN2 lysates, whereas in bound and for [32P]phosphate-associated radioactivity in 100 Al of 100 wild-type lysates it was only marginally reduced. This result nM Ran-[y-32P]GTP. indicates that there is no detectable Ran-specific nucleotide- RanGAPi is associated with the cytoplasmic side of the nu- clear envelope. The two antagonistic Ran-regulators RanGAPl and RCC1, strictly separated by the , and the GTPase Ran, shuttling between these compartments, might constitute the basic features of a molecular mechanism for directed 50 nucleo-cytoplasmic mRNA transport. In this scenario, Ran-GDP that entered the nucleus by diffusion or specific transport is converted to its GTP-bound form by nuclear RCC1. The activated Ran could then interact with mRNAs probably through as-yet-unknown effector molecules. Candi- "~255 dates for such effector proteins, ranging from 23.5 to 300 kDa, have been identified in different organisms by their ability to associate specifically with the GTP-bound form of Ran in an 0 overlay assay (28, 29, 32). Interestingly, at least one of these 0 20 40 60 complexes, composed of Ran-GTP and the 23.5-kDa Ran- Time. min binding protein RanBP1 that contains an RNA-binding motif FIG. 5. Ran-specific guanine nucleotide-exchange activity in crude (28), is sensitive to RanGAP-stimulated disintegration after extracts of tsBN2 cells (0) and in wild-type BHK21 cells (O) at the hydrolysis of Ran-bound GTP (32). Thus, the postulated restrictive temperature. Recovery of exchange activity due to reduc- mRNA-effector-Ran-GTP complex could be dissociated by tion of the temperature to 25°C during the exchange assay was not RanGAP-induced hydrolysis on the cytoplasmic side of the significant, as determined for the 10-min value (data not shown). Downloaded by guest on September 25, 2021 Biochemistry: Bischoff et at Proc. NatL Acad Sci. USA 92 (1995) 1753 exchange factor other than RCC1. It cannot be ruled out that 9. Kadowaki, T., Goldfarb, D., Spitz, L. M., Tartakoff, A. M. & a temperature-sensitive cytoplasmic nucleotide-exchange ac- Ohno, M. (1993) EMBO J. 12, 2929-2937. tivity could result from posttranslationally modified or differ- 10. Bischoff, F. R., Coutavas, E., D'Eustachio, P., Ponstingl, H., Ren, M. & Rush, M. (1995) in Guidebook to the Small , eds. entially spliced RCC1. However, polyclonal antibodies to Sambrook, J. & Tooze, J. (Oxford Univ. Press, Oxford), in press. RCC1, which should also recognize RCC1 derivatives, exclu- 11. Bischoff, F. R. & Ponstingl, H. (1991) Nature (London) 354, sively stain the nucleus of tsBN2 cells (31), making it unlikely 80-82. that any such hypothetical RCC1 subspecies is located in the 12. Nishimoto, T., Eilen, E. & Basilico, C. (1978) Cell 15, 475-483. cytoplasm. 13. Clark, K. L. & Sprague, G. F. (1989) Mo. Cell. Biol. 9,2682-2694. To understand the role of Ran in nuclear transport in more 14. Vijayraghavan, U., Company, M. & Abelson, J. (1989) Genes Dev. detail, the locations at which RanGTPase changes its infor- 3, 1206-1216. mation status by nucleotide exchange and by stimulation of 15. Matsumoto, T. & Beach, D. (1991) Cell 66, 347-360. 16. Bischoff, F. R., Klebe, C., Kretschmer, J., Wittinghofer, A. & GTP hydrolysis should be known, including low activities other Ponstingl, H. (1994) Proc. Natl. Acad. Sci. USA 91, 2587-2591. than RCC1 and RanGAP1. Yet we failed to detect any such 17. Klebe, C., Bischoff, F. R., Ponstingl, H. & Wittinghofer, A. activities by the assays described. If it can be verified that Ran (1995) Biochemistry 34, in press. is activated by nucleotide exchange in the nucleus and inacti- 18. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular vated by GTPase stimulation in the cytoplasmic compartment Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, exclusively, one might test more comprehensive models in Plainview, NY). which mRNA export from and protein import into the nucleus 19. Melchior, F., Weber, K. & Gerke, V. (1993) Moi. Biol. Cell 4, is coupled by the requirement for an additional transport 569-581. factor, vital for both processes. In this case, Ran-GTP- 20. Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991-1995. dependent export could be indispensable for recycling this 21. DeGregori, J., Russ, A., von Melchner, H., Rayburn, H., Pri- factor to the cytoplasm, where it is needed for an import yaranjan, P., Jenkins, N. A., Copeland, N. G. & Ruley, H. E. reaction not necessarily requiring cytoplasmic Ran-GTP. (1994) Genes Dev. 8, 265-76. 22. Traglia, H. M., Atkinson, N. S. & Hopper, A. K. (1989) Mo. Cell. F.R.B. and H.K contributed equally to this work. We thank Jurgen Bio. 9, 2989-2999. Kretschmer for competent technical assistance, Takeharu Nishimoto 23. Atkinson, N. S., Dunst, R. W. & Hopper, A. K. (1985) Mo. Cell. for providing the tsBN2 cell line, and Liz Muller and R6isin Deane for Bio. 5, 907-915. critical comments on the manuscript. This work was supported by 24. Hopper, A. K., Traglia, H. M. & Dunst, R. W. (1990) J. 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