Elf1p, a Member of the ABC Class of ATPases, Functions as a mRNA Export Factor in Schizosacchromyces pombe
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m205415200
ISSN1083-351X
AutoresLibor Kozák, Ganesh Gopal, Jin Ho Yoon, Zuben E. Sauna, Suresh V. Ambudkar, Anjan Thakurta, Ravi Dhar,
Tópico(s)ATP Synthase and ATPases Research
ResumoRae1p and Mex67p/Tap are conserved mRNA export factors. We have used synthetic lethal genetic screens inSchizosaccharomyces pombe to identify mutations in genes that are functionally linked to rae1 and mex67in mRNA export. From these screens, we have isolated mutations in a putative S. pombe homologue of theCandida albicans elf1 gene. The elf1 ofS. pombe is not an essential gene. When elf1mutations are combined with rae1-167 mutation, growth and mRNA export is inhibited in the double mutants. This inhibition can be suppressed by the multicopy expression of mex67suggesting that Mex67p can substitute for the loss of Elf1p function. Elf1p is a non-membrane member of the ATP-binding cassette (ABC) class of ATPase and the GFP-Elf1p fusion localizes to the cytoplasm. Elf1p, expressed and purified from Escherichia coli, binds and hydrolyzes ATP. A mutant Elf1p that carries a glycine to aspartic acid (G731D) mutation within the Walker A domain of the second ATP site retains the ATP binding but loses its ATPase activity in vitro. This mutant protein no longer functions in mRNA export. Taken together, our results show that Elf1p functions as a mRNA export factor along with Rae1p and Mex67p in S. pombe. Rae1p and Mex67p/Tap are conserved mRNA export factors. We have used synthetic lethal genetic screens inSchizosaccharomyces pombe to identify mutations in genes that are functionally linked to rae1 and mex67in mRNA export. From these screens, we have isolated mutations in a putative S. pombe homologue of theCandida albicans elf1 gene. The elf1 ofS. pombe is not an essential gene. When elf1mutations are combined with rae1-167 mutation, growth and mRNA export is inhibited in the double mutants. This inhibition can be suppressed by the multicopy expression of mex67suggesting that Mex67p can substitute for the loss of Elf1p function. Elf1p is a non-membrane member of the ATP-binding cassette (ABC) class of ATPase and the GFP-Elf1p fusion localizes to the cytoplasm. Elf1p, expressed and purified from Escherichia coli, binds and hydrolyzes ATP. A mutant Elf1p that carries a glycine to aspartic acid (G731D) mutation within the Walker A domain of the second ATP site retains the ATP binding but loses its ATPase activity in vitro. This mutant protein no longer functions in mRNA export. Taken together, our results show that Elf1p functions as a mRNA export factor along with Rae1p and Mex67p in S. pombe. nuclear envelope nuclear pore complex ribonucleoprotein ATP-binding cassette green fluorescent protein temperature sensitive elongation-like factor 4′,6-diamidino-2-phenylindole 4-morpholineethanesulfonic acid open reading frame reverse transcriptase ethyl methanesulfonate Edinburgh minimal medium In the eukaryotic cell, the nuclear envelope (NE)1 separates the nucleus from the cytoplasmic compartment. Embedded within the NE are the nuclear pore complexes (NPC) through which nucleo-cytosolic exchanges take place. While small molecules of molecular mass 40 kDa and under can diffuse passively through the NPC, receptor proteins interact with the NPC to mediate the transport of macromolecules (1Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1651) Google Scholar, 2Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). The mature mRNAs are exported out of the nucleus as ribonucleoprotein (RNP) complexes. Concurrent with transcription and splicing, the coordinated assembly of export-competent RNP complexes takes place in the nucleus by association of proteins with the maturing transcripts (3Zenklusen D. Stutz F. FEBS Lett. 2001; 498: 150-156Crossref PubMed Scopus (60) Google Scholar, 4Maniatis T. Reed R. Nature. 2002; 416: 499-506Crossref PubMed Scopus (912) Google Scholar, 5Reed R. Hurt E. Cell. 2002; 108: 523-531Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). In the cytoplasm, the RNP complexes are disassembled to release the mRNA and soluble export factors; the latter return to the nucleus for participating in the next round of mRNA export. The major components of the mRNA export machinery appear to be evolutionarily conserved. Genetic and biochemical approaches have led to the identification of several conserved export factors, Tap/Mex67p, Rae1p/Gle2p, Gle1, and Dbp5/RHA from yeasts to metazoans (6Segref A. Sharma K. Doye V. Hellwig A. Huber J. Luhrmann R. Hurt E. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (432) Google Scholar, 7Katahira J. Strasser K. Podtelejnikov A. Mann M. Jung J.U. Hurt E. EMBO J. 1999; 18: 2593-2609Crossref PubMed Scopus (339) Google Scholar, 8Brown J.A. Bharathi A. Ghosh A. Whalen W. Fitzgerald E. Dhar R. J. Biol. Chem. 1995; 270: 7411-7419Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 9Bharathi A. Ghosh A. Whalen W.A. Yoon J.H., Pu, R. Dasso M. Dhar R. Gene (Amst.). 1997; 198: 251-258Crossref PubMed Google Scholar, 10Kraemer D. Dresbach T. Drenckhahn D. Eur. J. Cell Biol. 2001; 80: 733-740Crossref PubMed Scopus (20) Google Scholar, 11Murphy R. Wente S.R. Nature. 1996; 383: 357-360Crossref PubMed Scopus (202) Google Scholar, 12Snay-Hodge C.A. Colot H.V. Goldstein A.L. Cole C.N. EMBO J. 1998; 17: 2663-2676Crossref PubMed Scopus (227) Google Scholar, 13Tseng S.S. Weaver P.L. Liu Y. Hitomi M. Tartakoff A.M. Chang T.H. EMBO J. 1998; 17: 2651-2662Crossref PubMed Scopus (220) Google Scholar). The molecular mechanism of Mex67p/Tap function in mRNA export is understood in some detail, but how Rae1p/Gle2p function is largely unknown (5Reed R. Hurt E. Cell. 2002; 108: 523-531Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 14Conti E. Izaurralde E. Curr. Opin. Cell Biol. 2001; 13: 310-319Crossref PubMed Scopus (217) Google Scholar). Nonetheless, there is a close reciprocal relationship between Rae1p and Mex67p in the two yeast systems that may indicate their functional homology and mechanistic similarity. Mex67/Tap are non-β karyopherin receptors. They associate with nucleoporins, bind mRNA, and interact with the RNA-binding protein, Aly/Yra1p, to mediate mRNA export (3Zenklusen D. Stutz F. FEBS Lett. 2001; 498: 150-156Crossref PubMed Scopus (60) Google Scholar, 5Reed R. Hurt E. Cell. 2002; 108: 523-531Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Rae1p, on the other hand, is an NPC-associated, conserved WD-domain protein that is also required for cell-cycle progression at the G2/M boundary (8Brown J.A. Bharathi A. Ghosh A. Whalen W. Fitzgerald E. Dhar R. J. Biol. Chem. 1995; 270: 7411-7419Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). While the hRae1p has been shown to bind mRNA and to shuttle between the nucleus and the cytoplasm, these properties have not been demonstrated for its yeast homologues (10Kraemer D. Dresbach T. Drenckhahn D. Eur. J. Cell Biol. 2001; 80: 733-740Crossref PubMed Scopus (20) Google Scholar, 15Pritchard C.E. Fornerod M. Kasper L.H. van Deursen J.M. J. Cell Biol. 1999; 145: 237-254Crossref PubMed Scopus (193) Google Scholar). In S. pombe, Rae1p, but not Mex67p, is essential for growth and mRNA export (16Yoon J.H. Love D.C. Guhathakurta A. Hanover J.A. Dhar R. Mol. Cell. Biol. 2000; 20: 8767-8782Crossref PubMed Scopus (72) Google Scholar). A temperature sensitive (ts) rae1-167 mutant rapidly accumulates poly(A)+ RNA in the nucleus at restrictive temperatures above 30 °C (8Brown J.A. Bharathi A. Ghosh A. Whalen W. Fitzgerald E. Dhar R. J. Biol. Chem. 1995; 270: 7411-7419Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Moreover,spmex67 expressed from a multicopy plasmid can partially suppress the mRNA export defect of rae1-167 mutation up to a restrictive temperature of 32 °C. However, in S. cerevisiae, Mex67p is essential for growth and mRNA export whereas Gle2p is not. These and other observations suggest that at least in S. pombe and likely in S. cerevisiae, the mRNA export machinery may operate more than one mRNA export pathway, consisting of Rae1p-specific, Mex67p-specific, and some shared export factors. Overall, the nuclear export of mRNA is expected to be an energy consuming process, but no specific step with a link to energy metabolism has been identified yet. Translocation of protein cargo by transportin, an importin β-type receptor, has been shown to occur in the absence of Ran or energy (17Ribbeck K. Kutay U. Paraskeva E. Gorlich D. Curr. Biol. 1999; 9: 47-50Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). However, several mRNA export factors that possess ATP-hydrolyzing activity have been identified, and the role of energy in the mRNA export process cannot be ruled out. In the nucleus, the splicing factor Sub2/UAP56, a DECD box ATPase, associates with the spliceosome and is involved in recruiting the RNA-binding protein, Yra1/Aly (18Strasser K. Hurt E. Nature. 2001; 413: 648-652Crossref PubMed Scopus (222) Google Scholar, 19Luo M.-J. Zhou Z. Magni K. Christofrides C. Rappsilber J. Mann M. Reed R. Nature. 2001; 413: 644-647Crossref PubMed Scopus (300) Google Scholar, 20Jensen T.H. Boulay J. Rosbash M. Libri D. Curr. Biol. 2001; 11: 1711-1715Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Both Sub2/UAP56 and Yra1/Aly are part of a conserved protein complex TREX, which is believed to couple transcription and mRNA export processes (21Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodriguez-Navarro S. Rondon A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (632) Google Scholar). The essential, shuttling mRNA export factor, Dbp5p/RHA (RNA helicase A), a DEAD box ATP-dependent RNA helicase, may play a role in the release of mRNA and/or in the disassembly of the RNP in the cytoplasm (12Snay-Hodge C.A. Colot H.V. Goldstein A.L. Cole C.N. EMBO J. 1998; 17: 2663-2676Crossref PubMed Scopus (227) Google Scholar). However, how ATP hydrolysis is involved in the functioning of Sub2/Uap56 or Dbp5/RHA is not known. Recently, through genetic interactions with mutations in SUB2, another ATP-dependent DNA, RNA helicase, Rad3p, was implicated in the nuclear export of mRNA (20Jensen T.H. Boulay J. Rosbash M. Libri D. Curr. Biol. 2001; 11: 1711-1715Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Rix7p, a member of theATPases associated with a variety of cellularactivities (AAA) family of ATPases in S. cerevisiae, has been shown to be an essential protein whose function is required for the assembly and nuclear export of 60 S ribosomal subunits (22Gadal O. Strauss D. Braspenning J. Hoepfner D. Petfalski E. Philippsen P. Tollervey D. Hurt E. EMBO J. 2001; 20: 3695-3704Crossref PubMed Scopus (78) Google Scholar). Since the export-competent RNP goes through several maturation steps, ATPases could function as conformational switches in unidirectional movement, assembly/disassembly of the RNA-protein complexes, and in the unwinding of double-stranded RNA. In this study, we have described the identification, genetic, and biochemical characterization of the putative S. pombehomologue of the Candida albicans Elf1p (23Sturtevant J. Cihlar R. Calderone R. Microbiology. 1998; 144: 2311-2321Crossref PubMed Scopus (18) Google Scholar). In S. pombe, elf1 is not essential. The rae1-167mutation is linked to mutations in elf1 gene for growth and mRNA export suggesting that Elf1p functions as an mRNA export factor when the function of Rae1p is compromised. The growth and mRNA export-defect associated with elf1 andrae1-167 double mutants can be suppressed by multicopy expression of mex67, suggesting that Mex67p can compensate for the loss of Elf1p function in mRNA export. Elf1p is a member of the ABC class of ATPases. The ABC ATPases are distinguished from other ATPases because in addition to the ATP-binding Walker A and B domains, they also contain a characteristic dodecapeptide linker region between the Walker motifs known as the ABC signature or the C-domain (24Gottesman M.M. Ambudkar S.V. J. Bioenerg. Biomembr. 2001; 33: 453-458Crossref PubMed Scopus (309) Google Scholar).E. coli-purified Elf1p binds and hydrolyzes ATP in vitro, and this enzymatic activity is essential for its function in mRNA export. Basic cell culture techniques were as described (25Moreno S. Klar A. Nurse P. Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3104) Google Scholar, 26Alfa C. Fantes P. Hyams J. Mcleod M. Warbrick E. Experiments with Fission Yeast. Cold Spring Harbor Press, Cold Spring Harbor, NY1993Google Scholar). The strains used in this work are listed in TableI. Appropriately supplemented Edinburgh minimal medium (EMM) medium was used to express genes from the pREP plasmids containing the nmt promoter (27Maundrell K. Gene (Amst.). 1993; 123: 127-130Crossref PubMed Scopus (922) Google Scholar). Thenmt promoter was repressed by the addition of 10 μm thiamine in EMM medium (28Forsburg S.L. Nucleic Acids Res. 1993; 21: 2955-2956Crossref PubMed Scopus (396) Google Scholar).Table IS. pombe strains used in this studyStrainGenotype(s)Sourcewild typeh− leu1–32 ura4D18Yoon (29Yoon J.H. Whalen W.A. Bharathi A. Shen R. Dhar R. Mol. Cell. Biol. 1997; 17: 7047-7060Crossref PubMed Scopus (30) Google Scholar)wild typeh+ leu1–32 ura4D18Yoon (29Yoon J.H. Whalen W.A. Bharathi A. Shen R. Dhar R. Mol. Cell. Biol. 1997; 17: 7047-7060Crossref PubMed Scopus (30) Google Scholar)rae1–167h−leu1–32 ura4D18 rae1–167Yoon (29Yoon J.H. Whalen W.A. Bharathi A. Shen R. Dhar R. Mol. Cell. Biol. 1997; 17: 7047-7060Crossref PubMed Scopus (30) Google Scholar)rae1–167h+ leu1–32 ura4D18 rae1–167Yoon (29Yoon J.H. Whalen W.A. Bharathi A. Shen R. Dhar R. Mol. Cell. Biol. 1997; 17: 7047-7060Crossref PubMed Scopus (30) Google Scholar)Δelf1h−leu1–32 ura4D18 elf1::ura4This studyΔelf1h− leu1–32 ura4D18 elf1::kanThis studyelf1–1h− leu1–32 ura4D18 elf1–1This studyelf1–21h−leu1–32 ura4D18 elf1–21This studyΔelf1Δmex67h− leu1–32 ura4D18 elf1::ura4 mex67::kanThis studyelf1–21Δmex67h−leu1–32 ura4D18 elf1–21mex67::kanThis studyΔmex67h− leu1–32 ura4D18 mex67::kanYoon (16Yoon J.H. Love D.C. Guhathakurta A. Hanover J.A. Dhar R. Mol. Cell. Biol. 2000; 20: 8767-8782Crossref PubMed Scopus (72) Google Scholar)Δnpp106h− leu1–32 ura4D18 npp106::ura4Yoon (29Yoon J.H. Whalen W.A. Bharathi A. Shen R. Dhar R. Mol. Cell. Biol. 1997; 17: 7047-7060Crossref PubMed Scopus (30) Google Scholar)Δnup184h− leu1–32 ura4D18 npp106::ura4Yoon (29Yoon J.H. Whalen W.A. Bharathi A. Shen R. Dhar R. Mol. Cell. Biol. 1997; 17: 7047-7060Crossref PubMed Scopus (30) Google Scholar)Δelf1Δnpp106h−leu1–32 ura4D18 elf1::kan npp106::ura4This studyΔelf1Δnup184h−leu1–32 ura4D18 elf1::kan nup 184::ura4This studyelf1–1 rae1–167h− leu1–32 ura4D18 elf1–1 rae1–167/pRep81X-rae1This studyelf1–21 rae1–167h− leu1–32 ura4D18 elf1–21 rae1–167/pRep81X-rae1This studyΔelf1 rae1–167h− leu1–32 ura4D18 elf1::ura4 rae1–167/pRep81X-rae1This studyelf1–1 Δmex67h−leu1–32 ura4D18 elf1–1 mex67::kan/pRep81X-mex67This studyrae1–167 Δmex67h−leu1–32 ura4D18 rae1–167 mex67::kan/pRep81X-rae1Yoon (16Yoon J.H. Love D.C. Guhathakurta A. Hanover J.A. Dhar R. Mol. Cell. Biol. 2000; 20: 8767-8782Crossref PubMed Scopus (72) Google Scholar) Open table in a new tab Synthetic Lethal Screen—rae1-167/pREP81X-rae1+ cells were mutagenized with ethyl methanesulfonate (EMS) as described previously (29Yoon J.H. Whalen W.A. Bharathi A. Shen R. Dhar R. Mol. Cell. Biol. 1997; 17: 7047-7060Crossref PubMed Scopus (30) Google Scholar). Δspmex67::kan/pREP81X-mex67+strain (1 × 108 cells) was mutagenized with 300 μg/ml of nitrosoguanidine (NG) for 1 h, with a 30% survival rate. After three washes with 0.9% solution of sodium chloride, cells were grown for 3 h in 1 ml of EMM medium without leucine. Then the cells were plated onto EMM agar plates without leucine and grown at 27 °C. Colonies were replica-plated onto EMM agar plates without leucine containing phloxin B in the presence (mex67repressed) and absence (mex67 expressed) of thiamine (10 μm) and grown at 27 °C. Synthetic lethal mutants were identified as those colonies that turned red in the presence of thiamine but remained pink in the absence of thiamine. The synthetic lethal strain SL21 containing a cognate sl21 mutation was isolated from the rae1-167 sl21/pREP81X-rae1strain and SL1 with its cognate sl1 mutation was isolated from the Δspmex67 sl1/pREP81X-mex67 strain. SL21 (rae1-167 sl21/pREP81X-rae1+) and SL1 (Δspmex67 sl1/pREP81X-mex67+) were transformed with a partial Sau3A genomic library of S. pombe cloned into the SalI site of pUR19 (30Berbet N. Muriel W.J. Carr A.M. Gene (Amst.). 1992; 114: 59-66Crossref PubMed Scopus (219) Google Scholar). Transformants that could grow at 27 °C in the presence of thiamine were isolated. Plasmids isolated from these transformants were enriched via E. coli transformation and retransformed into SL strains. Plasmids that rescued synthetic lethality of SL strains were sequenced. A 4.9-kb insert in a plasmid (26-3-1) contained the fullelf1 gene. Genomic DNA from SL21 and SL1 were isolated, and the full-length coding region of the elf1-21 andelf1-1 alleles were sequenced. The nonsense mutation CAA to TAA at amino acid 863 (Q863X) was the only change present in the elf1-21 allele, and the missense mutation GGT to GAT at amino acid 731 (G731D) was the only change present in theelf1-1 allele. The pREP81X-elf1 vector was constructed by generating a PCR product that inserted a SalI site upstream of the initiation codon and a BamHI site downstream of the termination codon, using the genomic library clone pUR19-elf1 (26-3-1) as a template. TheSalI-BamHI-digested PCR product was then cloned into pREP81X cut with XhoI and BamHI. The green fluorescent protein (GFP) fusion vectors were constructed as follows. GFP was fused to Elf1p at the N terminus by first creating aBamHI site immediately downstream of the initiation codon ofelf1 on the pUR19-elf1 plasmid. ABamHI-GFP-BamHI fragment was inserted into theBamHI site. Proper orientation of GFP was checked by PCR and by restriction fragment analysis. The resulting p5′-GFP-elf1 plasmid was capable of complementing both the SL1 and SL21 synthetic lethality. In a similar way for tagging the GFP at the C terminus of Elf1p, aSalI site was created immediately upstream of the termination codon on the pUR19-elf1 plasmid. ASalI-GFP-SalI fragment was inserted into theSalI site. The plasmids p5′GFP-elf1-1 (G731D) and p5′GFP-elf1-21(Q863X) were created from the normal p5′-GFP-elf1 plasmid by site-directed mutagenesis with the QuikChange site-directed mutagenesis kit (Stratagene) according to the instruction manual. The mutations were confirmed by sequencing. For expression inE. coli, elf1 and elf1-1 were amplified by PCR as NdeI-XhoI fragments and cloned into the E. coli expression vector pET16b (Novagen) to express polyhistidine-tagged proteins under the control of the T7 promoter. Single transformants were first patched on EMM agar plates in the absence of thiamine (−B1) with appropriate auxotrophic selection and then grown in 10 ml in liquid until the stationary phase. 10-fold dilutions (1 × 107 cells/ml) were plated in the presence or absence of thiamine (final concentration of B1 was 10 μm) on EMM plates without leucine and/or uracil as appropriate. Transmission electron micrographs of sections of S. pombe cells were taken following procedures as described before (29Yoon J.H. Whalen W.A. Bharathi A. Shen R. Dhar R. Mol. Cell. Biol. 1997; 17: 7047-7060Crossref PubMed Scopus (30) Google Scholar). In situ hybridization method was as described previously (31Amberg D.C. Goldstein A.L. Cole C.N. Genes Dev. 1992; 6: 1173-1189Crossref PubMed Scopus (298) Google Scholar) with the following modifications. Oligo(dT)50 carrying an α-digoxygenin at the 3′-end was used as the hybridization probe and fluorescein isothiocyanate-antidigoxygenin or rhodamine-antidigoxygenin as appropriate was used for detecting the hybridization probe by fluorescence microscopy. DAPI was used for observing the DNA. For expression of His-Elf1p and His-Elf1-1p in E. coli, pET16b containing the respective inserts were transformed into BL21 (DE3). Cultures were grown in 150 ml of LB at 27 °C and induced by addition of 0.3 mmisopropyl-1-thio-β-d-galactopyranoside. The cell pellet was resuspended in 5 ml of lysis buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 20% glycerol, and 2.5 mmβ-mercaptoethanol). The buffer also contained EDTA-free protease inhibitor mix (Roche Molecular Biochemicals). Cells were lysed by French Press, and the cell debris was removed by centrifugation at 14,000 × g for 30 min. The supernatant was loaded onto a TALON column (CLONTECH) pre-equilibrated with the lysis buffer. Binding of the His-tagged protein was performed for 45 min at 4 °C on a turning wheel in the presence of 10 mmimidazole. Bound Elf1p was washed with lysis buffer containing 100 mm imidazole and eluted with the same buffer containing 500 mm imidazole. The eluted protein was later dialyzed into a buffer without imidazole. Protein estimation was performed using the BioRad protein assay system. Purified Elf1p or Elf1-1p (1.5–3 μg) was analyzed by Coomassie Blue staining on a 4–20% polyacrylamide gel. Purified Elf1p or Elf1-1p were added to ATP binding buffer (40 mm MES-Tris, pH 6.8, 50 mm KCl, 5 mm sodium azide, 2 mmEGTA, 2 mm dithiothreitol, and 10 mmMgCl2) containing 10 μm8-azido-[α-32P]ATP in the dark on ice and incubated for 10 min. The samples were then photocross-linked by UV illumination at 365 nm on ice as described (32Sauna Z.E. Ambudkar S.V. J. Biol. Chem. 2001; 276: 11653-11661Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Cold ATP (12.5 mm) was added to displace excess non-covalently bound radionucleotide. The samples were then run in denaturing 8% Tris-glycine gels, dried, and exposed to Bio-Max MR films at −70 °C for 12–24 h. As described before (33Li Y. Altman S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 441-444Crossref PubMed Scopus (30) Google Scholar), ATPase activity of Elf1p was detected in a reaction mixture containing 50 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 1 mm dithiothreitol, 0.1 mm ATP, and 0.1 μCi of [α-32P]ATP. The reaction volume was 20 μl. The mixture was incubated at 37 °C for 30 min and hydrolysis stopped by addition of 1 μl of 0.4 m EDTA. The samples were then placed on ice. 3 μl of the reaction mixture were spotted on polyethyleneimine TLC plates and air-dried. The chromatograph was developed with 1 m LiCl and 1 m acetic acid solution. After drying, the TLC plate was exposed to Kodak X-OMAT AR film for 20 s to 30 min. We previously reported the use of a synthetic lethal screen to identify genes linked torae1 in the mRNA export process (29Yoon J.H. Whalen W.A. Bharathi A. Shen R. Dhar R. Mol. Cell. Biol. 1997; 17: 7047-7060Crossref PubMed Scopus (30) Google Scholar, 34Whalen W.A. Yoon J.H. Shen R. Dhar R. Genetics. 1999; 152: 827-838Crossref PubMed Google Scholar). In that screen, a rae1-167 (ts) mutant strain expressingrae1+ from a thiamine-repressiblenmt81 promoter on the pREP81X vector was mutagenized with EMS (27Maundrell K. Gene (Amst.). 1993; 123: 127-130Crossref PubMed Scopus (922) Google Scholar, 28Forsburg S.L. Nucleic Acids Res. 1993; 21: 2955-2956Crossref PubMed Scopus (396) Google Scholar). The synthetic lethal mutants were isolated as those colonies that could grow in the presence of Rae1p expression (−B1) but were unable to grow when expression of Rae1p was repressed (+B1). Mutations defining three complementation groups, SL64, SL27, and SL21 were identified. The corresponding synthetic lethal genes for sl64 and sl27 were identified as npp106 (scNIC96) andnup184 (scNUP184), respectively (29Yoon J.H. Whalen W.A. Bharathi A. Shen R. Dhar R. Mol. Cell. Biol. 1997; 17: 7047-7060Crossref PubMed Scopus (30) Google Scholar, 34Whalen W.A. Yoon J.H. Shen R. Dhar R. Genetics. 1999; 152: 827-838Crossref PubMed Google Scholar). For this study, we isolated a genomic clone corresponding to thesl21 that complemented the growth and mRNA export defect of SL21 (rae1-167 sl21/pREP81X-rae1+) cells under synthetic lethal conditions (see below). Sequence analysis of the genomic clone revealed an intronless 1057-amino acid ORF (SPAC3C7.08c in the S. pombe data base) with the predicted molecular mass of a 116.7-kDa protein. A purified DNA fragment within the open reading frame rescued the cognate sl21 mutation in SL21. Sequence analyses of two independent PCR-amplified ORF fragments using SL21 genomic DNA as template revealed a single mutation that introduced an ochre termination codon in place of a glutamine at position 863 (Q863X). A Blast search of the protein data base showed that the protein had significant homology with Elf1p, a protein from C. albicans (GenBankTMAF038153) and with a hypothetical ORF (GenBankTMZ73582) fromS. cerevisiae, both putative members of the ABC ATPase family (23Sturtevant J. Cihlar R. Calderone R. Microbiology. 1998; 144: 2311-2321Crossref PubMed Scopus (18) Google Scholar). Accordingly, the S. pombe gene was namedelf1 (spelf1) and the mutant strain (sl21) was renamed elf1-21. A similar genetic screen was used to identify mutations in genes that are synthetically lethal with Δmex67 (16Yoon J.H. Love D.C. Guhathakurta A. Hanover J.A. Dhar R. Mol. Cell. Biol. 2000; 20: 8767-8782Crossref PubMed Scopus (72) Google Scholar). One of the synthetic lethal gene mutations mapped within the elf1 gene was the mutant gene named elf1-1. Sequence analysis of theelf1-1 mutation showed that the glycine residue at position 731 of the wild type Elf1p was converted to an aspartic acid residue (G731D). An alignment of S. pombe Elf1p and its putative homologues from S. cerevisiae and C. albicans generated by the ClustalW algorithm is shown in Fig.1. These proteins share overall organizational features of the ABC ATPases. Their central and C-terminal regions are highly conserved with a less conserved N-terminal region. The central region contains two ATP binding domains, each consisting of a Walker A, Walker B, and a distinctive C-domain (Fig. 1, boxed and marked). A common feature of the ABC family of ATPases is that a single ATP binding domain is generally sufficient to bind ATP, but both are required for ATP hydrolysis (24Gottesman M.M. Ambudkar S.V. J. Bioenerg. Biomembr. 2001; 33: 453-458Crossref PubMed Scopus (309) Google Scholar). Interestingly, the elf1-1 mutation (G731D) maps within the second conserved Walker A domain (GPNGAGKS to GPNGADKS). Additionally, ATP hydrolysis by the ATPases is Mg2+-dependent, and the aspartate residue in the Walker B domain is essential for Mg2+ binding. The Elf1-21p lacks the second Walker B and the linker region (C-domain) region due to a translational termination codon at position 863. Interestingly, protein database searches did not reveal any putative homologues in higher eukaryotic cells, suggesting that other proteins may substitute for Elf1p. Elf1p also showed a lower level of homology to a family of non-membrane ABC ATPases whose members include the translational elongation factors, Ef3p from S. pombe,S. cerevisiae, and the fungi. Like Ef3p of S. cerevisiae, Elf1p of S. pombe also does not contain the hydrophobic sequences known to be required for membrane anchoring (35Chakraburtty K. Res. Microbiol. 2001; 152: 391-399Crossref PubMed Scopus (31) Google Scholar). However, S. pombe elf3 expressed from a multicopy plasmid did not complement the growth and mRNA export defect of SL21 cells, suggesting that Ef3p is likely not a functional homologue of Elf1p (data not shown). RNA-dependent helicases (S. cerivisiae DBP5) also contain ATPase activity (13Tseng S.S. Weaver P.L. Liu Y. Hitomi M. Tartakoff A.M. Chang T.H. EMBO J. 1998; 17: 2651-2662Crossref PubMed Scopus (220) Google Scholar), but Elf1p did not UV-cross-link with poly(A)+ RNA in vivo and S. pombe dbp5, expressed from a multicopy plasmid, did not functionally complement Elf1p function(s) (data not shown). Thus, Elf1p is not likely to function as an RNA helicase. To determine whether elf1 is an essential gene, most of its coding sequences (amino acids 1–1042) were replaced in the diploid strain by either the ura4+ or the kanamycin gene, respectively. Dissection of the tetrads showed that all four spores formed colonies, and the uracil prototrophy or the resistance to G418 segregated 2:2. The disruption of elf1 in the genome was confirmed by Southern blot hybridization of the genomic DNA digested with restriction enzymes (data not shown). The Δelf1 cells grew, albeit slightly slower than the wild type cells in a range of temperatures (21–36 °C, see below), suggesting that the null was not temperature sensitive for growth. Thus, the S. pombe elf1 gene is not essential for growth. Elf1p was tagged separately at the C and N terminus with GFP to determine its cellular localization. Both GFP fusions were functional and complemented the synthetic lethality of the Δelf1 rae1-167 mutation (data not shown), and both localized to the cytoplasm when expressed in the wild type or in the Δelf1 strain (Fig. 2, data not shown for the C-terminal GFP fusion). Similarly, the Elf1-1p (G731D) and Elf1-21p (Q863X) tagged with GFP at the N terminus also predominantly localized to the cytoplasm (Fig. 2). However, about 5–10% of cells expressing GFP-Elf1-21p also showed diffuse staining in the nucleus. We do not know if the Elf1p fusion protein shuttles between the nucleus and the cytoplasm; it did not accumulate in the nucleus following addition of leptomycin, an inhibitor of the Crm1p export receptor. The growth of elf1-21 cells was comparable to that of wild type, rae1-167, and Δmex67 cells, although Δelf1 and elf1-1 cells grew slightly slower (Fig 3A). None of the mutant strains showed any mRNA export defect when poly(A)+ RNA was detected in these cells by in situ hybridization (Fig. 3B)
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