An RNA-binding Respiratory Component Mediates Import of Type II tRNAs into Leishmania Mitochondria
2006; Elsevier BV; Volume: 281; Issue: 35 Linguagem: Inglês
10.1074/jbc.m604126200
ISSN1083-351X
AutoresSaibal Chatterjee, Pratik Home, Saikat Mukherjee, Bidesh Mahata, Srikanta Goswami, Gunjan Dhar, Samit Adhya,
Tópico(s)Viral Infections and Immunology Research
ResumoTransport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import. Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import. Mitochondria from a large number of species, including protists, higher plants, some invertebrates, and eutherian mammals do not contain sufficient numbers of functional tRNA genes and therefore import cytoplasmic tRNAs to support the translation of organellar mRNAs (reviewed in Refs. 1Bhattacharyya S.N. Adhya S. RNA Biol. 2004; 1: 84-88Crossref PubMed Scopus (30) Google Scholar and 2Schneider A. Marechal-Drouard L. Trends Cell Biol. 2000; 10: 509-513Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Mitochondrial tRNA import is especially important in kinetoplastid protozoa such as Leishmania and Trypanosoma that lack all mitochondrial tRNA genes (3Simpson A.M. Suyama Y. Dewes H. Campbell D. Simpson L. Nucleic Acids Res. 1989; 17: 5427-5445Crossref PubMed Scopus (145) Google Scholar, 4Hancock K. Hajduk S.L. J. Biol. Chem. 1990; 265: 19203-19215Abstract Full Text PDF Google Scholar). The precise manner in which polyanionic tRNA molecules cross the double mitochondrial membrane, is largely unresolved. In yeast, the mitochondrial protein import pore, as well as cytosolic carrier proteins, appears to be involved (5Tarassov I. Entelis N. Martin R.P. J. Mol. Biol. 1995; 245: 315-323Crossref PubMed Scopus (100) Google Scholar, 6Tarassov I. Entelis N. Martin R.P. EMBO J. 1995; 14: 3461-3471Crossref PubMed Scopus (113) Google Scholar). On the other hand, biochemical studies in kinetoplastid protozoa have revealed that these organisms have specialized mechanisms for import of tRNA that are distinct from those for protein import (7Nabholz C.E. Horn E.K. Schneider A. Mol. Biol. Cell. 1999; 10: 2547-2557Crossref PubMed Scopus (28) Google Scholar) but involve direct interaction of tRNAs with membrane-bound proteins (8Mahapatra S. Adhya S. J. Biol. Chem. 1996; 271: 20432-20437Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Leishmania and Trypanosoma mitochondria recognize sequence/structure motifs (import signals) in distinct domains of individual tRNAs (9Bhattacharyya S.N. Mukherjee S. Adhya S. Mol. Cell. Biol. 2000; 20: 7410-7417Crossref PubMed Scopus (15) Google Scholar, 10Esseiva A.C. Marechal-Drouard L. Cosset A. Schneider A. Mol. Biol. Cell. 2004; 15: 2750-2757Crossref PubMed Google Scholar), and the former rapidly select oligoribonucleotide-containing motifs matching those in importable tRNAs from a random sequence pool of high complexity (11Bhattacharyya S.N. Chatterjee S. Adhya S. Mol. Cell Biol. 2002; 22: 4372-4382Crossref PubMed Scopus (38) Google Scholar). However, there are differences in the intrinsic efficiencies of transfer of individual RNAs through the inner, as opposed to the outer membrane, in vitro; some, designated as type I RNAs, are imported efficiently into the matrix, whereas others (type II) are not. The potential problem of an imbalanced matrix tRNA pool is solved by the unique phenomenon of allosteric regulation; type I RNAs stimulate the inner membrane transfer of type II RNAs, whereas type II RNAs inhibit transfer of type I (11Bhattacharyya S.N. Chatterjee S. Adhya S. Mol. Cell Biol. 2002; 22: 4372-4382Crossref PubMed Scopus (38) Google Scholar, 12Goswami S. Chatterjee S. Bhattacharyya S.N. Basu S. Adhya S. Nucleic Acids Res. 2003; 31: 5552-5559Crossref PubMed Scopus (15) Google Scholar). On the basis of these interactions, tRNATyr, tRNAArg, and tRNATrp have been identified as type I, and tRNAIle, tRNAVal, and tRNAMet-e have been identified as type II, respectively (11Bhattacharyya S.N. Chatterjee S. Adhya S. Mol. Cell Biol. 2002; 22: 4372-4382Crossref PubMed Scopus (38) Google Scholar, 12Goswami S. Chatterjee S. Bhattacharyya S.N. Basu S. Adhya S. Nucleic Acids Res. 2003; 31: 5552-5559Crossref PubMed Scopus (15) Google Scholar, 13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar); in vitro evolution experiments suggest that many more tRNA species belong to these categories (11Bhattacharyya S.N. Chatterjee S. Adhya S. Mol. Cell Biol. 2002; 22: 4372-4382Crossref PubMed Scopus (38) Google Scholar). The "ping-pong" model (14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar) postulates that the two types of tRNA bind to different receptors; binding of type I tRNA to its receptor induces a conformational change that is transmitted to the type II receptor, opening up its tRNA-binding site. Type II tRNA loading in turn induces an allosteric transition resulting in the destabilization of the type I complex. Recently, a multi-protein complex (the RNA import complex (RIC)) 2The abbreviations used are: RIC, RNA import complex; Tet, tetracycline; BN, Blue Native; RT, reverse transcription; ORF, open reading frame; UCR, ubiquinol cytochrome c reductase; BU, 5-bromouridine.2The abbreviations used are: RIC, RNA import complex; Tet, tetracycline; BN, Blue Native; RT, reverse transcription; ORF, open reading frame; UCR, ubiquinol cytochrome c reductase; BU, 5-bromouridine. that induces translocation of tRNAs through artificial or biological membranes was isolated from Leishmania inner membrane (14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar, 15Bhattacharyya S.N. Adhya S. J. Biol. Chem. 2004; 279: 11259-11263Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 16Mahata B. Bhattacharyya S.N. Mukherjee S. Adhya S. J. Biol. Chem. 2005; 280: 5141-5144Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In a reconstituted liposome system, RIC retains the characteristic type I-type II interactions (14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar). Two tRNA-binding proteins with the properties of type I and type II receptors are associated with this complex (14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar). We have recently identified the type I receptor and showed that, in vivo, it is required for import of type I as well as type II tRNAs (13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar). We now report the identification of a subunit of this complex that binds type II tRNAs and is essential for the import of these tRNAs in vivo. Sequence Analysis and Homology Modeling—Affinity-purified RNA Import Complex from Leishmania tropica inner mitochondrial membranes (14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar) was resolved into its subunits by SDS-PAGE. The Coomassie Blue-stained 21-kDa band of RIC8 was trypsinized, and the peptides were subjected to liquid chromatography/mass spectrometry and tandem mass spectrometry analysis at the W. M. Keck Biomedical Mass Spectrometry Laboratory (University of Virginia). Sequenced peptides were matched against the NCBI and Leishmania major data bases (www.genedb.org/genedb/leish/index.jsp) to identify the corresponding genes using the BLAST or Sequest program. Selected sequences were aligned using ClustalW (www.ebi.ac.uk/clustalw/index.html). Secondary structure predictions were made by the PredictProtein server (www.cubic.bioc.columbia.edu/predictprotein/). Automated homology modeling of the Leishmania protein was performed by the SWISS-MODEL server (www.expasy.ch/swissmod/SWISS-MODEL.html), using the crystallographic structure of subunit 6 (Protein Data Bank code 1ntm) of bovine ubiquinol cytochrome c oxidase (Complex III) as template. The results were viewed in the Deep View/Swiss Pdb Viewer v. 3.7, and ray-traced images were generated using the POV-Ray 3.6 package. Cloning of RIC8A Gene—Sense and antisense primers (supplemental Table S1) corresponding to the open reading frame of RIC8A were used to amplify the intact or truncated gene from L. tropica strain UR6 genomic DNA. The sense primer was designed to have a BamHI restriction site at the 5′ end in frame with the coding sequence of the insert; the antisense primer had an inframe stop codon followed by a SalI site. The amplified fragment was cloned into TA vector pTZ57R (MBI Fermentas). Other genes were cloned and expressed similarly (13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar). Preparation of Recombinant Proteins and Antibodies—The RIC8A open reading frame was transferred as a BamHI-EcoRI fragment from pTZ57R to the pGEX4T-1 vector (Amersham Biosciences) downstream of, and in-frame with, the glutathione S-transferase gene. Recombinant plasmids were expressed in Escherichia coli BL21, and fusion protein was extracted from inclusion bodies and digested with thrombin, and the recombinant protein was recovered by gel electrophoresis, as described (13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar). To refold the protein, the soluble SDS extract was diluted 5-fold into TETN250 buffer (14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar) containing 0.1% Triton X-100 and incubated for 2 h at 4°C before assay. Electrophoretically pure recombinant protein was used to raise polyclonal antibody in BALB/c mice. Blue Native (BN) PAGE—Inner membrane mitochondrial complexes of L. tropica or the human HepG2 cell line were resolved as previously described (13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar). Briefly, mitochondria were extracted with BAM buffer (50 mm BisTris-HCl, pH 7.0, 0.75 m ϵ-aminocaproic acid, 2% dodecyl maltoside) for 45 min at 4 °C, and the extract was concentrated to ∼10 μl/200 μgof mitochondria by centrifugal ultrafiltration in a Microcon 30 unit (Amicon). Coomassie Blue G-250 (0.5%) was added to the extract before electrophoresis on 6% Blue Native gels (21Schagger H. Methods Enzymol. 1996; 264: 555-566Crossref PubMed Google Scholar). For two-dimensional analysis, protein bands of the first dimension were denatured with 0.125 m Tris-HCl, pH 6.8, 1% SDS, 1% β-mercaptoethanol for 40 min at 37 °C and then subjected to SDS-PAGE. Preparation of Radiolabeled tRNA—32P-Labeled tRNAs of high and low specific activity were prepared by T7 polymerase-mediated run-off transcription as described (11Bhattacharyya S.N. Chatterjee S. Adhya S. Mol. Cell Biol. 2002; 22: 4372-4382Crossref PubMed Scopus (38) Google Scholar, 13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar). Import Assays—Purified RIC (100 ng) was incorporated into phosphatidylcholine vesicles (50 μg lipid) and incubated with 32P-labeled tRNA (5 nm) and 4 mm ATP, and uptake was analyzed by RNase protection, as previously described (14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar). For immuno-inhibition experiments, proteoliposomes were preincubated with antiserum (1:50) for 30 min on ice. Where indicated, low specific activity effector tRNAs were present at one-tenth the concentration of the high specific activity substrate tRNA. Western and Northwestern Blots—Native complexes resolved by BN PAGE were denatured in situ before blot transfer, as described (13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar). The blots were probed with 1:100 dilution of antiserum and developed by the alkaline phosphatase colorimetric method. For Northwestern blots, the membrane was probed with 32P-labeled tRNA (17Ghosh A. Ghosh T. Ghosh S. Das S. Adhya S. Nucleic Acids Res. 1994; 22: 1663-1669Crossref PubMed Scopus (17) Google Scholar). Binding Assays—Indicated amounts of purified, recombinant refolded RIC8A were incubated with 32P-labeled tRNAIle (10–100 fmol) in 10-μl reactions containing binding buffer (10 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 2 mm dithiothreitol, 0.1 m KCl) for 30 min on ice and then electrophoresed on native gradient polyacrylamide gels (14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar) to resolve the ribonucleoprotein from free tRNA. Dried gel bands were quantified by liquid scintillation counting. Liposome binding assays were carried out under similar conditions with 250 ng of purified RIC incorporated into 80 μg of lipid and 1–250 fmol of tRNAIle, the vesicles were washed, and bound RNA was recovered for electrophoresis. Scatchard analysis was carried out by titrating a fixed amount of RIC8A (50 fmol) or liposome-bound RIC with tRNAIle. The plot of bound/free tRNA against the bound tRNA concentration yields a best fit curve with a slope of –1/Kd, where Kd is the dissociation constant for the complex. The total receptor concentration, [R]0, is the intercept on the x axis. Photochemical Cross-linking—T7 RNA polymerase transcripts were doubly labeled with [α-32P]UTP and 5-bromo-UTP and cross-linked to protein as previously described (14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar). Briefly, RNA was incubated with affinity-purified RIC or mitochondrial complexes and then UV-irradiated. Cross-linked RNA·protein complexes were immunoprecipitated after dissociation of the subunits with SDS and resolved by urea-PAGE (14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar). Northern Blotting—Total promastigote RNA was electrophoresed on a 5% acrylamide, 8 m urea gel, electroblotted on to a Hybond N+ membrane, and probed with radiolabeled RIC8A coding region. Conditional Knockdown—Details of the construction of L. tropica 13–90, a host containing constitutively expressed T7 RNA polymerase and Tet repressor genes, and of the targeting vector pGET, have been described (13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar). Knockdown vector pGET(AS)RIC8A was constructed by inserting the RIC8A gene between the HindIII and BamHI sites of pGET (i.e. in the reverse orientation with respect to the inducible T7 promoter). L. tropica 13–90 was transfected with pGET(AS)RIC8A and transformants selected on semi-solid agar containing G418, hygromycin, and 2.5 μg/ml phleomycin. The clones were grown in medium 199 containing the same antibiotics. The cultures were induced with 1 μg/ml tetracycline, and cell growth was monitored. Intracellular parameters were measured at 48 h, the time point at which cessation of growth was first observed. RT-PCR—Uninduced or induced cells were harvested, lysed, and separated into soluble (cytosolic) and particulate (mitochondrial) fractions (9Bhattacharyya S.N. Mukherjee S. Adhya S. Mol. Cell. Biol. 2000; 20: 7410-7417Crossref PubMed Scopus (15) Google Scholar). The crude mitochondria were treated with DNase and RNase before RNA isolation. RNA from 102–105 cells was denatured at 95 °C and reverse transcribed with Superscript II (Invitrogen) and the appropriate primer, as follows: (1Bhattacharyya S.N. Adhya S. RNA Biol. 2004; 1: 84-88Crossref PubMed Scopus (30) Google Scholar) for antisense RNA, the sense primer from the 5′ end of the coding region of RIC8A; (2Schneider A. Marechal-Drouard L. Trends Cell Biol. 2000; 10: 509-513Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) for mRNA or tRNA, the antisense primer complementary to the 3′ end of the gene (supplemental Table S1). The second primer was then added, and the cDNA was amplified with Taq DNA polymerase. To obtain proportional PCR signals, RT-PCR was performed with different amounts of input RNA. Import Reconstitution—Liposomes were reconstituted with mitochondrial extracts from knockdown cells and recombinant import factor as described (13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar). Briefly, refolded RIC8A (8 ng in 2 μl) and concentrated mitochondrial extract (5 × 106 cell equivalent in 8 μl) were incubated with liposomes (50 μg of lipid in 10 μl) for 1 h at 4°C and then assayed for import as above. RNA End Labeling—Total mitochondrial tRNA was dephosphorylated with shrimp alkaline phosphatase, ethanol-precipitated, and 5′-labeled with [γ-32P] ATP in the presence of T4 polynucleotide kinase. Mitochondrial Assays—Mitochondrial translation assays by [35S]methionine labeling of promastogotes in presence of cycloheximide, oxygen uptake measurements, and cytochrome oxidase cytochemical assays were performed as described (13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar). Homology of an RIC Subunit with a Complex III Component—On resolution of RIC by SDS-PAGE, a protein of 21 kDa (previously designated as RIC21p; now renamed RIC8) was observed that was present in the stoichiometry of 2–3/complex (Ref. 14Bhattacharyya S.N. Chatterjee S. Goswami S. Tripathi G. Dey S.N. Adhya S. Mol. Cell Biol. 2003; 23: 5217-5224Crossref PubMed Scopus (30) Google Scholar; see Fig. 3B). This protein was subjected to mass spectrometric sequencing, and the tryptic peptides were searched for in the L. major data base (18Ivens A.C. Peacock C.S. Worthey E.A. Murphy L. Aggarwal G. Berriman M. Sisk E. Rajandream M.-A. Adlem E. Aert R. Anupama A. Apostolou Z. Attipoe P. Bason N. Bauser C. Beck A. Beverley S.M. Bianchettin G. Borzym K. Bothe G. Bruschi C.V. Collins M. Cadag E. Ciarloni L. Clayton C. Coulson R.M.R. Cronin A. Cruz A.K. Davies R.M. De Gaudenzi J. Dobson D.E. Duesterhoeft A. Fazelina G. Fosker N. Frasch A.C. Fraser A. Fuchs M. Gabel C. Goble A. Goffeau A. Harris D. Hertz-Fowler C. Hilbert H. Horn D. Huang Y. Klages S. Knights A. Kube M. Larke N. Litvin L. Lord A. Louie T. Marra M. Masuy D. Matthews K. Michaeli S. Mottram J.C. Müller-Auer S. Munden H. Nelson S. Norbertczak H. Oliver K. O'Neil S. Pentony M. Pohl T.M. Price C. Purnelle B. Quail M.A. Rabbinowitsch E. Reinhardt R. Rieger M. Rinta J. Robben J. Robertson L. Ruiz J.C. Rutter S. Saunders D. Schäfer M. Schein J. Schwartz D.C. Seeger K. Seyler A. Sharp S. Shin H. Sivam D. Squares R. Squares S. Tosato V. Vogt C. Volckaert G. Wambutt R. Warren T. Wedler H. Woodward J. Zhou S. Zimmermann W. Smith D.F. Blackwell J.M. Stuart K.D. Barrell B. Myler P.J. Science. 2005; 309: 436-442Crossref PubMed Scopus (1137) Google Scholar) to retrieve complete open reading frames (ORFs). This resulted in the recovery of 10 ORFs, of which six were discarded as possible contaminants because of low protein coverage (less than 10%) or abnormally small size (less than 10 kDa). Of the remaining four (ORF-A, ORF-B, ORF-C, and ORF-D), ORF-C has sequence similarity with the iron-sulfur protein of mitochondrial succinate dehydrogenase (Complex II), ORF-B is a mitochondrial protein associated with a ribonucleoprotein (19Bringaud F. Peris M. Zen K.H. Simpson L. Mol. Biochem. Parasitol. 1995; 71: 65-79Crossref PubMed Scopus (41) Google Scholar), and ORF-D does not have sequence similarity with any known protein. 3S. Chatterjee and S. Adhya, unpublished data. These four ORFs were expressed separately in E. coli, and antibody was raised against each recombinant protein. Of these four antibodies, only anti-RIC8A (ORF-A) inhibited import in vitro. 3S. Chatterjee and S. Adhya, unpublished data. We therefore focused on the role of RIC8A, one of the major constituents of the 21-kDa band. BLAST analysis of RIC8A (systematic gene name LmjF35.0100 in the L. major data base) shows sequence similarities (28–30% identity) to the subunit 6b (or in the case of yeast, subunit 7) of respiratory Complex III (ubiquinol cytochrome c reductase or UCR) from a wide variety of species (Fig. 1A and supplemental Fig. S1). The L. tropica coding region is identical to that from L. major. 3S. Chatterjee and S. Adhya, unpublished data. In Leishmania and the related kinetoplastid protozoa Trypanosoma cruzi and Trypanosoma brucei, the corresponding predicted proteins are nearly identical in length (201–202 amino acid residues) and sequence (89 and 84% identity between Leishmania and the other two species). In other species such as man, UCR6b is smaller (14 kDa; see Fig. 1D). The sequence similarity between RIC8A and UCR6b is confined to the C-terminal 100 or so residues of the former. This similarity was confirmed by homology modeling of the secondary structure (Fig. 1B), which shows a four-helical bundle in the C-terminal region of RIC8A that is superimposable on the crystallographic structure of UCR6 (20Gao X. Wen X. Esser L. Quinn B. Yu L. Yu C.A. Xia D. Biochemistry. 2003; 42: 9067-9080Crossref PubMed Scopus (198) Google Scholar). The N-terminal extension does not show significant similarity to any known protein but is predicted to contain 1) several α-helices, one of which includes a periodic repeat of basic amino acids (between residues 42 and 61); and 2) a short cleaved mitochondrial targeting sequence, with the mature N terminus at Met-10 (as indicated by peptide sequencing). In Leishmania, there is a single gene for this protein (LmjF35.0100; henceforth designated as RIC8A/UCR6b) on chromosome 35 (17Ghosh A. Ghosh T. Ghosh S. Das S. Adhya S. Nucleic Acids Res. 1994; 22: 1663-1669Crossref PubMed Scopus (17) Google Scholar). A single 0.77-kb mRNA is expressed in promastigotes (Fig. 1C), Moreover, a single 21-kDa protein is present predominantly in the mitochondrial fraction (Fig. 1D). Type II tRNA Binding by RIC8A/UCR6b—To assess the tRNA binding activity of RIC8A, Western blots of bacterially expressed, gel-purified RIC8A, after suitable renaturation treatments in situ, were probed with radiolabeled tRNAs. Under these conditions, RIC8A interacted with tRNAIle, tRNAVal, and tRNAMet-e (all type II tRNAs), but with none of the type I tRNAs (Fig. 2A). The tRNA·protein complex was detected by gel shift assays using purified RIC8A, which confirmed the specificity for type II tRNA (Fig. 2B). Scatchard analysis (Fig. 2C) yielded a dissociation constant (Kd) of 0.42 nm at 4 °C for the tRNAIle·RIC8A complex (Table 1). Thus, RIC8A has an intrinsic affinity for type II tRNAs.TABLE 1Affinity of RIC8A for tRNAIleProteintRNATyr effectorKd[R]0nmnmFree RIC8A-0.422.28Complex-bound RIC8A-26.240.16Complex-bound RIC8A+5.291.75 Open table in a new tab To determine the location of the tRNA-binding site, the N- and C-terminal domains (residues 1–82 and 83–202, respectively) were separately expressed in E. coli, and the purified proteins were analyzed by Northwestern blotting using tRNAIle as probe. This experiment showed that only the N-terminal domain contains tRNA binding activity (Fig. 2D). Presence of RIC8A/UCR6b in Two Mitochondrial Complexes—In view of the facts that RIC8A/UCR6b is the product of a single gene, has structural similarity with UCR6b, binds tRNA, and is associated with the import complex, it is possible that RIC8A/UCR6b is a bi-functional protein with roles in both tRNA import and electron transport. To address this question, we resolved the mitochondrial inner membrane respiratory complexes of Leishmania by Blue Native gel electrophoresis (21Schagger H. Methods Enzymol. 1996; 264: 555-566Crossref PubMed Google Scholar) (Fig. 3A, left panel). As observed by us and others in the kinetoplastid protozoa Leishmania, Crithidia, and Phytomonas, (13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar, 22Horvath A. Berry E.A. Maslov D.A. Science. 2000; 287: 1639-1640Crossref PubMed Scopus (62) Google Scholar, 23Horvath A. Kingan T.G. Maslov D.A. J. Biol. Chem. 2000; 275: 17160-17165Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 24Speijer D. Breek C.K.D. Muijsers A.O. Hartog A.F. Berden J.A. Albracht S.P.J. Samyn B. Van Beeumen J. Benne R. Mol. Biochem. Parasitol. 1997; 85: 171-186Crossref PubMed Scopus (55) Google Scholar, 25Speijer D. Muijsers A.O. Dekker H. de Haan A. Breek C.K.D. Albracht S.P.J. Benne R. Mol. Biochem. Parasitol. 1996; 79: 47-59Crossref PubMed Scopus (23) Google Scholar, 26Maslov D.A. Nawathean P. Scheel J. Mol. Biochem. Parasitol. 1999; 99: 207-221Crossref PubMed Scopus (40) Google Scholar), four complexes were discernible, corresponding to Complexes III, IV, and V and the largest complex, specific to kinetoplastid protozoa, that has been identified as the import complex (13Goswami S. Dhar G. Mukherjee S. Mahata B. Chatterjee S. Home P. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8354-8359Crossref PubMed Scopus (43) Google Scholar). Immunoblotting of the resolved complexes revealed the presence of RIC8A/UCR6b in RIC and Complex III (Fig. 3A, right panel). The subunits of complex III and RIC were separated by denaturing electrophoresis in the second dimension. Most of the subunits in the two complexes are distinct, but Western blot analysis showed that the same 21-kDa RIC8A/UCR6b is shared by both (Fig. 3B), as expected of a bi-functional protein. Because RIC8A/UCR6b is shared by RIC and Complex III, we inquired whether both complexes can bind tRNA. When the mixture of native respiratory complexes obtained by detergent extraction of mitochondria was exposed to tRNAIle (type II) doubly labeled with 32P and 5-bromouridine (BU), a photoactivable nucleoside analogue, and then UV-irradiated, only RIC among the mitochondrial complexes was tagged; cross-linking required the presence of type I tRNA (Fig. 3C). In contrast, none of the human respiratory complexes interacted with tRNA under any condition (Fig. 3C). After dissociation of the
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