tRNA-triggered ATP Hydrolysis and Generation of Membrane Potential by the Leishmania Mitochondrial tRNA Import Complex

Abstract

Translocation of tRNAs across mitochondrial membranes is a receptor-mediated active transport process requiring ATP. A large tRNA import complex from the inner membrane of Leishmania mitochondria catalyzes translocation into phospholipid vesicles. In this reconstituted system, the import substrate tRNATyr(GUA) specifically stimulated hydrolysis of ATP within the vesicles, with the subsequent generation of a membrane potential by pumping out of protons, as shown by the protonophore-sensitive uptake of the potential-sensitive dye rhodamine 123. Generation of membrane potential was dependent on ATP hydrolysis, and inhibited by oligomycin, recalling the proton-translocation mechanism of the respiratory F1-F0-ATPase. For translocation of tRNA, ATP could be replaced by low pH of the medium, but proton-dependent import was resistant to oligomycin. Moreover, ATP hydrolysis, generation of membrane potential and tRNA uptake were inhibited by carboxyatractyloside, a specific inhibitor of mitochondrial ATP-ADP translocase, implying an ATP requirement within the vesicles. These observations imply a gating mechanism in which tRNA, on binding to its receptor, triggers the energetic activation of the complex, leading to the opening of import channels. Translocation of tRNAs across mitochondrial membranes is a receptor-mediated active transport process requiring ATP. A large tRNA import complex from the inner membrane of Leishmania mitochondria catalyzes translocation into phospholipid vesicles. In this reconstituted system, the import substrate tRNATyr(GUA) specifically stimulated hydrolysis of ATP within the vesicles, with the subsequent generation of a membrane potential by pumping out of protons, as shown by the protonophore-sensitive uptake of the potential-sensitive dye rhodamine 123. Generation of membrane potential was dependent on ATP hydrolysis, and inhibited by oligomycin, recalling the proton-translocation mechanism of the respiratory F1-F0-ATPase. For translocation of tRNA, ATP could be replaced by low pH of the medium, but proton-dependent import was resistant to oligomycin. Moreover, ATP hydrolysis, generation of membrane potential and tRNA uptake were inhibited by carboxyatractyloside, a specific inhibitor of mitochondrial ATP-ADP translocase, implying an ATP requirement within the vesicles. These observations imply a gating mechanism in which tRNA, on binding to its receptor, triggers the energetic activation of the complex, leading to the opening of import channels. In many species across the phylogenetic scale, the mitochondrial genome is incomplete with respect to the number of tRNA genes required for translation of the organellar mRNAs (1Schneider A. Marechal-Drouard L. 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The mechanism of translocation of these highly negatively charged macromolecules across the hydrophobic and electrostatic barriers of two mitochondrial membranes (of which the inner membrane is negatively charged in respiring mitochondria due to selective proton expulsion from the matrix) is a matter of considerable interest but poorly understood. Particularly, the bioenergetic inputs required to overcome these barriers have not been precisely defined. Using in vitro systems, it was shown that tRNA import in yeast, Leishmania and Trypanosoma mitochondria is ATP-dependent (7Tarrasov I. Entelis N.S. Nucleic Acids Res. 1992; 20: 1277-1281Crossref PubMed Scopus (55) Google Scholar, 8Mahapatra S. Ghosh T. Adhya S. Nucleic Acids Res. 1994; 22: 3381-3386Crossref PubMed Scopus (46) Google Scholar, 9Rubio M.A. Liu X. Yuzawa H. Alonzo J.D. Simpson L. RNA (N. Y.). 2000; 6: 988-1003Crossref PubMed Scopus (44) Google Scholar, 10Yermovsky-Kammerer A.E. Hajduk S.L. Mol. Cell. Biol. 1999; 19: 6253-6259Crossref PubMed Google Scholar) and requires ATP hydrolysis (11Mukherjee S. Bhattacharyya S.N. Adhya S. J. Biol. Chem. 1999; 274: 31249-31255Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). ATP could have a direct role in inducing conformational changes leading to opening of import channels. Alternatively, the mitochondrial F1-F0-ATPase, or some other ATPase dedicated to import, could generate a proton gradient that actually drives import. A role of protons was indicated by the inhibitory effect of protonophores (11Mukherjee S. Bhattacharyya S.N. Adhya S. J. Biol. Chem. 1999; 274: 31249-31255Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), which dissipate proton gradients, but whether protons are sufficient to induce import remains an open question. Finally, it is unclear whether proton gradient generation and tRNA translocation are separate or coupled processes and whether tRNA itself plays an active role in proton translocation. We have recently reported the isolation of a multiprotein complex (the RNA import complex or RIC) 1The abbreviations used are: RIC, RNA import complex; DTT, dithiothreitol; PEI, polyethyleneimine; TLC, thin layer chromatography; STE, sucrose-Tris-EDTA; ANT, adenine nucleotide translocator; AMPPCP, adenosine 5′-(β,γ-methylene)triphosphate; CCCP, m-chlorocarbonylcyanide phenylhydrazone. that is sufficient to induce import of tRNAs into artificial phospholipid vesicles (12Bhattacharyya 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). This reconstituted system retains all the properties of import in intact mitoplasts, including ATP dependence and sensitivity to respiratory uncouplers and inhibitors (12Bhattacharyya 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 were identified within this complex by photo-cross-linking and immunochemistry, a 45-kDa protein that binds tRNATyr directly and another 21-kDa protein that binds tRNAIle only in the presence of tRNATyr, suggesting allosteric changes within RIC leading to modulation of tRNA affinities (12Bhattacharyya 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, 13Bhattacharyya S.N. Chatterjee S. Adhya S. Mol. Cell. Biol. 2002; 22: 4372-4382Crossref PubMed Scopus (38) Google Scholar, 14Goswami S. Chatterjee S. Bhattacharyya S.N. Basu S. Adhya S. Nucleic Acids Res. 2003; 31: 5552-5559Crossref PubMed Scopus (15) Google Scholar). In the present study, we have used the RIC-reconstituted liposome system to study the bioenergetic transactions leading to import. Isolation of Mitochondria and Purificaton of RIC—Mitochondia were prepared from Leishmania tropica strain UR6 and purified by Percoll gradient centrifugation (8Mahapatra S. Ghosh T. Adhya S. Nucleic Acids Res. 1994; 22: 3381-3386Crossref PubMed Scopus (46) Google Scholar). Mitoplasts were prepared by digitonin treatment as described previously (11Mukherjee S. Bhattacharyya S.N. Adhya S. J. Biol. Chem. 1999; 274: 31249-31255Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). RIC was purified from mitochondrial inner membranes as described (12Bhattacharyya 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, mitochondrial inner membranes were extracted with 0.5% sodium deoxycholate in presence of 0.4 m KCl, and the extract was subjected to affinity chromatography using as ligand, the D arm of tRNATyr, which contains an import signal (14Goswami S. Chatterjee S. Bhattacharyya S.N. Basu S. Adhya S. Nucleic Acids Res. 2003; 31: 5552-5559Crossref PubMed Scopus (15) Google Scholar, 16Mahapatra S. Ghosh S. Bera S.K. Ghosh T. Das A. Adhya S. Nucleic Acids Res. 1998; 26: 2037-2041Crossref PubMed Scopus (42) Google Scholar). The affinity-purified fraction was used for ATPase activity assays or for proteoliposome reconstitution. Preparation of Proteoliposomes—Empty liposomes were prepared (12Bhattacharyya 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) by suspending phosphatidyl choline (250 μg) in 50 mm HEPES-KOH, pH 7.5, 10 mm MgAc2, 2 mm DTT, 10% glycerol, 0.25% deoxycholate and dialyzing the suspension against 5 mm HEPES-KOH, pH 7.5, 5 mm MgAc2, 20 mm KCl for 18 h at 4 °C. Aggregated material was removed by centrifugation at 2300 × g and the supernatant, containing mainly unilamellar vesicles, was used. For reconstitution, liposomes (25 μg) and RIC (100 ng) were incubated in the same buffer without deoxycholate for 1 h before import or ATPase assays. Import Substrates—32P-Labeled tRNATyr(GUA) or tRNAGln(CUG) was prepared by T7 RNA polymerase transcription as described previously (15Adhya S. Ghosh T. Das A. Bera S.K. Mahapatra S. J. Biol. Chem. 1997; 272: 21396-21402Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Import Assays—Proteoliposomes (equivalent to 100 ng of RIC) were incubated with 100 fmol of substrate RNA in 20 μl of import buffer (10 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm DTT, and 4 mm ATP) at 37 °C for 15 min, then RNases A and T1 were added at final concentrations of 2.5 μg/ml and 50 units/ml, respectively, and RNase digestion continued at 37 °C for 15 min. The proteoliposomes were then washed with 3 ml of liposome washing buffer (5 mm HEPES-KOH, pH 7.5, 2 mm DTT, 5 mm EDTA) and the vesicles recovered by centrifugation at 120,000 × g for 30 min at 4 °C. RNA was isolated from the liposomes as described previously (12Bhattacharyya 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, 16Mahapatra S. Ghosh S. Bera S.K. Ghosh T. Das A. Adhya S. Nucleic Acids Res. 1998; 26: 2037-2041Crossref PubMed Scopus (42) Google Scholar). ATPase Assay—RIC (10 ng) or proteoliposomes reconstituted with 10 ng of RIC (25 μg of phopholipid) was incubated in a 5-μl reaction with 1 mm [γ-32P]ATP (specific activity 2000 cpm/pmol) in 10 mm Tris-HCl, pH 8.0, 10 mm MgAc2, and 2 mm DTT, in the presence or absence of low specific activity (5000 cpm/pmol) tRNA (5 nm) as indicated. Inhibitors were preincubated with RIC or proteoliposomes on ice for 15 min prior to addition of the assay mix. After incubation at 37 °C for 10 min, 1 μl of each reaction was mixed with 1 μl of 1% SDS, deoxycholate, or Triton X-100 and spotted on prewashed polyethyleneimine (PEI) cellulose thin layer chromatography (TLC) plates (Sigma), essentially as described previously (17Böhme E. Schultz G. Methods Enzymol. 1974; 38: 27-38Crossref PubMed Scopus (53) Google Scholar). The plates were developed by sequential runs in 0.1 m acetic acid and 1 m LiCl and autoradiographed. Radioactive spots were quantified by liquid scintillation counting or densitometry. Apparent Km values were estimated from rate curves as the concentration of ATP or tRNATyr for half-maximal activity (pmol of ATP hydrolyzed per min). To determine the site of ATP hydrolysis, proteoliposomes were washed with isotonic sucrose-Tris-EDTA (STE; Ref. 8Mahapatra S. Ghosh T. Adhya S. Nucleic Acids Res. 1994; 22: 3381-3386Crossref PubMed Scopus (46) Google Scholar) and separated from free, i.e. external ATP by centrifugal ultrafiltration with a Microcon 30 ultrafilter (Amicon) at 16,000 × g, and resuspended in 10 μl of STE for TLC. Rhodamine 123 Uptake Assay—RIC-proteoliposomes (10 μg of lipid and 3 ng of RIC) were incubated with 0.05% rhodamine 123 in 10 μl of import buffer with 4 mm ATP for 15 min at 37 °C. Wherever indicated, liposomes were preincubated with inhibitors at 4 °C for 15 min. The proteoliposome suspension was mixed with an equal volume of STE containing 20% glycerol and observed under a Leica DMIRB inverted confocal microscope with laser excitation at 488 nm and emission at 540–668 nm. Images at ×1000 magnification were analyzed and quantified by the Leica SP2 confocal software. The mean fluorescence intensity per particle was calculated as the product fav × nf, where fav = average intensity of fluorescence in stained particles, and nf = fraction of particles staining above background. A tRNA-stimulated ATPase Activity in RIC—To determine whether the ATP-hydrolytic activity required for import is an integral part of, or distinct from, RIC, the purified complex was incubated with [γ-32P]ATP and radiolabeled Pi separated by thin layer chromatography. At high concentrations of RIC, a nonspecific ATPase activity was observed (data not shown). However, at low RIC concentrations, ATP hydrolysis was stimulated ∼16-fold by the import substrate tRNATyr (Fig. 1A). In contrast, tRNAGln(CUG), which is not imported in vivo or in vitro, had only a 4-fold effect (Fig. 1A); total Escherichia coli tRNA had no effect (data not shown). The preference of ATPase stimulatory activity for an importable tRNA indicates the relevance of this ATPase to import. From rate curves (Fig. 1C), the apparent KmtRNA(Tyr) for ATP hydrolysis was 0.98 nm, which is close to the value of 1.16 nm for RIC-mediated import (12Bhattacharyya 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). Similarly, KmATP for the ATPase was 1.32 mm, compared with 1.35 mm for import (12Bhattacharyya 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 examine further the relationship between the RIC ATPase and import, the complex was incorporated into liposomes. Incubation of the proteoliposomes with ATP resulted in tRNATyr-stimulated ATP hydrolysis (Fig. 1B), indicating retention of the activity in a membrane-bound form of the enzyme. However, the degree of stimulation of membrane-bound ATPase by tRNATyr was 2-fold compared with 16-fold for the soluble complex. This could be due to partial denaturation, with concomitant exposure of the ATPase site and loss of allosteric regulation, on insertion of the complex into the membrane. Import both in vitro (11Mukherjee S. Bhattacharyya S.N. Adhya S. J. Biol. Chem. 1999; 274: 31249-31255Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 12Bhattacharyya 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) and in transfected cells (14Goswami S. Chatterjee S. Bhattacharyya S.N. Basu S. Adhya S. Nucleic Acids Res. 2003; 31: 5552-5559Crossref PubMed Scopus (15) Google Scholar) has been shown to be sensitive to oligomycin, a specific inhibitor of the mitochondrial F1-F0-ATPase. Oligomycin apparently exerts its effect by reducing the permeability of the F0 portion of the enzyme to protons (18Pringle M.J. Kenneally M.K. Joshi S. J. Biol. Chem. 1990; 265: 7632-7637Abstract Full Text PDF PubMed Google Scholar). The ATPase activity of RIC was resistant to oligomycin, both in the free (Fig. 1D) and membrane-bound (Fig. 1E) forms. Thus, the oligomycin-sensitive step in import must be after the initial ATP hydrolysis step. ATP Hydrolysis Inside the Liposome—tRNA-stimulated ATP hydrolysis by membrane-bound RIC could occur on either side of the liposome membrane. The site of hydrolysis was determined in two ways. First, we had previously observed that import in intact mitochondria (11Mukherjee S. Bhattacharyya S.N. Adhya S. J. Biol. Chem. 1999; 274: 31249-31255Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) or mitoplasts 2S. N. Bhattacharyya and S. Adhya, unpublished data. is sensitive to carboxyatractyloside, a specific inhibitor of the adenine nucleotide translocator (ANT) on the inner membrane, suggesting the requirement of transport of ATP into the matrix. In the RIC-liposome system, tRNA uptake was similarly sensitive to carboxyatractyloside (Fig. 3C). In the same system, ATP hydrolysis was also inhibited by the drug, but the ATPase activity of the free RIC was unaffected (Fig. 1, F and G). These results are consistent with the presence of ANT, or an ANT-like carrier, in the complex and further indicate that upon insertion of RIC into the membrane the ATPase active site is exposed to the inner compartment. In a second experiment, we directly checked for the presence of labeled ATP and Pi within the vesicles as follows. RIC-liposomes were incubated with [γ-32P]ATP and separated from free ATP by ultrafiltration. When the liposomes were directly run on TLC of the was the with or no free Pi the (Fig. However, Pi was upon treatment of the liposomes with Triton X-100 (Fig. In a experiment, free RIC was similarly incubated with ATP and ATP Pi was in the (Fig. the of RIC itself binding or of these This the uptake and hydrolysis of ATP within the and Generation of dye rhodamine 123 is into respiring mitochondria of cells in a membrane Cell Biol. PubMed Scopus Google Scholar). We have previously observed uptake of rhodamine 123 into isolated Leishmania mitochondria in presence of ATP (11Mukherjee S. Bhattacharyya S.N. Adhya S. J. Biol. Chem. 1999; 274: 31249-31255Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The of the RIC-liposome system the of a assay of membrane potential generation by rhodamine 123 or RIC-reconstituted liposomes were incubated with tRNA and ATP and uptake of rhodamine 123 by confocal fluorescence of the vesicles labeled in the system (Fig. Uptake was dependent on the presence of RIC, and ATP could not be by the AMPPCP, a requirement for ATP tRNATyr could not be replaced by the Moreover, uptake was inhibited by the m-chlorocarbonylcyanide which proton this that the membrane potential is by RIC-mediated pumping of Uptake was also sensitive to oligomycin, a proton of ATP transport by carboxyatractyloside uptake of rhodamine implying the requirement of ATP hydrolysis within the vesicles. These results indicate the generation of a membrane potential by a proton activity of Import of import of tRNA in mitochondria (11Mukherjee S. Bhattacharyya S.N. Adhya S. J. Biol. Chem. 1999; 274: 31249-31255Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) and in transfected cells (14Goswami S. Chatterjee S. Bhattacharyya S.N. Basu S. Adhya S. Nucleic Acids Res. 2003; 31: 5552-5559Crossref PubMed Scopus (15) Google Scholar) has been shown to be sensitive to protonophores and that dissipate either or both of the or of the gradient by proton translocation in respiring In RIC-reconstituted ATP-dependent uptake of tRNA was also sensitive to CCCP, and (Fig. A and This that it is the proton gradient by ATP hydrolysis, ATP hydrolysis that the for import but not an role for ATP. we checked whether ATP be replaced by protons in the RIC-reconstituted Uptake assays were in of the pH of the liposomes At an external pH of was no uptake in absence of ATP (Fig. but as the external pH was tRNA uptake at an external pH of it the value with ATP. uptake was similarly sensitive to and (Fig. The in presence of high external the (the membrane of the which for the without Thus, these indicate the requirement of both for RIC-mediated import. between ATP-dependent and proton-dependent import was observed with respect to to oligomycin. ATP-dependent import was sensitive to (Fig. proton-dependent import was resistant (Fig. This indicates in the the oligomycin-sensitive step between ATP hydrolysis and i.e. at the step of proton with concomitant generation of a membrane potential The binding of substrate tRNA to its on the RIC has on the binding of other a tRNA binding of a tRNA, a tRNA the binding of a tRNA (12Bhattacharyya 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, 13Bhattacharyya S.N. Chatterjee S. Adhya S. Mol. Cell. Biol. 2002; 22: 4372-4382Crossref PubMed Scopus (38) Google Scholar, 14Goswami S. Chatterjee S. Bhattacharyya S.N. Basu S. Adhya S. Nucleic Acids Res. 2003; 31: 5552-5559Crossref PubMed Scopus (15) Google Scholar). induce the opening of the import (12Bhattacharyya 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). In tRNA specifically an ATPase activity (Fig. and a membrane potential (Fig. results are consistent with a of within the complex to tRNA substrate tRNA binds to its ATP is into the a translocase, which is or to the adenine nucleotide binding to the activation of an ATPase which catalyzes ATP hydrolysis within the ATP hydrolysis is by pumping of protons an oligomycin-sensitive from to the membrane, in the generation of a membrane of the proton gradient to opening of the import by this the requirement for the membrane the tRNA is the import external protons are required at this step. The protons to the on the RNA or the to translocation of the The ATPase we as to be but distinct from, the mitochondrial respiratory and the a of the two Ref. M. T. Y. H. Y. J. Biol. 2000; PubMed Google Scholar). In all ATP hydrolysis is coupled to proton translocation across the membrane. Moreover, proton from the in which ATP hydrolysis is and both and in membrane-bound protons in an oligomycin-sensitive are also particles the of the of have been observed with purified RIC (12Bhattacharyya 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). N. S. S. and S. Adhya, unpublished data. the of an RIC with to the of which is also present in M. T. Y. H. Y. J. Biol. 2000; PubMed Google Scholar). the and to a of multiprotein dedicated to of the RIC is the with which it tRNA translocation across addition or with membrane vesicles, whether liposomes (12Bhattacharyya 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) or mitoplasts or intact mitochondria is and by as is with respiratory S. D. a Scholar), is This a insertion of the complex into Moreover, membrane insertion of the complex be This is indicated by the that in the reconstituted ATP hydrolysis and the activity is sensitive to carboxyatractyloside (Fig. the soluble ATPase is implying that or all of the ATPase active are to the insertion be to ATPase The mechanism of insertion is is insertion a lipid of the in Leishmania and Trypanosoma Mol. 1997; PubMed Scopus Google Scholar). is that the mitochondrial inner membrane contains a mechanism for a membrane potential that is of respiratory we have observed that with intact mitochondria or mitoplasts, respiratory such as are to ATP as an for import. N. S. and S. Adhya, unpublished data. the as to whether the proton gradient by RIC is dedicated to import or whether it be under to other inner membrane transport The present study the presence of within the RIC that to be to mitochondrial in ATP and an oligomycin-sensitive proton and an ATP be to the proteins to the relationship between tRNA import and We Tripathi for the confocal