Dual Functions of �-Ketoglutarate Dehydrogenase E2 in the KrebsCycle and Mitochondrial DNA Inheritance in Trypanosoma brucei

Steven E. Sykes,a Stephen L. Hajdukb

Department of Cellular Biology, University of Georgia, Athens, Georgia, USAa; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia,USAb

The dihydrolipoyl succinyltransferase (E2) of the multisubunit �-ketoglutarate dehydrogenase complex (�-KD) is an es-sential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally reg-ulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. Wefound that despite the absence of a functional �-KD, bloodstream form (BF) trypanosomes express �-KDE2, which local-ized to the mitochondrial matrix and inner membrane. Furthermore, �-KDE2 fractionated with the mitochondrial ge-nome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for �-KDE2 in kDNA maintenance was re-vealed in �-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showedpronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation ofcells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochon-drial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that�-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribu-tion of kDNA networks to daughter cells at cytokinesis.

The �-keto acid dehydrogenases are multienzyme assembliesthat catalyze the decarboxylation of their respective �-keto

acids, which subsequently produce acyl-coenzyme A (CoA) andNADH (1). This family of high-molecular-mass (greater than 1MDa) complexes consists of pyruvate dehydrogenase (PDH),branched-chain �-keto acid dehydrogenase (BCKAD), and �-ketoglutarate dehydrogenase (�-KD). Each complex is composedof an �-keto acid dehydrogenase subunit (E1), an acyltransferasesubunit (E2), and a dihydrolipoamide dehydrogenase subunit(E3). Multiple copies of E2 form the cores of these multimericcomplexes, and the remaining components (E1 and E3) surroundthis structure (1). In each case, the highly conserved E2 has asimilar quaternary structure with a covalently attached lipoic acidprosthetic group that swings from one active site to the next (2, 3).

Although E2 has a vital role in �-KD metabolism, the enzymehas also been shown to associate with prokaryotic genomes andthe mitochondrial DNA (mtDNA) of multiple eukaryotic organ-isms (4–9). mtDNA is organized into stable protein-DNA unitscalled nucleoids attached to the mitochondrial inner membraneand is involved in genome replication, segregation, and mainte-nance (10, 11). Proteins isolated from mtDNA nucleoids fromhigher eukaryotes revealed the association of the E2 enzymes fromthe PDH and BCKAD complexes (6). Additionally, �-KD E2 (�-KDE2) associates with the mtDNA nucleoid of Saccharomycescerevisiae and is required for mtDNA maintenance (4, 5).

The protozoan parasite Trypanosoma brucei possesses a dis-tinctive mitochondrial genome termed the kinetoplast DNA(kDNA). The kDNA is a large network structure composed ofthousands of catenated DNA minicircles (�1 kb) and approxi-mately 50 maxicircles (�20 kb). Maxicircles contain the codinginformation for components of the oxidoreductase complexes,ATP synthase, and rRNAs, while minicircles encode small guideRNAs (gRNAs) necessary for posttranscriptional editing of max-icircle-encoded mRNAs (12).

Mitochondrial metabolism and ATP production are develop-

mentally regulated in T. brucei. In mammals, the bloodstreamdevelopmental forms of trypanosomes (BF) utilize high rates ofglycolysis to produce ATP and pyruvate but lack functional Krebscycle enzymes and mitochondrial respiratory complexes neededfor oxidative phosphorylation (13). Thus, all ATP is produced byglycolytic substrate level phosphorylation. In the midgut of theinsect vector, the tsetse fly, trypanosomes rapidly differentiate intothe procyclic developmental form (PF) and develop a fully func-tional mitochondrion capable of converting proline to succinateand transferring reducing equivalents to NADH via the Krebs cy-cle enzymes (14).

Despite the lack of conventional oxidative phosphorylation inthe BF, the kDNA must be faithfully replicated and segregated,since it encodes enzymes needed in the PF and other developmen-tal stages in the tsetse fly (15–18). A number of structural proteinsand replication enzymes necessary for kDNA replication and seg-regation have been identified (19, 20), and the mechanism ofkDNA replication has been described in detail (21). During themitochondrial S phase, minicircles are released from the kDNAnetwork by a type II topoisomerase and relocate to the kinetofla-gellar zone (KFZ), a region between one face of the kDNA disk andthe basal bodies of the flagellum (22). Next, free minicircles bindthe universal binding protein (UBP) at discrete foci in the KFZwhere replication initiates, giving rise to replicative intermediatescontaining theta structures (23, 24). Replicating minicircles mi-grate to antipodal sites on the elongated kDNA network wheresingle-strand gaps from lagging-strand replication are filled and

Received 27 September 2012 Accepted 30 October 2012

Published ahead of print 2 November 2012

Address correspondence to Stephen L. Hajduk, shajduk@bmb.uga.edu.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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the newly replicated minicircles, containing a single gap at theorigin of replication, are reattached to the network (11, 25–27).RNA interference (RNAi) knockdown or conditional knockoutsof several of the key replicative enzymes results in a rapid loss ofminicircles (27–29). Complete kDNA replication results in a net-work with twice the number of minicircles and maxicircles thatundergoes lateral elongation and finally segregation to daughtercells, ensuring equal inheritance of minicircles and maxicircles.

While the mechanism of network segregation is poorly under-stood, the association of the kDNA with a specialized portion ofthe mitochondrial membrane, immediately adjacent to the basalbodies of the flagellum, is required for kDNA segregation (11, 30).The association of the kDNA with the basal bodies has beendefined morphologically as a filamentous network of proteinsknown as the tripartite attachment complex (TAC) (11). The TACmaintains a physical connection between the kDNA and flagellumand fractionates with a larger flagellum-kDNA complex (FKC)upon cell solubilization with nonionic detergents (11, 31). TheTAC extends from the basal bodies of the flagellum to the outermitochondrial membrane (exclusion zone filaments) and contin-ues across the inner mitochondrial membrane to the KFZ face ofthe kDNA (unilateral filaments). To date, only two components ofthe TAC have been identified. (i) p166 was the first protein com-ponent identified in the TAC (30). This large nuclear-encodedprotein was initially discovered during an RNAi library screen forkDNA replication/segregation defects (30). p166 contains an N-terminal mitochondrial import signal, as well as a short trans-membrane domain, and associates with the unilateral filaments ofthe TAC. The RNAi knockdowns of p166 resulted in alteredkDNA structure due to asymmetric segregation of the kDNA, butno effect of kDNA replication was observed (30). (ii) Alternativelyedited protein 1 (AEP-1), encoded by an alternatively edited cy-tochrome oxidase III (COIII) mRNA, is a chimeric protein with aunique N-terminal 60-amino-acid DNA binding domain and fiveC-terminal transmembrane domains of COIII (32). AEP-1 asso-ciates with the unilateral filaments of the TAC, where it serves as amaintenance factor for kDNA (33).

The structural organization of the kDNA network has givenrise to unique mechanisms of replication, segregation, and in-heritance of this mitochondrial genome. Central to these pro-cesses is the FKC, which ensures segregation of the newly rep-licated kDNA networks and distribution to daughter cells atcytokinesis. Considering the complexity of the FKC, it is likelythat both structural and motor proteins, necessary for segrega-tion and inheritance, remain to be defined (11, 21). In thisstudy, we report the expression of �-KDE2 in the oxidative-phosphorylation-deficient BF T. brucei. We show that �-KDE2is expressed and localizes to the BF mitochondria, despite thelack of �-KD activity. Surprisingly, we found that �-KDE2 lo-calized to antipodal sites on the kDNA network and is a stablecomponent of the FKC. RNAi knockdown of �-KDE2 results inincreased numbers of dyskinetoplastic cells with a correspond-ing increase of cells with two kinetoplasts, consistent with arole for �-KDE2 in the distribution of kDNA at cytokinesis andnot in either segregation or replication. Finally, depletion of�-KDE2 results in collapse of the mitochondrial membranepotential in dyskinteoplastic cells and a reduction in total mi-tochondrial volume. These data demonstrate the importanceof �-KDE2 as a bifunctional protein necessary for the mainte-nance of the kDNA and mitochondria in T. brucei.

MATERIALS AND METHODSCell culture. Procyclic-form T. brucei was grown in Cunningham’s (SM)medium supplemented with fetal bovine serum (FBS) (Gemini Bioprod-ucts, West Sacramento, CA). Bloodstream form T. brucei was maintainedin HMI-9 medium containing FBS and Serum Plus medium supplement(SAFC Biosciences, Lenexa, KS). The �-KDE2 RNAi cell line was culturedin HMI-9 medium that was supplemented with tetracycline-free FBS.

Construction of �-KDE2-PTP and �-KDE2-RNAi cell lines.Primers 5=-GGGCCCAAGATAAACTTCGAAGAGGGCAC-3= and 5=-GCGGCCGCGGCGAGGTCGAGCACAATA-3= against the �-KDE2(Tb11.01.3550) open reading frame (ORF) amplified 912 bp of PF- and BF-667 genomic DNA, which was subsequently cloned into the pC-PTP-NEOexpression vector (34). The fragment was digested with ApaI and NotI forinsertion into the vector. Constructs were linearized with a unique restrictionsite for transfection into the two T. brucei cell types. For the�-KDE2 RNAi cellline, primers 5=-CCCTCGAGGCTCACGACATTCAACGAGA-3= and 5=-CCAAGCTTTCTGTGGTGGGTTGACGATA-3= were used to amplify apartial �-KDE2 sequence (425 bp) from BF-9013 genomic DNA that wasligated into the inducible pZJM RNAi vector (35). NotI was used to lin-earize the construct for transfection. All bloodstream constructs weretransfected using the Lonza nucleofector system (Lonza, Walkersville,MD). The procyclic �-KDE2-PTP construct was transfected using theBio-Rad electroporation system (Bio-Rad, Hercules, CA).

Fractionation of mitochondrial proteins. Cultured PF T. brucei(TRUE667) and BF T. brucei (TRUE667) cells, isolated from infectedSprague-Dawley rats, were hypotonically lysed, and mitochondria werepurified by a previously described method (36). Subcellular fractionationwas performed as discussed previously with minor modifications (32, 37).Matrix proteins were purified by incubating mitochondria in 0.5% (vol/vol) Triton X-100, 20 mM HEPES-NaOH (pH 7.6) with 1� Completeprotease inhibitor cocktail (Roche Indianapolis, IN) for 45 min on ice.Insoluble material was separated from the matrix fraction by centrifuga-tion at 12,000 � g for 10 min at 4°C. The membrane fraction was collectedafter purification of the matrix proteins by incubation of the insolublefraction in 2% (wt/vol) n-dodecyl-�-D-maltoside (Sigma, St. Louis, MO),50 mM NaCl, 50 mM imidazole, 2 mM 6-aminohexanoic acid, 1 mMEDTA, and 1� Complete EDTA-free protease inhibitor cocktail, pH 7, at4°C for 1 h on ice. An insoluble fraction was collected by centrifugation at13,000 � g for 20 min at 4°C, and the soluble fraction was saved forsubsequent analysis. The total cell, cytosolic, total mitochondrial, matrix,and membrane fractions of PF and BF T. brucei were denatured in areducing SDS loading buffer, applied equivalently (2 � 106 cells) to gels,and resolved by SDS-PAGE. The gels were either stained with Coomassieblue or analyzed by Western blotting.

Extraction of FKCs. We used the published method for purifyingFKCs from T. brucei (11). For SDS-PAGE analysis, these complexes wereextracted from 6 � 107 BF cells, and a fraction of these structures weretreated with DNase (Roche) for 1 h on ice to remove the kDNA. FKCsisolated from 1 � 107 cells were loaded on gels, resolved, stained withCoomassie blue, and analyzed by Western blotting.

Western blot analysis. Protein blots were blocked in 5% (wt/vol)milk-TBST (150 mM NaCl, 10 mM Tris-HCl, pH 8, 0.05% [vol/vol]Tween 20) and incubated overnight with the following primary antibod-ies: polyclonal rabbit HSP-70 (1:3,000; Abcam, Cambridge, MA), mono-clonal mouse MTP-70 (1:500), monoclonal mouse iron sulfur protein(ISP) (1:2,000), monoclonal mouse trypanosome alternative oxidase(TAO) (1:100), monoclonal rat YL1/2 (1:5,000; Abcam), and polyclonalperoxidase anti-peroxidase soluble complex (PAP) (1:5,000; Sigma). Theblots were washed three times and incubated with a goat anti-rabbithorseradish peroxidase (HRP) secondary antibody (1:5,000) for 1 h.

BrdU analysis. The bromodeoxyuridine (BrdU) assay was performedas previously described (38, 39) with few modifications. BF cells (2 � 106)were incubated with 50 �M 5-bromo-2-deoxyuridine (Sigma) and 50 �M2-deoxycytidine (Sigma) at 37°C for 30 and 480 min. The cells werewashed in medium, dried on a slide, and fixed in methanol (�20°C) for 30

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min. After fixation, the cells were rehydrated in phosphate-buffered saline(PBS) and pretreated with 2.0 M HCl for an additional 30 min. The slideswere neutralized in PBS (three 5-min washes) and prepared for immuno-fluorescence assay. For FKC localization, slides were fixed with methanolfor 10 min and treated with ice-cold 2% paraformaldehyde on the slide for5 min. Next, the slides were heated at 90°C in 10 mM sodium citrate (pH6.0) for 10 min. The slides were removed from the bath, allowed to cool toroom temperature (27°C), and washed with PBS for immunofluorescenceassay.

Immunofluorescence microscopy. Log-phase cultured �-KDE2-PTPBF and PF cells were incubated with MitoTracker (Life Technologies,Grand Island, NY), washed, equilibrated with medium for 30 min, andattached to poly-L-lysine slides for 30 additional minutes. The cells werefixed with 0.5% paraformaldehyde for 1 min, washed in PBS, and perme-abilized with ice-cold 0.05% Triton X-100 in PBS for 5 min at 4°C. Theslides were washed and blocked using 20% fetal bovine serum in PBS.Cells were incubated with protein C polyclonal antibodies (1:200) dilutedin blocking buffer and remained in primary antibody for 1 h. The slideswere washed, and the cells were incubated with appropriate secondaryantibody (1:500) for 30 min. For FKCs, intact and DNase-treated struc-tures were dried on a slide and fixed with methanol (�20°C) for 10 min.The slides were blocked with 0.5% bovine serum albumin (BSA) in PBSfor 30 min, and the following primary antibodies were diluted in theblocking buffer and added: monoclonal rat YL1/2 (1:1,000) and poly-clonal rabbit protein C (1:200). The slides were washed in blocking buffer,and an appropriate secondary antibody (1:500) was used. BF �-KDE2RNAi cells were washed in medium, dried on the slide, and fixed in meth-anol for 10 min. BrdU-treated FKCs were blocked in 0.5% BSA in PBS for30 min and incubated with polyclonal rabbit protein C (1:200) and mono-clonal mouse BrdU (1:100) primary antibodies diluted in blocking bufferfor 90 min. The slides were washed in blocking buffer and incubated inappropriate secondary antibody (polyclonal, 1:500; monoclonal, 1:100)for 1 h. After the secondary antibody, all slides were rinsed in PBS andcoated with 4=,6=-diamidino-2-phenylindole (DAPI) containing the anti-fade reagent ProlongGold (Life Technologies). Images were acquired us-ing a Zeiss Axio Observer inverted microscope equipped with anAxioCam HSm and evaluated with AxioVision v4.6 software (Zeiss).

Activity of the �-KD. Purified intact PF and BF mitochondria weresolubilized in buffer containing 2% (wt/vol) n-dodecyl-�-D-maltoside, 50mM NaCl, 50 mM imidazole, 2 mM 6-aminohexanoic acid, 1 mM EDTA,and 1� Complete EDTA-free protease inhibitor cocktail on ice for 1 h andcentrifuged at 13,000 � g for 20 min. Soluble mitochondrial homogenate(40 �g) was incubated in 1 ml of �-KD assay buffer (50 mM Tris-HCl, pH7.6, 0.1 mM CaCl2, 0.05 mM EDTA, 0.3 mM thiamine pyrophosphate, 1mM MgCl2, 3 mM �-ketoglutarate, 3 mM NAD�, and 0.75 mg/ml ofcoenzyme A at 25°C). The activity of �-KD was monitored spectrophoto-metrically by the production of NADH per minute at 340 nm.

Northern and Southern analyses. All radiolabeled probes were pre-pared using a Prime-It random primer-labeling kit (Stratagene, SantaClara, CA). For Northern analysis, total RNA was extracted from bothdevelopmental stages using TriPure Isolation Reagent (Roche). Tran-scripts were separated on a 7% formaldehyde-1% agarose gel, blotted to amembrane, and evaluated with radiolabeled probes generated from ORF-specific sequences (�-KDE1, �-KDE2, E3, and �-tubulin). Probes werehybridized in a mixture containing 50% (vol/vol) formamide, 5� SSC(1� SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5� Denhardt’ssolution (Sigma), 1% (wt/vol) SDS, and 100 �g/ml salmon sperm DNA(Life Technologies) at 55°C overnight. Blots were washed twice at 30-minintervals in 0.2� SSC containing 0.1% SDS at 68°C. Total genomic DNAwas extracted from induced �-KDE2 RNAi cells every 24 h for Southernanalysis. Undigested and digested (XbaI and HindIII) DNA was resolvedwith agarose, transferred to a blot, and analyzed for kDNA content. Toevaluate minicircle sequences, probes were derived from a cloned frag-ment composed of a predicted origin of replication (ori) and a bent helicalregion conserved in all minicircles. Maxicircle probes were generated

from a PCR product using primers against the preedited 9S genomic se-quence. All Southern probes were hybridized in a solution containing50% formamide (Sigma), 3� SSC, 1� Denhardt’s solution, 20 �g/mlsalmon sperm DNA, 5% dextran sulfate, and 2% SDS at 42°C overnight.Blots were washed twice in a solution containing 3� SSC-0.5% SDS at55°C for 30 min, exposed to a storage phosphor screen (Molecular Dy-namics), and analyzed on a Storm-860 PhosphorImager (GE Healthcare).

RESULTSExpression of �-KDE2 in BF T. brucei. Replication, segregation,and the inheritance of the kDNA network require the assembly ofa novel apparatus, the FKC, to facilitate equal distribution of thekDNA to daughter cells. We reasoned that dual-functioning pro-teins might be part of the FKC and might participate in kDNAmaintenance. To address this possibility, we looked at the level ofexpression of known nuclear-encoded mitochondrial enzymes inBF trypanosomes lacking mitochondrial oxidative phosphoryla-tion. A similar analysis of mitochondrial encoded proteins led tothe identification of an alternatively edited COIII mRNA that en-coded a component of the FKC (32, 40). The developmental reg-ulation of nuclear-encoded mitochondrial proteins in T. bruceioccurs principally at the level of RNA stability and, to a lesserextent, by differential protein stability (41, 42). High-throughputRNA sequencing (RNA-Seq) provides an accurate evaluation ofthe life cycle-dependent expression of mitochondrial proteins(43–46). Genome-wide analysis of mRNA levels indicated that theexpression of �-KDE1, �-KDE2, and E3 was not developmentallyregulated in BF and PF trypanosomes (45, 46). Unlike �-KDE1and �-KDE2, the E3 subunit is a component of four distinct mul-tienzyme mitochondrial complexes encoded by a single trypano-some gene (47). Therefore, sequence analysis alone cannot be usedto determine whether E3 mRNAs encode subunits of the �-KD orother enzyme complexes (46). To verify the RNA-Seq results, totalRNA from procyclic and bloodstream trypanosomes (1 � 107 cellequivalents) was analyzed by Northern blot hybridization withprobes specific for �-KDE1 (�3.0 kb), �-KDE2 (�1.1 kb), and E3(�1.4 kb) (Fig. 1A). Hybridization was normalized relative to therRNA ethidium stain for each lane (Fig. 1A). Consistent with pub-lished RNA-Seq results, the mRNAs of �-KDE1, �-KDE2, and E3were expressed in both BF and PF trypanosomes (45) (Fig. 1A).

�-KD activity is absent in BF T. brucei. Since previous studieshave shown that BF T. brucei lacks metabolically active Krebs cycleenzymes, the presence of �-KDE1 and �-KDE2 mRNA expressionin both BF and PF trypanosomes was unexpected. However, E3has previously been shown to be essential in BF trypanosomes(47). We therefore next examined these cells for �-KD activity.This mitochondrial enzyme complex specifically converts �-keto-glutarate to succinyl-CoA and subsequently reduces NAD� toNADH. This dinucleotide conversion can be measured spectro-photometrically for �-KD activity when �-ketoglutarate is addedas a substrate. At 340 nm, the procyclic mitochondrial lysates, butnot BF mitochondrial lysates, showed increasing production ofNADH (Fig. 1B). To determine whether the lack of �-KD activityin BF trypanosomes was due to an endogenous inhibitor of �-KDin the BF mitochondrial fraction, equal amounts of BF and PFmitochondrial lysates were mixed and added to the same �-KDassay mixture. Since the total protein concentration during thisanalysis was kept constant, a 2-fold reduction in the NADH pro-duced (�mol/min) was expected if no enzyme antagonists werepresent (Fig. 1B). Lack of inhibition by the BF lysates indicates that

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�-KDE1, �-KDE2, and E3, while expressed in BF mitochondria,do not assemble into a functional enzyme complex, suggestingalternative functions for these proteins in BF trypanosomes. Evi-dence for the bifunctionality of �-KDE2 in other organisms led usto examine the function of the protein in BF trypanosomes (10).

�-KDE2 distribution in the mitochondria of trypanosomes.Krebs cycle-associated �-KDE2 is present in a soluble complexwithin the mitochondrial matrix. However, a fraction of E2 trans-ferases (�-KDE2, PDHE2, and BCKADE2) has also been shown tobe associated with DNA nucleoids attached to the mitochondrialmembranes of other cells (5–7). To evaluate the localization of�-KDE2 in T. brucei, a C-terminally epitope-tagged version of theprotein (�-KDE2-PTP) was constructed (34, 48). Expression ofthe BF and PF (�-KDE2-PTP) tagged transcripts from a singleallele was confirmed by Northern analysis, which identified 1.5-kb(�-KDE2-PTP mRNA) and 1.1-kb (endogenous �-KDE2 mRNA)bands in both cell types (Fig. 2A). Though inserted into the en-dogenous �-KDE2 locus, �-KDE2-PTP expression was approxi-mately 2-fold higher than that of wild-type (WT) �-KDE2 andis possibly a result of increased mRNA stability due to the 3= un-translated region (UTR) sequences flanking the PTP coding se-quences (Fig. 2A) (34).

We next examined the intracellular localization of �-KDE2-PTP by immunofluorescence microscopy (Fig. 2B). Using an an-tibody against the protein C epitope, �-KDE2-PTP distributionwas compared with MitoTracker Red staining (Fig. 2B). Withinthe large and highly branched mitochondria of the PF trypano-somes, the �-KDE2-PTP and MitoTracker staining were superim-posed (Fig. 2B, top). Similarly, while the BF mitochondrion wasreduced to a single, largely unbranched tubular structure, Mi-toTracker and �-KDE2-PTP colocalized throughout theorganelle (Fig. 2B, bottom), despite the lack of �-KD activity(Fig. 1B and 2B).

Subcellular fractionation studies confirmed the mitochondriallocalization of �-KDE2 in both BF and PF T. brucei. In Westernblot studies, total cell lysates from nontransfected BF and PF cells(WT) did not contain proteins that cross-react with the antibody(�-protein A) used to detect �-KDE2-PTP. Total BF and PF pro-tein from �-KDE2-PTP-expressing cells (TC) and cellular frac-

tions enriched in cytosol (CY), total mitochondria (TM), mito-chondrial matrix (MA), and mitochondrial membrane (ME) wereresolved by SDS-PAGE and analyzed by Western blotting withantibodies for marker proteins (Fig. 2C and D). �-KDE2-PTP wasexclusively detected in mitochondrial fractions relative to the cy-tosolic HSP-70 and the mitochondrial matrix protein MTP-70.Examination of the submitochondrial distribution of �-KDE2-PTP revealed that approximately 25% localized with the mito-chondrial membrane markers, Rieske iron-sulfur protein (ISP)for PF and the alternative oxidase (TAO) for BF (Fig. 2C and D).These results confirmed the localization of �-KDE2-PTP to themitochondria of PF and BF T. brucei and suggested similar sub-mitochondrial localization in the two developmental stages. Tofurther evaluate the alternative function(s) of �-KDE2 in trypano-some mitochondria, we focused our studies on BF trypanosomes,where the �-KD enzymatic activity is absent.

�-KDE2 associates with the FKC. The kDNA network of try-panosomes is anchored to the base of the flagellum by the TAC.This association with the mitochondrial membrane is reminiscentof the attachment of the mitochondrial nucleoid in diverse organ-isms. Since �-KDE2 is distributed in both the mitochondrial ma-trix and membranes, it was not possible to determine whetherthere was a specific association with the kDNA using intact cells.To release the mitochondrial matrix and membrane-associated�-KDE2-PTP, cells mildly fixed with paraformaldehyde weretreated with low concentrations of Triton X-100 (0.25%) to createcell ghosts. This treatment permeabilizes many subcellular organ-elles, including the mitochondrion, while leaving the DNA-asso-ciated cytoskeleton intact (31) (Fig. 3A). The cell ghosts were sub-sequently examined by immunofluorescence microscopy usinganti-protein C and anti-tyrosinated �-tubulin (tyr-�-tubulin) an-tibodies to visualize �-KDE2-PTP and the basal bodies of the fla-gellum, respectively. The nucleus and kinetoplast were stainedwith DAPI (Fig. 3A). The majority of the �-KDE2-PTP signal wasdepleted in these preparations, leaving a prominent structure inclose proximity to the kDNA and basal bodies. Three-dimensionalreconstruction (3D-R) images further support the close proximityof �-KDE2-PTP to the kDNA and basal body (Fig. 3A).

Since kDNA replication involves the detachment, duplication,

FIG 1 Stage-specific mRNA expression of �-KD subunits and complex activity in developmental stages. (A) Northern analysis of �-KDE1, �-KDE2, and E3 inPF and BF T. brucei. RNA from 1 � 107 cells was probed for �-KDE1, �-KDE2, and E3 transcripts. Ethidium bromide (EtBr) stain for rRNAs is shown for eachlane. (B) Activity of �-KD in BF and PF T. brucei. Purified mitochondria were solubilized in nonionic detergents, and 40 �g of PF or BF mitochondrial proteinwas added to the assay medium for the conversion of NAD� to NADH. For the mixing experiment, 20 �g of PF and 20 �g of BF homogenates were combinedand added to the same reaction mixture. Each assay was measured at 340 nm at 27°C.

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and reattachment of minicircles, the kDNA network grows later-ally until it is twice the size of the kDNA network in nonreplicatingcells. The newly replicated kDNA network segregates, and alongwith a newly formed flagellum, is distributed to daughter cells atcytokinesis. Late in the kDNA replication cycle, �-KDE2-PTP dis-tributes with each lobe of the kDNA network in close proximity tothe old and newly formed flagella (Fig. 3B). In this image, a singleunfixed cell treated with detergent has two flagella, one nucleus,

and a single V-shaped kDNA network that has completed kDNAreplication and is just beginning network scission (11). Interest-ingly, �-KDE2-PTP foci are associated with this bilobed kDNAgenome, with a single point located on each lobe.

When cells were treated with detergent and CaCl2, there was ageneral depolymerization of the subpellicular microtubules, leav-ing the kDNA associated with the flagellum via the TAC (Fig. 3C)(11). �-KDE2-PTP was localized to either a single point on the

FIG 2 Localization and subcellular fractionation of �-KDE2-PTP. A partial �-KDE2 sequence was ligated into the C-terminal PTP expression vector (pC-PTP-NEO) and transfected into BF and PF T. brucei. (A) Northern analysis of �-KDE2-PTP transcript expression in PF and BF developmental stages. RNA from 5 �106 cells was evaluated with radiolabeled probes against the �-KDE2 sequence. EtBr stain for rRNAs is shown for each lane. (B) Cellular localization of�-KDE2-PTP by immunofluorescence microscopy. �-KDE2-PTP PF and BF cells were stained for DNA with DAPI, mitochondria with MitoTracker, and�-KDE2-PTP with antibodies against the protein C epitope. (C) Subcellular fractionation of procyclic-form �-KDE2-PTP cells. Lysates from total PF-667 (WT)cells and fractionated PF �-KDE2-PTP cells (TC) were resolved by SDS-PAGE and analyzed by Western blotting using antibodies against HSP-70 (cytosolicmarker), MTP-70 (mitochondrial matrix marker), ISP (mitochondrial membrane marker), and protein A epitope (�-KDE2-PTP). (D) Subcellular fractionationof BF �-KDE2-PTP cells. Lysates from total BF-667 (WT) cells and BF �-KDE2-PTP cells (TC) were fractionated and analyzed as for panel C with TAO as themitochondrial membrane marker. The positions of the nucleus (n), kDNA (k), heat shock protein 70 (HSP-70), mitochondrial heat shock protein 70 (MTP-70),ISP, and TAO are indicated. Abbreviation are used for cells and organelle fractions in panels C and D: total cell protein for nontransfected (WT) and�-KDE2-PTP transfected (TC) T. brucei 667, cytosolic protein (CY), total mitochondrial protein (TM), mitochondrial matrix protein (MA), and mitochondrialmembrane protein (ME).

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kDNA (Fig. 3C, top) or at two points (Fig. 3C, bottom) in asyn-chronous populations. The orientation of the 3D-R images fur-ther supports the close association of �-KDE2-PTP with thekDNA and also better resolves the two discrete �-KDE2-PTPpoints (Fig. 3C, bottom). The heterogeneity in �-KDE2-PTP fociwas likely due to cell cycle differences, with a singular localizationassociated with kDNA networks that had recently completed rep-lication and segregation (Fig. 3B and C). These results show that�-KDE2-PTP is closely associated with the FKC and cofraction-ates with the TAC.

�-KDE2-PTP maintains an antipodal distribution through-out kDNA replication. Many proteins that maintain the kDNA inT. brucei are organized around the network in specialized regions(21). Antipodal sites, which are structural projections that flankthis genome, house many of the replication proteins and are the

regions where newly replicated minicircles reattach to the net-work. The thymidine analogue BrdU is a synthetic base that isincorporated into the free replicating kDNA minicircles during Sphase. Initial reattachment of these minicircles to the networkleads to labeling of the antipodal sites (38). To determine whether�-KDE2-PTP localized to the antipodal sites, cells were incubatedwith BrdU for 30 min, fixed, treated with HCl, and stained withDAPI and antibodies for BrdU. Trypanosomes in early S phaseshowed two sites of BrdU staining flanking the kDNA disk andrevealed an antipodal distribution of minicircles (Fig. 4A). Addi-tionally, FKCs prepared from cells treated with BrdU for 30 minrevealed colocalization of these minicircles with �-KDE2-PTP atthe antipodal sites (Fig. 4B). When BrdU labeling was extended to480 min, replicated BrdU-labeled DNA was distributed through-out the kDNA network, but the position of �-KDE2-PTP re-

FIG 3 �-KDE2-PTP associates with the FKC. (A) (Top) Mildly fixed BF �-KDE2-PTP cells were incubated with 0.25% Triton X-100 to create cell ghosts andstained with DAPI and antibodies against tyr-�-tubulin and the protein C epitope of �-KDE2-PTP. (Bottom) Magnified 3D-R images near the kDNA. (B)Cytoskeletons were created by incubating bloodstream �-KDE2-PTP cells in 0.25% Triton X-100. These structures were stained with DAPI and antibodiesagainst the PFR and �-KDE2-PTP. (C) BF �-KDE2-PTP cells were solubilized in 0.5% Triton X-100, and microtubules were depolymerized with 1 mM Ca2� tocreate FKCs. FKCs were stained with DAPI and antibodies against the tyr-�-tubulin and �-KDE2-PTP. Magnified 3D-R images are shown for each panel. Thepositions of the nucleus (n), kDNA (k), flagellum (f), basal bodies (bb), and paraflagellar rod (PFR) are indicated.

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mained constant. In cells that had completed kDNA replicationand segregation, we observed a single �-KDE2-PTP staining fo-cus, as has been described for other proteins localized to theantipodal sites (49).

�-KDE2-PTP is stably linked to mitochondrial structural el-ements. The antipodal localization of �-KDE2-PTP led us to askwhether the protein stably interacts within the FKC by directbinding to the kDNA or to TAC elements surrounding the ge-nome. To determine if �-KDE2-PTP directly associates with thekDNA, purified FKC preparations were treated with DNase, andTM, FKC, and DNase-treated FKC (FKC�D) fractions were re-solved by SDS-PAGE and analyzed by Western blotting (Fig. 4C).The blots were probed with antibodies against TAO, tyr-�-tubu-lin, and �-KDE2-PTP. The membrane protein TAO was present

in TM but was lost during FKC purification. Both �-KDE2-PTPand the basal body marker tyr-�-tubulin remained associatedwith the FCK following DNase treatment (Fig. 4C). Treatmentwith DNase caused mild dilution of the FKC samples, since asimilar reduction in signal intensity was observed in the tyr-�-tubulin and �-KDE2-PTP FKC�D fractions. The association of�-KDE2-PTP with the FKC was further confirmed by immuno-fluorescence microscopy of the FKC�D preparations. No kDNAwas detectable by DAPI staining, yet both tyr-�-tubulin and�-KDE2-PTP were retained in the DNase-treated FKC (Fig. 4D).Together, these results show that �-KDE2-PTP, though localizedto the antipodal sites, is directly associated with the FKC and in-dicates that the antipodal replication/reattachment sites interactwith this complex.

FIG 4 Antipodal localization and stable association of �-KDE2-PTP with the FKC. (A) BF �-KDE2-PTP T. brucei cells were incubated with BrdU for 30 min,fixed, and acid treated. The cells were stained with DAPI and probed with a monoclonal antibody against BrdU to show the distribution of recently replicatedminicircle kDNA. A magnified image of newly replicated BrdU-labeled minicircles reattached to the kDNA network is shown in the merged image. (B) FKCs wereisolated from cells treated with BrdU for 30 min and 480 min, heat treated, and stained with antibodies against BrdU and �-KDE2-PTP. (C) Proteins from TM,FKCs, and FKC�D from bloodstream form �-KDE2-PTP cells were resolved by SDS-PAGE and analyzed by Western blotting. The proteins were probed withantibodies against TAO, tyr-�-tubulin, and the protein A domain of �-KDE2-PTP. (D) Immunofluorescence microscopy of DNase-treated FKCs stained withDAPI and antibodies against tyr-�-tubulin and the protein C domain of �-KDE2-PTP. The positions of the nucleus (n), kDNA (k), flagellum (f), and basal bodies(bb) are indicated.

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RNAi knockdown of �-KDE2 decreases the growth rate andinfluences kDNA distribution. The localization of �-KDE2-PTPto the FKC in BF T. brucei suggested the protein had an alternativefunction in kDNA maintenance. To examine this secondary func-tion for �-KDE2, we developed an RNAi cell line by cloning apartial �-KDE2 nucleotide sequence into the inducible pZJM vec-tor (35). Northern analysis of this induced cell line revealed com-plete loss of detectable �-KDE2 mRNA 24 h after induction(Fig. 5A). Loss of �-KDE2 mRNA levels was accompanied by a

26-fold reduction in cell numbers by day 4 (Fig. 5B). The effect ofRNAi knockdown of �-KDE2 on the morphology of the kDNAnetwork was evaluated by DAPI staining (Fig. 5C and D). At thetime of induction, over 85% of the cells contained one kinetoplastand one nucleus (1k1n), with a small fraction of cells containing0k1n, 2k1n, and 2k2n (Fig. 5C). By day 3, the percentage of dys-kinetoplastic cells (0k1n) lacking detectable kDNA staining hadincreased to 27% of the total cells (Fig. 5D, E, and F). A similarincrease in the number of cells with two kinetoplasts (2k1n and

FIG 5 Effect of �-KDE2 RNAi knockdown in BF T. brucei. BF T. brucei cells were transfected with the inducible RNAi vector containing 425 bp of �-KDE2sequence. (A) Northern analysis showing the reduction of �-KDE2 mRNA levels by 24 h. The blot was evaluated with radiolabeled probes against �-KDE2 and�-tubulin sequences. dsRNA, double-stranded RNA. (B) Effects of �-KDE2 knockdown on cell growth. Cells were grown in the presence (�) or absence (�) of1 �g/ml doxycycline and monitored for changes in proliferation. (C and D) The numbers of kinetoplasts and nuclei were examined on day 0 and 3 dayspostinduction by DAPI staining. Dyskinetoplastic cells are indicated (d). (E) Examples of DAPI-stained (1k1n), dyskinetoplastic (0k1n), 2k1n, and 2k2n cells. (F)Quantitation of induced and uninduced cell types on day 3 of RNAi. A total of 400 DAPI-stained cells were analyzed.

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2k2n) was observed. The increased fraction of dyskinetoplastictrypanosomes and cells containing two kinetoplasts suggestedthat �-KDE2 may be required for kDNA inheritance by daughtercells following segregation of the kDNA.

kDNA abundance is not affected by �-KDE2 mRNA deple-tion. Many of the enzymes localized to the kDNA antipodal sitesare involved in kDNA replication. RNAi knockdown of these rep-lication proteins leads to kDNA loss (27, 50). In order to evaluatethe effect of �-KDE2 RNAi knockdown on kDNA replication, theabundance of free replicating minicircles was determined in totalgenomic DNA isolated from induced RNAi cells over a 3-day pe-riod. Free minicircles, separated by agarose gel electrophoresis toresolve the covalently closed and nicked/gapped circles, were an-alyzed by Southern blotting (Fig. 6A). Hybridization with a probespecific for a conserved sequence found on all minicircles revealedno change in the abundance of both closed and nicked/gappedconformations. Additionally, the abundance of kDNA network-associated minicircles and maxicircles were also assayed, and nodifferences were observed (Fig. 6B). These results suggest that theoverall abundance of kDNA is unaltered by RNAi knockdown of�-KDE2, consistent with the coordinate increase of both dyskin-etoplastic trypanosomes and cells containing two kinetoplasts.Together, these studies showed that �-KDE2 was necessary forinheritance of the kDNA by daughter cells at cytokinesis and notfor replication or segregation of the kDNA.

Dyskinetoplastic T. brucei cells have truncated mitochon-dria with decreased membrane potential. In the BF of T. brucei,the mitochondrial ATP synthase functions as an ATPase, driv-ing the movement of protons across the mitochondrial mem-brane and establishing a membrane potential (17, 51). A singlemaxicircle gene encoding the mRNA for subunit A6, numerousminicircle gRNAs for the editing of A6 pre-mRNA, and nuclear-encoded editing enzymes are all necessary for the successfulproduction of the functional ATPase (52). An expected conse-quence of the generation of dyskinetoplastic trypanosomes by�-KDE2 RNAi is the loss of A6 expression and the inability of

these cells to maintain a mitochondrial membrane potential(Fig. 7). �-KDE2 RNAi cells were incubated with MitoTrackerRed on day 3 of induction, washed, fixed with methanol, andprepared for immunofluorescence analysis with an antibodyagainst the mitochondrial membrane protein TAO and DAPI.All cells containing a DAPI-stainable kinetoplast had overlap-ping signals for both MitoTracker and TAO. Cells lacking astainable kDNA network also lacked MitoTracker staining, in-dicating a loss of membrane potential (Fig. 7A). These dyskin-etoplastic cells also showed a dramatic reduction in mitochon-drial volume based on TAO staining. A 14-fold reduction inlength was observed in extreme cases. Examples of uninduced(�RNAi) and induced (�RNAi) cells with mitochondrionlength in micrometers are shown in Fig. 7B. Quantitation re-vealed that 100% of the kinetoplastids on day 3 contained mi-tochondria that were more than 5 �m in length (Fig. 7C). Alsoon day 3, dyskinetoplastic trypanosomes contained mitochon-dria that were between 2 and 8 �m (77%), and only 23% were1 �m or less (Fig. 7C). Together, the RNAi knockdown studiesshowed that �-KDE2 was necessary for inheritance of thekDNA by daughter cells and that the production of dyskineto-plastic trypanosomes resulted in the rapid loss of mitochon-drial membrane potential and reduced mitochondrial volume.

DISCUSSION

Whether structural or enzymatic, most proteins are encoded by asingle gene and have a single function. However, there are a num-ber of proteins that are multifunctional, with seemingly unrelatedactivities. In mammals, alternative splicing provides an importantmechanism for generation of protein diversity, with literally thou-sands of splice variants originating from a single gene (53). Try-panosomes lack conventional cis-splicing, but alternative trans-splicing at the 5= ends of nuclear-encoded mRNAs and alternativeediting of mitochondrial mRNA have recently been recognized asmechanisms for increasing the variety of proteins (32, 33, 40, 45).Expanded functional diversity can also be accomplished without

FIG 6 Effects of �-KDE2 RNAi on minicircle and maxicircle abundance. (A) Total genomic DNA was fractionated by agarose gel electrophoresis and analyzedby Southern blotting over a 3-day period after induction of �-KDE2 RNAi for changes in free replicating minicircle levels. (B) Southern blot analysis of XbaI- andHindIII-digested total genomic DNA for variations in the amounts of kDNA-associated minicircles and maxicircles. Both blots were hybridized with radiolabeledprobes for minicircle and maxicircle sequences. Hybridization with a �-tubulin probe was used as a loading control.

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differences in protein sequence or posttranslational modification,but rather, by simply having multiple activities associated with thesame protein. These “moonlighting proteins” also provide addi-tional complexity to the limited number of proteins encoded bythe eukaryotic genome. Seven of the eight Krebs cycle enzymeshave alternative functions in eukaryotes. Three of these proteinshave been identified as mtDNA maintenance factors in differentorganisms, suggesting a strong evolutionary trend for metabolicenzymes to be involved in genome preservation (4, 54, 55). Here,we report the moonlighting activity of �-KDE2 and define itsfunction in the inheritance of the mitochondrial genome of Afri-can trypanosomes.

The BF of T. brucei lacks �-KD activity yet expresses all threesubunits of �-KD. The enzymatic activities of �-KD were ex-

pected to fractionate with the mitochondrial matrix; however,�-KDE2 is also associated with mitochondrial membranes (Fig.2). In yeast, �-KDE2 also associates with the mtDNA nucleoid atthe mitochondrial membrane and is necessary for maintenance ofthe genome (4, 5). Similarly, we found that �-KDE2 in T. bruceiassociates with the FKC apparatus. The association of �-KDE2with the FKC is not mediated by direct binding to kDNA, butrather, the protein is physically linked to the FKC in a DNase-resistant manner. Despite apparent similarities in the distributionof �-KDE2 in yeast and trypanosomes, the structure of the kDNAnetwork and its mechanism of replication impose unique require-ments. Immunofluorescence microscopy localized �-KDE2 todiscrete positions on the kDNA within the FKC. The mechanismof kDNA replication requires a spatial order of the genome, rep-

FIG 7 Mitochondrial function and morphology are altered in dyskinetoplastic cells. (A) Fluorescence microscopy analysis of �-KDE2 RNAi cells. Day 3-inducedcells were stained for DNA with DAPI and mitochondria with MitoTracker Red and probed with antibodies against TAO. Dyskinetoplastic cells are indicated (d).(B) Mitochondrial morphology of �-KDE2 RNAi cells. Shown are observed changes in mitochondrion lengths of dyskinetoplastic cells (�m) by TAO staining.(C) Quantification of observed mitochondrion lengths for trypanosomes with and without kinetoplasts on day 3. A total of 400 stained cells were analyzed. DIC,differential interference contrast microscopy.

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lication enzymes, and segregation apparatus. The KFZ and theantipodal sites represent defined functional zones surroundingthe kDNA and are involved in replication, segregation, and main-tenance of the trypanosome mitochondrial genome. Pulse-label-ing cells with BrdU allowed the identification of the antipodal sitesand showed that �-KDE2 localizes there. Initially, localization of�-KDE2 to the site of minicircle reattachment following replica-tion led us to believe that �-KDE2 might be directly involved inkDNA replication. Moreover, RNAi knockdown of �-KDE2mRNA resulted in a dramatic increase in cells lacking kDNA stain-ing with DAPI. However, we also observed a corresponding in-crease in cells with two kDNA networks, and Southern blot hy-bridization confirmed that the abundance of kDNA was unalteredduring RNAi knockdown of �-KDE2 expression despite an in-creased number of dyskinetoplastic cells. Together, these resultsindicate that �-KDE2 is not directly involved in kDNA replicationor segregation, but rather, is necessary for the distribution of thereplicated kDNA networks to daughter cells at cytokinesis. Wepropose that unequal distribution of the replicated kDNA net-works gave rise to a dyskinetoplastic daughter cell and a daughtercell with two kinetoplasts.

The antipodal distribution of �-KDE2 is static throughout thecell cycle, suggesting a structural versus a catalytic function for�-KDE2 at the FKC of T. brucei. In asynchronous populations,�-KDE2 is associated with one or both antipodal sites on thekDNA. We have found �-KDE2 on a bilobed kDNA (preparti-tioned), suggesting that the �-KDE2 stably associated as a singlesite on each lobe of the replicating kDNA network. Complete seg-regation of this “V-shaped” bilobed network produces daughterkDNA networks with a single podal site containing �-KDE2. Thisis similar to the results seen in the analysis of the distribution ofmitochondrial topoisomerase II in the related organism Crithidiafasciculata (49). Furthermore, while BrdU analysis of kDNA rep-lication revealed an initial colocalization of newly replicatedminicircles and �-KDE2 at the antipodal sites, continuous uptakeof BrdU led to a uniform distribution of the nucleotide analoguethroughout the kDNA network, whereas �-KDE2 remained staticat the antipodal sites (Fig. 4B).

A possible role for �-KDE2 within the FKC might involvekDNA binding and suggests that �-KDE2 could serve as abridge to the proteinaceous filaments linking the kDNA net-work to the mitochondrial membrane. We found that �-KDE2association with FKC resisted treatment with DNase, but addi-tional studies are needed to determine whether �-KDE2 hasDNA binding properties. Within the FKC, the unilateral fila-ments of the TAC attach to the mitochondrial membranes andextend through the KFZ to a single face of the kDNA disk (11).Only two unilateral filament proteins (p166 and AEP-1) havebeen reported (30, 33), and possible interactions with �-KDE2have not been determined. The resistance of �-KDE2 to deter-gent solubilization and DNase treatment suggests an associa-tion with unidentified proteins that make up the structuralskeleton in the mitochondria.

Two previously studied TAC proteins, p166 and AEP-1, local-ize to the kDNA/basal body region of T. brucei and purify with theFKC (30, 33). RNAi knockdown of p166 and expression of a dom-inant-negative mutant of AEP-1 showed that both are essential forkDNA maintenance and segregation. �-KDE2 RNAi had no effecton kDNA morphology or division but resulted in an increase ofdyskinetoplastic cells and a corresponding increase in cells with

two kinetoplasts. This suggests that in the absence of �-KDE2,newly replicated kDNA networks fail to partition into the newlyformed mitochondrion, leading to unequal kDNA distribution tothe daughter cells.

If �-KDE2 is necessary for kDNA inheritance, it is some-what surprising that the maximum percentage of dyskineto-plastic cells never exceeds approximately 35%. It is well estab-lished that BF trypanosomes lack a functional Krebs cycle andoxidative phosphorylation and that ATP is exclusively pro-duced during glycolysis. However, mitochondrial translation isrequired in BF T. brucei, suggesting an essential function isprovided by a mitochondrial gene product (15). It seems likelythat the depletion of �-KDE2 by RNAi is lethal to BF trypano-somes due to the moonlighting function of the protein inkDNA inheritance. In BF T. brucei, the formation of a protongradient across the mitochondrial membrane, necessary forimport of nuclear-encoded mitochondrial proteins, is depen-dent on the expression of a functional F1Fo ATPase. One essen-tial component of the F1Fo ATPase is the maxicircle-encodedA6 subunit. Since dyskinetoplastic trypanosomes lack the A6gene, they cannot maintain a proton gradient and lose the abil-ity to import proteins from the cytoplasm. This occurs rapidly,resulting in loss of membrane potential and reduced mito-chondrial volume within 3 days of RNAi induction.

The moonlighting function of �-KDE2 in kDNA distributionto daughter cells further underscores the importance of multi-functional proteins in the maintenance of the mitochondrial ge-nome in eukaryotes. Unexpectedly, we have also demonstratedthe essential role of mitochondrion-encoded proteins in the du-plication and inheritance of the intact mitochondrion in BF try-panosomes. The complexity of the FKC and kDNA replicationcycle has driven the diversification of mitochondrial proteins intrypanosomes, and as additional FKC-associated proteins areidentified, it is likely that new moonlighting and alternatively pro-cessed proteins will be discovered.

ACKNOWLEDGMENTS

We thank D. M. Engman (MTP-70 antibody), M. Chaudhuri (TAOantibody), K. Gull (PFR antibody), A. Gunzl (c-PTP-NEO plasmid),and P. Englund (pZJM plasmid) for the reagents used in this study. Wealso thank members of the Hajduk laboratory for discussions and crit-ical reading of the manuscript. We are particularly grateful to TorstenOchsenreiter for important insights into mechanisms of protein diver-sification.

This work was supported by NIH grant AI21401 to S.L.H.

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