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EUKARYOTIC CELL, Sept. 2009, p. 1418–1428 Vol. 8, No. 91535-9778/09/$08.00�0 doi:10.1128/EC.00132-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Downregulation of Mitochondrial Porin Inhibits Cell Growth andAlters Respiratory Phenotype in Trypanosoma brucei�†

Ujjal K. Singha, Shvetank Sharma, and Minu Chaudhuri*Department of Microbial Pathogenesis and Immune Response, Meharry Medical College, Nashville, Tennessee 37208

Received 6 May 2009/Accepted 10 July 2009

Porin is the most abundant outer membrane (OM) protein of mitochondria. It forms the aqueous channelon the mitochondrial OM and mediates major metabolite flux between mitochondria and cytosol. Mitochon-drial porin in Trypanosoma brucei, a unicellular parasitic protozoan and the causative agent of Africantrypanosomiasis, possesses a �-barrel structure similar to the bacterial OM porin OmpA. T. brucei porin(TbPorin) is present as a monomer as well as an oligomer on the mitochondrial OM, and its expression isdevelopmentally regulated. In spite of its distinct structure, the TbPorin function is similar to those of othereukaryotic porins. TbPorin RNA interference (RNAi) reduced cell growth in both procyclic and bloodstreamforms. The depletion of TbPorin decreased ATP production by inhibiting metabolite flux through the OM.Additionally, the level of trypanosome alternative oxidase (TAO) decreased, whereas the levels of cytochrome-dependent respiratory complexes III and IV increased in TbPorin-depleted mitochondria. Furthermore, thedepletion of TbPorin reduced cellular respiration via TAO, which is not coupled with oxidative phosphor-ylation, but increased the capacity for cyanide-sensitive respiration. Together, these data reveal that TbPorinknockdown reduced the mitochondrial ATP level, which in turn increased the capacity of the cytochrome-dependent respiratory pathway (CP), in an attempt to compensate for the mitochondrial energy crisis.However, a simultaneous decrease in the substrate-level phosphorylation due to TbPorin RNAi caused growthinhibition in the procyclic form. We also found that the expressions of TAO and CP proteins are coordinatelyregulated in T. brucei according to mitochondrial energy demand.

Trypanosoma brucei belongs to a group of parasitic protozoathat possess a single tubular mitochondrion with a concate-nated structure of mitochondrial DNA known as kinetoplast(30). T. brucei is the infectious agent of the disease Africantrypanosomiasis, which is spread from one mammal to anotherby the bite of the tsetse fly (53). During transmission from theinsect vector to the mammalian host and vice versa, the para-site undergoes various developmental stages accompanied bydramatic changes in mitochondrial activities (15). The blood-stream form that grows in mammalian blood uses glucose as itsenergy source and suppresses many mitochondrial activities.The bloodstream-form mitochondria lack cytochromes; thus,respiration in this form is solely dependent on the cytochrome-independent trypanosome alternative oxidase (TAO) (15). Incontrast, the procyclic form that lives in the insect’s midgutpossesses a developed mitochondrion with a full complementof the cytochrome-dependent respiratory system and a re-duced level of TAO. The procyclic-form mitochondria produceATP by both oxidative and substrate-level phosphorylations(7). On the other hand, the bloodstream-form mitochondria donot produce ATP but hydrolyze ATP to maintain the innermembrane (IM) potential (10, 33, 39, 48). Many of the mito-chondrial IM- and matrix-localized proteins in T. brucei arewell characterized (11, 29, 34, 43, 45). However, the mitochon-

drial outer membrane (OM) proteins in this group of parasiticprotozoa have been poorly explored.

Mitochondrial porin, which is also known as the voltage-dependent anion-selective channel (VDAC), is the most abun-dant protein in the OM (17, 28). The sizes and the secondarystructures of this protein are very similar among differentorganisms. The VDAC possesses a N-terminal �-helical domain,and the rest of the protein consists of a number of amphiphilic�-strands, which form a barrel-like structure that integratesinto the lipid bilayer (16, 17, 28). Recently, the three-dimen-sional structure of the human VDAC has been elucidated bynuclear magnetic resonance spectroscopy and X-ray crystallog-raphy, which showed a �-barrel architecture composed of 19�-strands and the N-terminal �-helix located horizontally mid-way in the pore (5). Saccharomyces cerevisiae and Neurosporacrassa VDACs also possess 16 to 19 �-strands, similar to themammalian VDAC (17).

The VDAC exists as different isomeric forms in different spe-cies (16, 19). In yeasts, there are two forms: VDAC1 and VDAC2.Only VDAC1 has the channel activity and is abundantly ex-pressed (22, 23). Animals have three isoforms: VDAC1 toVDAC3. These isoforms showed more than 80% sequencehomology among themselves. However, their expression levelsand tissue specificities are different (16). Plants also have mul-tiple isoforms of the VDAC with various expression levelsunder different pathological conditions (19). The VDAC playsa crucial role in regulated transport of ADP, ATP, Ca2�, andother metabolites in and out of mitochondria (17, 28, 41). TwoATP-binding sites found at the N- and C-terminal regions inthe VDAC are critical for its function (54). Downregulation ofVDAC expression disrupts mitochondrial energy production(22, 25). In contrast, overexpression of the VDAC in metazoa

* Corresponding author. Mailing address: Department of MicrobialPathogenesis and Immune Response, Meharry Medical College, Nash-ville, TN 37208. Phone: (615) 327-5726. Fax: (615) 327-6072. E-mail:[email protected].

† Supplemental material for this article may be found at http://ec.asm.org/.

� Published ahead of print on 17 July 2009.

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induces apoptosis, which can be blocked by compounds thatinhibit its channel activity (1, 47).

The OM of gram-negative bacteria also consists of varioustypes of porins (24, 32, 40). Based on their structures andfunctions, they are divided into five groups. OmpA belongs tothe small �-barrel integral membrane protein family, which iscomposed of eight �-strands. It is highly abundant and ubiq-uitous among most gram-negative bacteria (21). Other typesof porins include general porin OmpF, which consists of 16�-strands; substrate-specific porins, such as LamB or malto-porin, which contains 18 �-strands; receptor-type porin FhuA,the largest �-barrel, with 22 �-strands; and phospholipase Aor OMPLA, an integral membrane enzyme containing 12�-strands (21, 24, 32, 40). The OmpA plays important roles inbacterial conjugation, adhesion, invasion, and immune evasionand also acts as the receptor for several bacteriophagesthrough its surface-exposed loops (44).

Here, we show that the T. brucei mitochondrial porin(TbPorin) possesses a predicted �-barrel structure that has fewer�-strands than other mitochondrial porins but is similar tobacterial OmpA. TbPorin is crucial for mitochondrial energyproduction via both oxidative and substrate-level phosphory-lations. The depletion of TbPorin reduced cell growth of theprocyclic form as well as the bloodstream form. Furthermore,it reveals that depletion of mitochondrial ATP level by down-regulation of porin alters the electron flow via TAO and thecytochrome-dependent pathway (CP) as well as the levels ofproteins in these pathways.

MATERIALS AND METHODS

Cells. The procyclic form of the Trypanosoma brucei 427 cell line (29-13)resistant to hygromycin and neomycin (G418) and expressing the tetracyclinerepressor gene (Tetr) and T7 RNA polymerase were grown in SDM-79 medium(JRH Biosciences) containing 10% heat-inactivated fetal bovine serum and ap-propriate antibiotics (hygromycin, 50 �g/ml; G418, 15 �g/ml) (8). Bloodstream-form cells were maintained in HMI-9 medium supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals) and 10% Serum Plus (JRHBiosciences) (43). For the measurement of cell growth, the procyclic-form cellswere inoculated at a cell density of 2 � 106/ml to 3 � 106/ml, and the blood-stream-form cells were inoculated at a cell density of 1 � 105/ml in mediumcontaining appropriate antibiotics in the presence or absence of doxycycline.Cells were harvested at different time points of growth (0 to 9 days), and thenumber of cells were counted in a Neubauer hemocytometer. The log of thecumulative cell number was plotted against the time of incubation in culture.

Comparison of the predicted primary, secondary, and tertiary structures.Sequence comparison was performed using the ClustalW alignment program(20) in MacVector 10.0. The prediction of the secondary structure of porins wasperformed using PRED TM�� (3) and TMBPro (36) prediction tools availableonline. Structure alignment was performed using the iMol software program (4).

Generation of an inducible TbPorin RNAi-disrupted cell line and RNA anal-ysis. To prepare the construct for TbPorin double-stranded RNA expression, the534 bp fragment of the coding region of the TbPorin gene was PCR amplifiedfrom T. brucei genomic DNA by using high-fidelity Pfu polymerase (Stratagene).Sense and antisense primers containing the proper restriction sites at 5� endswere TbPorin For (5�-GCGGATCCCCACAAGGATGCGAAAGACCTAC-3�)and TbPorin Rev (5�-AGAAGCTTTTTGGCACACGAGCAGTGATAC-3�).The amplified product was cloned into the BamHI/HindIII sites of a tetracycline-inducible dual-promoter plasmid vector, p2T7Ti-177 (50). The construct forTbPorin RNA interference (RNAi) was verified by sequencing. The purifiedplasmid DNA was linearized by NotI. The linearized plasmid was used fortransfection into procyclic-form cells (T. brucei 427 29-13) expressing T7 poly-merase and tetracycline repressor proteins according to standard protocols (8).After transfection, the plasmid was integrated into 177-base-pair repeat regionsof the minichromosomes in T. brucei.

RNA was isolated from the procyclic trypanosomes grown for 4 days with orwithout doxycycline by using Trizol reagent (Invitrogen) according to the man-

ufacturer’s protocol and was concentrated by 2 M LiCl precipitation. For North-ern analysis, RNA was fractionated in formaldehyde-agarose gels (1%) andtransferred to nitrocellulose membranes (37). TbPorin and actin gene probeswere generated using a random-primer-labeling protocol (Invitrogen) from theTbPorin cDNA clone and the PCR-amplified genomic fragment of the T. bruceiactin gene. Hybridization was carried out in Rapid-Hyb buffer (Amersham) for16 h. The membranes were washed at 55°C with 0.1� SSC (150 mM NaCl, 15mM Na-citrate, pH 7.4) containing 0.1% sodium dodecyl sulfate (SDS) andexposed to X-ray film (37). The intensities of TbPorin mRNA bands werequantitated by an imaging densitometer (model GS-700; Bio-Rad) and normal-ized with the intensity of the corresponding actin mRNA.

Isolation of mitochondria. Mitochondria were isolated from the parasite afterlysis via nitrogen cavitation in isotonic buffer as described previously (13, 43).The isolated mitochondria were stored at a protein concentration of 10 mg/ml inSME buffer (250 mM sucrose, 20 mM MOPS-KOH, 2 mM EDTA) containing50% glycerol at �70°C. Before they were used, mitochondria were washed twicewith 9 volumes of SME buffer to remove glycerol.

Treatment of mitochondria. For alkali extraction, isolated mitochondria (100�g) were treated with 100 �l of 100 mM Na2CO3 at pH 11.0 for 30 min on ice(13, 43). The supernatant and pellet fractions were collected after centrifugationand analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immuno-blotting. Protease digestion of isolated mitochondria was performed by treatingmitochondria (50 �g) with various concentration of proteinase K (0 to 150�g/ml) in 100-�l reaction volumes in SME buffer for 30 min on ice. After thetreatment, proteinase K was inhibited by phenylmethylsulfonyl fluoride (2 mM).Mitochondria were reisolated, and the proteins were analyzed by immunoblotanalysis. Mitochondria were also treated with Triton X-100 (1%, vol/vol) incombination with proteinase K (150 �g/ml), and proteins were analyzed asdescribed above. For cross-linking, mitochondria (1 mg/ml) in SME buffer weretreated with different concentrations of EGS [ethylglycol bis(succinimidyl succi-nate); 0 to 0.4 mM] for 30 min at room temperature. After incubation, 100 mMglycine was added to a final concentration of 15 mM to quench the excesscross-linker. The mixture was then incubated for additional 15 min and preparedfor SDS-PAGE analysis. Mitoplasts were isolated after treatment of mitochon-dria with 3 volumes of water for 15 min on ice followed by reconstitution ofosmolarity with the addition of one-third of a volume of 2.4 M sucrose. Mito-plasts were pelleted by centrifugation. The supernatant and pellet fractions wereanalyzed for the release of cytochrome c (Cyt c) as described previously (2, 38).

Generation of TbPorin-specific antibodies and immunoblot analysis. Poly-clonal antibodies against the TbPorin protein were generated by Bethyl Labo-ratories, Inc., Montgomery, TX, by using the synthetic peptide consisting of thelast 14-amino-acid sequence of the C terminus. Briefly, the synthetic peptide wasaffinity purified by high-performance liquid chromatography and verified by massspectrometry. The TbPorin peptide was conjugated to keyhole limpet hemocy-anin and used to generate specific antibodies in rabbits. Antiserum againstTbPorin was affinity purified by using TbPorin peptide as the ligand. Preimmuneserum collected from the same rabbits was used as a control. Total cellularproteins and proteins from isolated mitochondria were analyzed by SDS-PAGE(10 or 15%) as described previously (27) and transferred to nitrocellulose mem-brane at 4°C in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% [vol/vol]methanol, pH 8.3) at 100 V (46). Blots were treated with TbPorin polyclonalantiserum and antiserum against T. brucei Cyt c1 (34), cytochrome oxidasesubunit 4 (COIV) (31), a homologue of mitochondrial OM protein carnitinepalmitoyl transferase (CPT) (D. P. Nierlich, D. G. Kuznair, J. Ghazvini, and L.Simpson, unpublished data), T. brucei serine/threonine protein phosphatase 5(TbPP5), a cytosolic protein (12), and mitochondrial Hsp70 (18) (all at 1/1,000dilution in blocking buffer). TAO (11) was detected with the correspondingmonoclonal hybridoma supernatants (1/50 dilution in TBST [10 mM Tris-HCl,150 mM NaCl, pH 8.0]). Monoclonal antibody against T. brucei �-tubulin (52)was used in 1/20,000 dilution in TBST. A peptide antibody was also generated(Bethyl Laboratories, Inc, Montgomery, TX) against T. brucei ATP/ADP carrierprotein (AAC) (Tb10.61.1820; amino acid residues 294 to 307) identified in a T.brucei gene database (www.geneDB.org). The purified anti-AAC antibody wasused at a 1/20,000 dilution for immunoblot analysis of T. brucei mitochondrialproteins. Blots were treated with the appropriate secondary antibody and devel-oped using an enhanced chemiluminescence detection system (ECL system;Amersham). The intensity of the bands was quantitated using an imaging den-sitometer (model GS-700; Bio-Rad) and normalized with the correspondingbands for tubulin.

Measurement of ATP. ATP concentration was measured using an ATP biolu-minescence assay kit (Invitrogen) as described previously (38). Mitochondria andmitoplasts were suspended in an ATP assay buffer (20 mM Tris-HCl, pH 7.4, 15mM KH2PO4, 0.6 M sorbitol, 10 mM MgSO4, 2.5 mg/ml fatty acid-free bovine

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serum albumin) at a concentration of 1 mg/ml. Substrates succinate, �-ketoglu-tarate (�-KG), and pyruvate with succinate were added to mitochondria sepa-rately at a final concentration of 5 mM and incubated at 27°C. The reaction wasstarted by the addition of ADP (55 �M). Duplicate samples were treated withantimycin A (2.7 �M) when succinate was used as the substrate. At different timepoints, samples (100 �l) were treated with 2.5 �l of 60% perchloric acid andimmediately subjected to a vortex. Samples were incubated on ice for 30 min andcentrifuged at 13,000 rpm for 5 min. Supernatants were collected in fresh tubes,and 20 �l of 1 N KOH was added to each tube to neutralize the acid. Sampleswere centrifuged again, and the resultant supernatants were used for estimationof the ATP concentration by luciferase ATP assay reagents according to themanufacturer’s protocol. Briefly, 10 �l of the samples were mixed with 40 �l of0.5 M Tris-acetate, pH 7.75, and 50 �l of luciferase reagent. The fluorescenceintensity was measured with a luminometer by using a 1-s delay and an integra-tion time of 1 to 10 s. The ATP concentration for each sample was assayed intriplicate.

Measurement of oxygen consumption. Oxygen consumption by whole cells andisolated mitochondria was measured in a closed chamber of 0.5 ml by an oxygenelectrode connected to an amplifier (YSI model 5300) and recorded on graphpaper as a trace (49). Procyclic cells were harvested and resuspended in SDM-79medium containing 10% fetal bovine serum. For each assay, 3 � 107 cells wereused. Salicylhydroxamic acid (SHAM), the inhibitor for alternative oxidase, andKCN, the inhibitor of cytochrome oxidase (15, 49), were used at concentrationsof 2 mM and 1 mM, respectively. The percent total oxygen consumption inhib-ited by either of these inhibitors is proportional to the electron flow through therespective pathway. The oxygen consumption inhibited by SHAM, in the pres-ence of KCN was measured as the capacity of the CP, and similarly, the amountof oxygen consumption inhibited by KCN in the presence of SHAM was con-sidered the capacity of the alternative pathway (AP).

Blue native PAGE (BN-PAGE). Mitochondrial proteins from the parental andporin knockdown (KD) cells were solubilized in 72 �l of ice-cold buffer Ncontaining 20 mM Tris, pH 7.0, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, 1mM phenylmethylsulfonyl fluoride, 1 �g/ml leupeptin, and 1% digitonin. Thesolubilized supernatants were clarified by centrifugation at 100,000 � g for 30min at 4°C. The supernatants were supplemented with 7.5 �l of sample buffer(750 mM amino caproic acid, 5% Coomassie blue) and were electrophoresed ona linear 6-to-13% polyacrylamide gradient gel (13, 14, 51). Protein complexeswere detected by immunoblot analysis. Molecular size marker proteins apofer-ritin (400 kDa), �-amylase (200 kDa), alcohol dehydrogenase (150 kDa), and

bovine serum albumin (66 kDa) were run on the same gel and visualized byCoomassie blue staining.

RESULTS

TbPorin possesses a �-barrel structure similar to bacte-rial OmpA. We identified by BLAST analysis the gene formitochondrial porin with duplicate copies (Tb927.2.2510 andTb927.2.2520) in the T. brucei genome database (6) by usingNeurospora crassa mitochondrial porin as the query. The twocopies of TbPorin are identical in their coding sequencesand tandemly arranged on chromosome 2 in T. brucei. Thepredicted size of the encoded protein is 29.1 kDa, and theisoelectric pH is 9.6. A ClustalW alignment of this proteinwith several other mitochondrial porins from fungi and hu-mans showed that TbPorin has 13 to 16% overall identity and27 to 32% similarity (Fig. 1; see Table S1 in the supplementalmaterial). A BLAST search in the NCBI protein database byusing TbPorin as the query showed a strong homology of thisprotein with six hypothetical proteins in Trypanosoma cruzi(64% identity and 74.4% similarity) and the hypothetical pro-tein in each Leishmania species (e.g., L. major [39% identityand 57% similarity], L. infantum, and L. braziliensis). TheBLAST search with TbPorin also showed homology with theporin-like proteins from Toxoplasma and Euglena spp.

The VDAC has two conserved nucleotide binding sites, oneat the C-terminal end and the other near the N-terminal region(54). In both regions, the conserved lysine residue is critical forATP binding. TbPorin also has the conserved lysines (K24 andK262) and several other conserved residues at the correspond-ing N- and C-terminal regions (Fig. 1). Thus, TbPorin pos-

FIG. 1. Analysis of the primary and secondary structures of TbPorin. ClustalW alignment of the predicted protein sequence of TbPorin withthose from other species. The predicted �-strands are indicated by dotted lines. Two conserved nucleotide binding sites are marked by solid lineson the top of the sequences. The accession numbers of the sequences are as follows: for TbPorin, Tb927.2.2520; Saccharomyces cerevisiae porin(SC Porin), NP014343; Neurospora crassa porin (Nc Porin), CAD71033; and Homo sapiens porin (Hu Porin), P21796.

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sesses characteristic sequences similar to those of other mito-chondrial porins.

Analysis of the secondary structure using PRED-TM�� pre-diction tools indicated that TbPorin possesses eight transmem-brane �-strands that alternate with nonmembranous loops.Both the N and C termini of TbPorin are predicted to becomposed of �-helices (Fig. 1). In contrast, the Saccharomycescerevisiae and human VDAC1s possess 12 and 15 predicted�-strands, respectively. The predicted C-terminal �-helix of theTbPorin is significantly longer than that of yeast and humanVDAC1s. The three-dimensional structure analysis using theTMBPro prediction program showed that, similar to yeast andhuman VDAC1s, the TbPorin protein sequence can be foldedinto a �-barrel structure (see Fig. S1A in the supplementalmaterial). However, the number of �-strands in TbPorin islower than those in S. cerevisiae and human VDACs. Theprediction of an exceptionally long C-terminal tail in TbPorinwas also observed using the TMBPro prediction tool. To-gether, the data suggest that TbPorin forms a �-barrel struc-ture with a diameter smaller than that in the VDACs of othereukaryotes. TbPorin also has both the N- and C-terminal �-he-lices possibly exposed in the intermembrane space of mito-chondria. A similar structure has been found for OmpA, amember of the membrane-integral �-barrel protein family(21). OmpA consists of eight �-strands and a C-terminal glob-ular domain that is not present in other types of porins (21).Superimposing the predicted structures of TbPorin and OmpAby using the iMol software clearly showed a good match(see Fig. S1B in the supplemental material), suggesting thatTbPorin is structurally similar to bacterial OmpA.

TbPorin is a mitochondrial OM protein. In order to inves-tigate the subcellular location of TbPorin, cell fractionationfollowed by immunoblot analysis was performed. T. brucei pro-cyclic cells were lysed in isotonic buffer. The cytosolic andmitochondrial fractions were separated by differential centrif-ugation as described previously (13, 43). Analysis of equalamounts of proteins from total lysate and cytosolic and mito-chondrial fractions by immunoblotting using specific antibod-ies revealed that TbPorin is highly enriched in the mitochon-drial fraction, similar to other mitochondrial proteins, e.g.,Hsp70 and Cyt c1 (Fig. 2A). As expected, the cytosolic proteinTbPP5 is exclusively present in the cytosolic fraction. Theseresults showed that TbPorin is localized in mitochondria. Al-kali extraction of the isolated mitochondria followed by immu-noblot analysis demonstrated that TbPorin is present in thealkali-resistant membrane pellet, similar to TAO, but a signif-icant proportion of Hsp70, a matrix protein, is found in thesoluble fraction (Fig. 2B), suggesting that TbPorin is mem-brane integrated. A proteinase K treatment of isolated T. bru-cei mitochondria followed by immunoblot analysis of mito-chondrial proteins showed that the TbPorin level decreaseswith an increasing concentration of proteinase K (Fig. 2C).The level of TbPorin is reduced 50% and 70 to 80% at pro-teinase K concentrations of 50 and 150 �g/ml, respectively.The CPT, a putative T. brucei mitochondrial OM protein, isdecreased significantly even at 25 and 50 �g/ml of proteinaseK. By contrast, the levels of mitochondrial IM proteins Cyt c1

and Tim17 and the matrix-localized protein Hsp70 are essen-tially protected during proteinase K digestion. Most of theseproteins are completely digested by proteinase K when mito-

chondrial membrane is solubilized with Triton X-100, showingthat these proteins are not inherently resistant to proteinase K.These results indicate that TbPorin is localized in the mito-chondrial OM.

TbPorin expression level is developmentally regulated. Mi-tochondrial proteins from the procyclic and bloodstream formswere analyzed by Western blotting using antibodies forTbPorin and a few other mitochondrial proteins in T. brucei, e.g.,AAC, Tim17, TAO, Cyt c1, and Hsp70. The steady-state ex-pression level of the TbPorin protein is six- to sevenfold higherin the procyclic form than in the bloodstream form of T. brucei(Fig. 3A). The levels of AAC and Tim17 in the procyclic-formmitochondria are twofold and fourfold higher than these levelsin bloodstream-form mitochondria, as reported previously(43). As expected, TAO is more abundant and Cyt c1 is absentin the bloodstream-form mitochondria (15). The levels ofHsp70 are similar between the procyclic- and bloodstream-form mitochondria. The transcript levels of TbPorin in the twodevelopmental stages reflect a pattern similar to that of the

FIG. 2. Subcellular localization of TbPorin. (A) Immunoblot anal-ysis of subcellular fractions by using TbPorin-, mitochondrial Hsp70(mHsp70)-, Cyt c1-, and TbPP5-specific antibodies as probes. Thedifferent fractions were total lysate (T), cytosol (C), and mitochondria(M). Ten-microgram proteins from each fraction were loaded intoeach lane. (B) Sodium carbonate extraction followed by immuno-blot analysis of mitochondrial proteins from the T. brucei procyclicform. After Na2CO3 treatment, mitochondria were reisolated bycentrifugation. The supernatant (S) and pellet (P) fractions from10-�g mitochondrial proteins were analyzed using TbPorin-, TAO-,and mitochondrial Hsp70 (mHsp70)-specific antibodies as probes.(C) Proteinase K digestion of mitochondria (10 �g) followed byimmunoblot analysis using TbPorin-, CPT-, Cyt c1-, Tim17-, andmitochondrial Hsp70 (mHsp70)-specific antibodies as probes. Con-centrations of proteinase K are indicated at the top. Mitochondriasamples were treated with Triton X-100 (1%) along with proteinaseK (150 �g/ml) as indicated. After digestion, mitochondria werereisolated by centrifugation and electrophoresed as described in thetext.



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protein levels. The procyclic form possesses five- to sixfoldmore TbPorin mRNA than the bloodstream form (Fig. 3B).

TbPorin is present as monomeric and oligomeric forms inmitochondria. It has been found that a single molecule of theVDAC is capable of forming a channel (55). However, onmitochondrial membranes, the monomeric form of the VDACalso assembled in dimeric to hexameric or higher oligomericforms in different species (16, 55). The chemical cross-linkingof proteins with EGS in isolated T. brucei mitochondria fol-lowed by SDS-PAGE and immunoblot analysis showed thatTbPorin is present primarily as a monomer and a fraction ofthe protein appears as dimers. The dimeric form is detectedmore with increasing concentrations of EGS from 0 to 0.4 mM.The trimeric and tetrameric forms are detected in the procy-clic-form mitochondria only at the concentrations of EGSabove 0.1 mM. TbPorin is mostly present as a monomer in thebloodstream-form mitochondria and forms a dimer only at orabove an EGS concentration of 0.3 mM (Fig. 4). Since thebloodstream form possesses six- to sevenfold less TbPorin thanthe procyclic-form mitochondria, the TbPorin monomers are

possibly more dispersed on the mitochondrial OM of thebloodstream form and thus could not be cross-linked at lowerconcentrations of EGS. Together, it showed that, similar toVDACs in other organisms, TbPorin is capable of formingmultimeric structures on mitochondrial membranes.

Depletion of TbPorin reduced cell growth in both procyclicand bloodstream forms. To explore the physiological role ofTbPorin, RNAi studies were performed. Induction of theTbPorin double-stranded RNA in the procyclic form re-duced its transcript level more than 95% within 2 days (Fig.5A). The TbPorin protein level was decreased about 50% and80% at days 4 and 6, respectively (Fig. 5B). Depletion ofTbPorin reduced cell growth in the procyclic form. A consis-tent difference in cell numbers was observed at day 4 andbeyond (Fig. 5C). We also performed TbPorin RNAi in thebloodstream form. A reduction in the TbPorin protein levelupon induction of the expression of porin double-strandedRNA was accompanied by inhibition of cell growth also in thebloodstream form (Fig. 5D and E). The cell number was sig-nificantly reduced at day 5 of doxycycline treatment, and it

FIG. 3. Expression levels of TbPorin in two developmental forms. (A) Mitochondrial proteins isolated from the procyclic and bloodstreamforms were analyzed by immunoblotting using TbPorin-, AAC-, Tim17-, TAO-, Cyt c1-, and mitochondrial Hsp70 (mHsp70)-specific antibodies asprobes. Amounts of proteins loaded in each lane are indicated at the top of the gels. (B) The transcript level of TbPorin was analyzed by Northernblotting of total RNA isolated from the procyclic and bloodstream forms. Actin and ethidium bromide-stained rRNA were used as the loadingcontrols. The results are representative of three sets of experiments.

FIG. 4. TbPorin is present in oligomeric forms. Mitochondria isolated from the procyclic and bloodstream forms were incubated with variousconcentrations of EGS (0 to 400 �M) for 30 min at room temperature. After incubation, the samples were treated with glycine at 15 mM to quenchthe excess cross-linker, and proteins were analyzed by SDS-PAGE and immunoblotting using TbPorin-specific antibody as the probe. The amountof mitochondrial protein loaded for the procyclic form (5 �g) was fourfold less than the amount loaded for the bloodstream form (20 �g).



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became stationary afterwards. These results clearly indicatethat TbPorin is critical for mitochondrial activities thus re-quired for normal cell growth in both forms. Recently, Pusniket al. reported that depletion of the VDAC affects cell growthof the procyclic form only in the absence of glucose (35). Theirdouble-knockout cell lines grew similarly to controls in regularprocyclic medium SDM-79. In contrast, our findings showedthat a partial depletion of TbPorin reduced cell growth even inthe presence of glucose. This discrepancy could be due tomistargeting of our RNAi construct, although it is unlikely. Wetook a PCR-based approach to demonstrate that the TbPorinRNAi construct is properly targeted at the 177-base-pair re-peat regions in the minichromosomes (see Fig. S2 in the sup-plemental material). Therefore, the differences between ourresults and those of Pusnik et al. could be due to differences inthe constituents of the semisynthetic SDM-79 medium. Also, itcould be possible that during the generation of their double-knockout cell line, cells gradually adapted to growing withoutmitochondrial porin by increasing the activity of other protein

channels on the OM. A similar observation has been reportedfor S. cerevisiae (23).

TbPorin is crucial for mitochondrial ATP production. Inorder to explore the reason for reduced cell growth due todepletion of TbPorin, we compared the energy productioncapacities of mitochondria isolated from porin KD and controlprocyclic cells. Different substrates, such as succinate, �-KG,and pyruvate with succinate, were used to compare in or-ganello ATP production via oxidative and substrate-level phos-phorylations as described previously (38). Succinate donateselectrons to the mitochondrial electron transport chain, thusproducing ATP by oxidative phosphorylation (7, 38), whereas�-KG and pyruvate with succinate generate ATP by substrate-level phosphorylation mediated by succinate dehydrogenaseand via the acetyl/succinyl coenzyme A transferase cycle, re-spectively (7, 38). We observed that depletion of TbPorin re-duced mitochondrial ATP production by all these substratesabout two- to fourfold relative to the control (Fig. 6). In thewild-type procyclic-form mitochondria, ATP production peaked

FIG. 5. TbPorin is required for cell growth. TbPorin RNAi-disrupted cell lines were allowed to grow in the presence and absence of doxycycline(1 �g/ml). Procyclic (A, B, and C)- and bloodstream (D and E)-form cells were harvested at different time points as indicated after induction withdoxycycline for RNA and protein analysis by Northern (A) and immunoblot (B and D) analyses, respectively. Actin and the ethidium bromide-stained rRNA were used as the controls for Northern blotting. TbPP5- and tubulin-specific antibodies were used to show equal loadings of thesamples for immunoblot analysis of the procyclic and the bloodstream forms, respectively. Proteins from 5 � 106 cells were loaded in each lanefor immunoblot analysis. The numbers of the procyclic (C)- and bloodstream (E)-form cells were counted at different time points during inductionand plotted against time. The results are representative of three independent experiments.



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within 2 to 5 min after the addition of ADP. After reaching themaximum point, ATP production either gradually dropped orstabilized as the substrate was utilized (Fig. 6A and C). Asexpected, the addition of ADP alone showed a negligible effecton ATP production (data not shown). In contrast to the con-trol, mitochondria isolated from TbPorin KD cells showed verylittle stimulation of ATP production after the addition of ADPand substrates. In these mitochondria, ATP production wasslightly increased at 2 min in the presence of succinate, �-KG,and pyruvate but could not reach the levels found in the con-trol. In the presence of antimycin, ATP production by succi-nate was inhibited in the control mitochondria as expected,indicating that ATP is produced by oxidative phosphorylation(Fig. 6A). The residual ATP production in porin KD mito-chondria was also decreased by antimycin. When mitochondriawere pretreated with water to rupture the mitochondrial OM,the ability to produce ATP upon the addition of succinate wassignificantly increased in porin KD samples (Fig. 6B). This

suggested that a reduction of metabolite flux through the OMin porin KD mitochondria is possibly the cause of reduction ofits ability to produce ATP. Together, these results showed thatdepletion of TbPorin reduced both oxidative and substrate-level phosphorylations in mitochondria because the substratesand ADP could not enter through the OM. A similar obser-vation has been made by other investigators (35).

Downregulation of TbPorin reduced the expression level ofTAO and increased cytochrome pathway proteins as well asthe levels of complexes III and IV. To understand the effect ofdepletion of TbPorin on mitochondrial proteins, the steady-state levels of several known IM- and matrix-localized proteinswere compared in TbPorin-depleted and control mitochondriaby semiquantitative immunoblot analysis. Interestingly, we foundthat TbPorin KD affects differentially the steady-state levels ofdifferent mitochondrial proteins. When the TbPorin level wasreduced about 50%, the steady-state level of TAO was reducedabout 20 to 30% relative to the control. By contrast, the levels

FIG. 6. TbPorin is crucial for mitochondrial energy production. ATP concentrations were measured using a luciferase-based ATP assay kit asdescribed in Materials and Methods. Mitochondria isolated from uninduced control and TbPorin KD cells grown in the presence of doxycyclinefor 4 days were incubated separately with substrates succinate (A and B), �-KG, and pyruvate with succinate (C). Succinate was added in duplicatesamples in the absence or presence of antimycin (2.7 �M). Mitoplasts were prepared from uninduced control and porin KD mitochondria asdescribed in Materials and Methods and incubated with succinate (panel B). The reaction was started with the addition of ADP (55 �M). Atdifferent time points (0 to 30 min), aliquots were collected. Proteins were precipitated by 60% perchloric acid, and the supernatants were used forthe luciferase assay. Relative light units (RLU) were plotted against time. Each assay was performed in triplicate.



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of COIV, Cyt c1, and Tim17 were increased approximately30%, 10%, and 5%, respectively, in comparison to the unin-duced control (Fig. 7A and B). The expression level of theAAC, a major IM protein, was slightly increased due to TbPorinRNAi (Fig. 7A and B). However, the level of the matrix-localized mitochondrial Hsp70 was unaltered, and the samewas observed for tubulin. These results showed that TbPorinis possibly not involved in mitochondrial protein biogenesisin general. However, depletion of TbPorin affects differentlythe levels of components of the cytochrome oxidase andTAO in the procyclic form.

Observing the changes in the levels of COIV and TAO, webecame interested in investigating the effect of TbPorin deple-tion on the levels of mitochondrial respiratory complexes. Themembrane protein complexes from TbPorin KD and controlmitochondria were solubilized by digitonin, separated by BN-PAGE, and probed with specific antibodies. Mitochondrialporin in other eukaryotes forms an oligomeric complex on theOM (16, 26, 55). We found that TbPorin forms a complex withan apparent mass of 212 kDa (Fig. 8). This complex was abol-ished in porin KD mitochondria. The same blot was probedwith antibodies against T. brucei COIV, Cyt c1, and TAO. Theantibodies for Cyt c1 and COIV detected complex III andcomplex IV, respectively, as expected (56). Interestingly, in theTbPorin KD mitochondria, the levels of both complex III andcomplex IV were increased in comparison to those in mito-chondria of the uninduced control cells. The increase was morepronounced for complex IV (Fig. 8). It has been shown previ-ously that during detergent solubilization of mitochondrialproteins, TAO forms an oligomeric complex (14). In theTbPorin KD mitochondria, the level of this TAO oligomer wasreduced (Fig. 8), as found for the TAO protein in the previ-ously described experiment (Fig. 7A). The experiment wasrepeated multiple times, and similar results were observed.Altogether, TbPorin KD decreased the level of TAO but in-

FIG. 7. TbPorin RNAi differentially affects the steady-state level ofmitochondrial proteins. (A) The TbPorin KD and uninduced control cellswere grown for 4 days in the presence of doxycycline, and mitochondriawere isolated as described in the text. Mitochondrial proteins (12.5 and6.25 �g) were analyzed by immunoblot analysis using antibody probes forTbPorin, TAO, Cyt c1, COIV, AAC, Tim17, mitochondrial Hsp70(mHsp70), and �-tubulin. (B) The intensities of the respective proteinbands were quantitated from three independent experiments by using animaging densitometer as described in Materials and Methods and nor-malized with the corresponding �-tubulin protein bands. Percent in-creases and decreases in the intensity in TbPorin KD cells relative to thecontrol were plotted for different proteins.

FIG. 8. Depletion of TbPorin increased the level of respiratory complexes III and IV. Mitochondria isolated from the uninduced control (Con) andTbPorin KD cells were solubilized with digitonin (1.0%). The solubilized supernatants were clarified by centrifugation at 100,000 � g. The samples withincreasing amounts of proteins were electrophorsed by BN-PAGE and immunoblotted with antibodies for porin, COIV, Cyt c1, and TAO. Molecular sizemarker proteins apoferritin dimer (800 kDa), apoferritin monomer (400 kDa), �-amylase (200 kDa), alcohol dehydrogenase (150 kDa), and bovine serumalbumin (66 kDa) were run on the same gel and visualized by Coomassie blue staining. The porin and TAO oligomeric complexes, the bc1 reductasecomplex, and the cytochrome oxidase complex are indicated on the side of the corresponding blot developed with porin-, TAO-, Cyt c1-, andCOIV-specific antibodies. The experiment was repeated more than three times, and consistent results were obtained.



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creased complex III and complex IV levels in the procyclic-form mitochondria.

Depletion of TbPorin reduced cellular respiration via APand increased the capacity of CP. The effect of TbPorin de-pletion on two terminal oxidases led us to determine its effecton cellular respiration. The procyclic-form cells respire bothvia a usual CP, which is coupled with oxidative phosphor-ylation, and also through non-proton motive TAO (15, 49).SHAM is the specific inhibitor of TAO, while KCN is theinhibitor of cytochrome oxidase. These two inhibitors wereused to estimate the electron flow via the AP and CP asdescribed previously (49). Induction of TbPorin RNAi reducedthe electron flow through the AP. After induction with doxy-cycline for 4 days, inhibition of cellular respiration by SHAMwas reduced to 30% in comparison to the uninduced controlvalue (Fig. 9A). This indicates that reduction of TbPorin re-duces the engagement of AP, which is not proton motive. Incontrast, the percent inhibition of total respiration by KCN wasabout 80%, and it was increased by 2 to 3% due to TbPorinKD. The overall rates of respiration of the wild-type andTbPorin KD cells were comparable. We also measured thecapacities of the AP and CP in both types of cells. The amountof oxygen consumption inhibited by SHAM in the presence ofKCN is considered the AP capacity, and similarly, the amountof oxygen consumption inhibited by KCN in the presence ofSHAM is considered the CP capacity (49). At day 4 of induc-tion for TbPorin RNAi, the AP capacity was clearly decreasedand the CP capacity was significantly increased relative tothose in the uninduced control (Fig. 9B). Thus, depletion ofTbPorin moves the electron flow partially from the AP to theCP as it alters the levels of AP and CP proteins.

DISCUSSION

We analyzed the structure and characterized the function ofmitochondrial porin in T. brucei. The predicted structure ofTbPorin is distinct in comparison to mitochondrial porins fromother species. TbPorin depletion reduced cell growth for theprocyclic form as well as the bloodstream form. TbPorin iscrucial for mitochondrial metabolite transport and thus essen-tial for ATP production in the procyclic form. We also dem-onstrated that cellular respiration via the AP and CP can beregulated by changes in metabolite flux through TbPorin in theprocyclic form.

TbPorin possesses a predicted �-barrel structure. Few struc-tural features in TbPorin are bioinformatically distinct relativeto mitochondrial porins from other species. In contrast to themitochondrial porins with 16 to 19 �-strands from fungi andhumans, TbPorin has only 8 predicted �-strands, a numberwhich is similar to the bacterial OM porins OmpA and OmpX(21). The bacterial OM is enriched with various integral �-bar-rel proteins. The OmpA family proteins, with a barrel structureof eight �-strands, are ubiquitous in most gram-negative bac-teria (21). Further analysis of the structure of TbPorin by X-raycrystallography and nuclear magnetic resonance spectroscopyis required to confirm this similarity. Since T. brucei divergedvery early during evolution (42), a bacterial porin on trypano-some mitochondria is not unexpected.

We found that TbPorin is localized on the mitochondrialOM as an integral membrane protein. It is known that theVDAC is functionally monomeric; however, it can cluster intight but regular groups. It exists in a variety of oligomericstates, from 2- to 20-mers (16, 55). The chemical cross-linking ofisolated mitochondria from the procyclic form demonstrated thatTbPorin is also present in oligomeric states. However, it primarilyexists in the monomeric and dimeric forms; the higher-orderedstructures, such as trimers and tetramers, are detected only inthe procyclic-form mitochondria at higher concentrations ofEGS. BN-PAGE analysis of digitonin-solubilized mitochon-drial supernatant showed that TbPorin is present in a complexwith an apparent molecular mass of 212 kDa. This could be thehexameric or heptameric form of TbPorin. However, the con-tribution of detergent micelle in this complex needs consider-ation. It is also possible that the complex is a hetero-oligomer,as VDAC-interacting partners are found in other eukaryotes(16, 22). Thus, the exact oligomeric state in this complex canonly be speculated. The steady-state level of TbPorin is re-duced about six- to sevenfold in the bloodstream form of T.brucei. In this form, a majority of TbPorin exists in the mono-meric state, indicating that its distribution is less clustered andmore dispersed on the OM. In the bloodstream form, mito-chondrial activities are suppressed (15). Thus, it can be con-sidered that a reduced level of metabolite flux via a reducedlevel of mitochondrial porin is sufficient to maintain mitochon-drial activities in the bloodstream form. The expression levelsof several other mitochondrial proteins, excluding TAO, arealso reduced in the bloodstream form. TAO is the only termi-nal oxidase in this form and is crucial for its respiration. Thus,it is greatly upregulated in comparison to that in the procyclicform.

To evaluate the function of TbPorin, we performed RNAi inthe procyclic form as well as the bloodstream form. A reduc-

FIG. 9. Depletion of TbPorin alters the respiratory capacity. Oxy-gen consumption by cells was measured by oxygen electrode and oxy-gen monitor as described in Materials and Methods. The uninducedcontrol and TbPorin KD cells were grown in the presence of doxycy-cline and harvested at day 4 for a respiration assay. (A) The inhibitionof oxygen consumption by KCN (1 mM) and SHAM (1 mM) is pre-sented as the percent total oxygen consumption by each type of cells.(B) The AP and CP capacities were measured by calculating theamount of oxygen consumption inhibited by the specific inhibitor ofone pathway in the presence of the inhibitor of the other pathway. Theresults represent three independent experiments. Student’s t test anal-ysis of different data sets showed that the results are significant with Pvalues less than 0.05. The initial concentration of oxygen in the cham-ber was 240 �mol/ml at 27°C.



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tion in the TbPorin protein level decreased cell growth in bothprocyclic and bloodstream forms, indicating that porin isrequired for mitochondrial activities in both developmentalstages. We also found that the mitochondrial ATP level isreduced about twofold in the procyclic form due to TbPorinRNAi. Furthermore, the capacity of isolated mitochondria toproduce ATP by oxidative and substrate-level phosphoryla-tions is significantly reduced due to depletion of TbPorin. Dis-ruption of the OM of the porin-depleted mitochondria in-creased ATP production when substrates were provided. Thus,similar to other eukaryote VDACs, TbPorin mediates themajor metabolite flux between the mitochondria and cy-tosol, which is necessary for production of ATP. The trans-port of ADP/ATP occurs in a reverse orientation in thebloodstream-form mitochondria, and this process is crucialfor maintaining the mitochondrial membrane potential. It isassumed that TbPorin is involved in the transport of nucle-otides and substrates also in the bloodstream-form mito-chondria. Therefore, depletion of TbPorin reduces cellgrowth in this form, too.

TbPorin RNAi differentially affects the steady-state level ofmitochondrial proteins in the procyclic form. Interestingly, wefound that the expression level of TAO is decreased and thoseof Cyt c1 and COIV were increased in mitochondria in whichTbPorin was partially depleted by RNAi. There was no changein the transcript level of these proteins in TbPorin KD cellsrelative to the control (data not shown), indicating that theregulation possibly occurs at the stage of protein synthesis orprotein stability. Using BN-PAGE analysis, we separated mi-tochondrial membrane complexes. We found that the level ofcomplex IV is significantly increased in TbPorin-depleted mi-tochondria, as seen for its component proteins. These resultsare consistent and correlated with the levels of the correspond-ing components. Furthermore, the increase in the level of themature cytochrome oxidase complex was also correlated withthe respiratory activity via the CP. In the procyclic form, themajor respiratory flux goes through the CP and 20 to 30% ofthe activity remains engaged via the AP (49). Respiration viathe AP does not produce ATP; thus, during the ATP crisisgenerated by TbPorin RNAi, the electron flow was reduced viathe AP. The results are consistent among multiple experimentsand also correlated with the reduction in the TAO proteinlevel. Simultaneously, the capacity of electron flow via the CPwas increased, as it may cause increased ATP production byoxidative phosphorylation. However, cells could not overcomethe ATP demand by this alteration, due to a simultaneouscollapse of the substrate-level phosphorylation by TbPorin KD.It has been demonstrated previously that the substrate-levelphosphorylation is crucial for the survival of the procyclic form(7, 9).

Besides the proteins that are involved in respiration, theexpression levels of some other mitochondrial proteins werealso altered due to TbPorin RNAi. The level of T. bruceiTim17, the IM protein translocator, was increased due toTbPorin depletion. The reason for this change is not clear atthe moment. However, it has been found in S. cerevisiae thatdepletion of mitochondrial porin (POR 1) increased the levelof the protein translocase of the mitochondrial OM complex(Tom) components, possibly to increase OM permeability dueto cellular needs (23). Since T. brucei Tom proteins have not

yet been characterized, we could not verify the effect of porindepletion on Tom proteins in T. brucei. It can be anticipatedthat in TbPorin-depleted mitochondria, the T. brucei Tim17level was increased due to a similar reason, such as compen-sation of the metabolite flow in and out of mitochondria.

Overall, we found that TbPorin is crucial for mitochondrialactivities in both forms. The substrate-level phosphorylation aswell as oxidative phosphorylation for cellular energy produc-tion in the procyclic form is dependent on continuous metab-olite flux via TbPorin. It has been reported that inhibition ofsubstrate-level phosphorylation alone caused inhibition in cellgrowth in this form (9). Therefore, it is expected that depletionof TbPorin would also affect cell proliferation in a similarmanner unless another protein(s) takes over its function in theabsence of TbPorin. Thus, our findings properly justifiedTbPorin function. Besides the effect on cell growth andmitochondrial ATP production due to TbPorin RNAi, wealso demonstrated here that regulation of the metaboliteflux through TbPorin regulates the electron flow mediatedby two terminal oxidases via increasing or decreasing thelevels of the component proteins and their assemblies. Inother eukaryotes, VDAC activity is known to be regulated bydifferent cellular components under different physiologicalconditions to modulate the function of mitochondria (16, 28).Therefore, it is likely that TbPorin plays a crucial role in reg-ulation of the metabolic pattern with changes in nutrients andother environmental factors that occurs in the parasite lifecycle. As mitochondrial OM proteins in this organism arepoorly characterized, the TbPorin-specific antibody will alsoserve as a useful marker for the OM. Altogether, this studyrevealed that TbPorin is critical for mitochondrial activities,although structurally it is closer to a prokaryotic porin.

ACKNOWLEDGMENTS

We thank George Cross and Elizabeth Wirtz for the pLew100 vectorand the procyclic 427 (29-13) and SM427 cell lines, Paul Englund forCrithidia Hsp70-specific antibody, Steve Hajduk for anti-Cyt c1, KeithGull for antitubulin antibody and the p2T7-177 RNAi vector, andMichael Izban, VaNae Hamilton, and Melanie Duncun for criticallyreviewing the manuscript.

The work was supported by NIH grants 3SO6GM08037-30S1 and1SC1GM081146.

REFERENCES

1. Abu-Hamad, S., S. Sivan, and V. Shosham-Barmatz. 2006. The expressionlevel of the voltage-dependent anion channel controls life and death of thecell. Proc. Natl. Acad. Sci. USA 103:5787–5792.

2. Allemann, N., and A. Schneider. 2000. ATP production in isolated mito-chondria of procyclic Trypanosoma brucei. Mol. Biochem. Parasitol. 111:87–94.

3. Bagos, P. G., T. D. Liakopoulos, I. C. Spyropoulos, and A. J. Hamodrakas.2004. PRED-TM��: a web-server for predicting the topology of �-barrelouter membrane proteins. Nucleic Acids Res. 32:W400–W404.

4. Baxevanis, A. D. 2008. Searching NCBI databases using Entrez, chapter 1. InCurrent protocols in bioinformatics, unit 1.3. Wiley, Hoboken, NJ.

5. Bayrhuber, M., T. Meins, M. Habeck, S. Backer, K. Giller, S. Villinger, C.Vonrhein, C. Griesinger, M. Zweckstetter, and K. Zeth. 2008. Structure ofthe human voltage-dependent anion channel. Proc. Natl. Acad. Sci. USA105:15370–15375.

6. Berriman, M., et al. 2005. The genome of the African trypanosome Trypano-soma brucei. Science 309:416–422.

7. Besteiro, S., M. P. L. Barrett, L. Riviere, and F. Bringaud. 2005. Energygeneration in the insect stages of of Trypanosoma brucei: metabolism in flux.Trends Parasitol. 21:185–191.

8. Biebinger, S., L. E. Wirtz, P. Lorenz, and C. Clayton. 1997. Vectors forinducible expression of toxic gene products in bloodstream and procyclicTrypanosoma brucei. Mol. Biochem. Parasitol. 85:99–112.



http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

9. Bochud-Allemann, N., and A. Schneider. 2002. Mitochondrial substrate levelphosphorylation is essential for growth of procyclic Trypanosoma brucei.J. Biol. Chem. 277:32849–32854.

10. Brown, S. V., P. Hosking, J. Li, and N. Williams. 2006. ATP synthase isresponsible for maintaining mitochondrial membrane potential in blood-stream form Trypanosoma brucei. Eukaryot. Cell 5:45–53.

11. Chaudhuri, M., W. U. Ajayi, and G. C. Hill. 1998. Biochemical and molec-ular properties of the Trypanosoma brucei alternative oxidase. Mol. Biochem.Parasitol. 95:53–68.

12. Chaudhuri, M. 2001. Cloning and characterization of a novel serine/threo-nine protein phosphatase type 5 from Trypanosoma brucei. Gene 266:1–13.

13. Chaudhuri, M., and F. E. Nargang. 2003. Import and assembly of Neuros-pora crassa Tom40 into mitochondria of Trypanosoma brucei in vivo. Curr.Genet. 44:85–94.

14. Chaudhuri, M., R. D. Ott, L. Saha, S. Williams, and G. C. Hill. 2005. Thetrypanosome alternative oxidase exists as a monomer in Trypanosoma bruceimitochondria. Parasitol. Res. 96:178–183.

15. Chaudhuri, M., R. D. Ott, and G. C. Hill. 2006. Trypanosome alternativeoxidase: from molecule to function. Trends Parasitol. 22:484–491.

16. Colombini, M., E. Blachly-Dyson, and M. Forte. 1996. VDAC, a channel inthe outer membrane. Ion Channels 4:169–202.

17. De Pinto, V., S. Reina, F. Guarino, and A. Messina. 2008. Structure of thevoltage dependent anion channel: state of the art. J. Bioenerg. Biomembr.40:139–147.

18. Effron, P. N., A. F. Torri, D. M. Engman, J. E. Donelson, and P. T. Englund.1993. A mitochondrial heat shock protein from Crithidia fasciculata. Mol.Biochem. Parasitol. 59:191–200.

19. Elkeles, A., K. M. Devos, D. Graur, M. Zizi, and A. Breiman. 1995. MutiplecDNAs of wheat voltage-dependent anion channels: isolation, differentialexpression, mapping and evolution. Plant Mol. Biol. 29:109–124.

20. Higgins, D. G., J. D. Thompson, and T. J. Gibson. 1996. Using CLUSTALfor multiple sequence alignments. Methods Enzymol. 266:383–402.

21. Khalid, S., P. J. Bond, T. Carpenter, and M. S. P. Sansom. 2008. OmpA:gating and dynamics via molecular dynamics simulations. Biochim. Biophys.Acta 1778:1871–1880.

22. Kmita, H., M. Budzinska, and M. O. Stobienia. 2003. Modulation of thevoltage-dependent anion-selective channel by cytoplasmic proteins from wildtype and the channel depleted cells of Saccharomyces cerevisiae. Acta Bio-chim. Pol. 50:415–424.

23. Kmita, H., N. Antos, M. Wojtkowska, and L. Hryniewiecka. 2004. Processesunderlying the upregulation of Tom proteins in S. cerevisiae mitochondriadepleted of the VDAC channel. J. Bioenerg. Biomembr. 36:187–193.

24. Koebnik, R., K. P. Locher, and P. Van Gelder. 2000. Structure and functionof bacterial outer membrane proteins: barrels in a nutshell. Mol. Microbiol.37:239–253.

25. Krauskopf, A., O. Eriksson, W. J. Craigen, M. A. Forte, and P. Bernardi.2006. Properties of the permeability transition in VDAC-/- mitochondria.Biochim. Biophys. Acta 1757:590–595.

26. Krimmer, T., D. Rapaport, M. T. Ryan, C. Mesinger, C. K. Kassenbrock, E.Blachly-Dyson, M. Forte, M. G. Douglas, W. Neupert, F. E. Nargang, and N.Pfanner. 2001. Biogenesis of porin of the outer mitochondrial membraneinvolves an import pathway via receptors and the general import pore of theTOM complex. J. Cell Biol. 152:289–300.

27. Laemmli, U. K. 1970. Cleavage of structural protein during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

28. Lemasters, J. J., and E. Holmuhamedov. 2006. Voltage-dependent anionchannel (VDAC) as mitochondrial governator—thinking outside the box.Biochim. Biophys. Acta 1762:181–190.

29. Liu, B., Y. Liu, S. A. Motyka, E. E. Agbo, and P. T. Englund. 2005. Fellowshipof the rings: the replication of kinetoplast DNA. Trends Parasitol. 21:363–369.

30. Lukes, J., H. Hashimi, and A. Zikova. 2005. Unexplained complexity of themitochondrial genome and transcriptome in kinetoplastid flagellates. Curr.Genet. 48:277–299.

31. Maslov, D. A., A. Zinkova, I. Kyselova, and J. Lukes. 2002. A putative novelnuclear-encoded subunit of the cytochrome coxidase complex in trypanoso-matids. Mol. Biochem. Parasitol. 125:113–125.

32. Niederweis, M. 2003. Mycobacterial porins—new channel proteins in uniqueouter membranes. Mol. Microbiol. 49:1167–1177.

33. Nolan, D. P., and H. P. Voorheis. 1992. The mitochondrion in bloodstream

forms of Trypanosoma brucei is energized by the electrogenic pumping ofprotons catalysed by the F1F0-ATPase. Eur. J. Biochem. 209:207–216.

34. Priest, J. W., and S. L. Hajduk. 1994. Developmental regulation of Trypano-soma brucei cytochrome c reductase during bloodstream to procyclic differ-entiation. Mol. Biochem. Parasitol. 65:291–304.

35. Pusnik, M., F. Charriere, P. Maser, R. Waller, M. J. Dagley, T. Lithgow, andA. Schneider. 2009. The single mitochondrial porin of Trypanosoma brucei isthe main metabolite transporter in the outer mitochondrial membrane. Mol.Biol. Evol. 26:671–680.

36. Randall, A., J. Cheng, M. Sweredoski, and P. Baldi. 2008. TMBPro: second-ary structure, beta contact and tertiary structure prediction of transmem-brane beta-barrel proteins. Bioinformatics 24:513–520.

37. Sambrook, J., E. Fritsch, and T. Maniatis. 1994. Molecular cloning: alaboratory manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY.

38. Schneider, A., N. Bouzaidi-Tiali, A. L. Chanez, and L. Bulliard. 2007. ATPproduction in isolated mitochondria of procyclic Trypanosoma brucei. Meth-ods Mol. Biol. 372:379–387.

39. Schnaufer, A., D. G. Clark-Walker, A. G. Steinberg, and K. Stuart. 2005. TheF1-ATP synthase complex in bloodstream stage trypanosomes has an un-usual and essential function. EMBO J. 24:4029–4040.

40. Schulz, G. E. 2002. The structure of bacterial outer membrane proteins.Biochim. Biophys. Acta 1565:308–317.

41. Shoshan-Barmatz, V., A. Israelson, D. Brdiczka, and S. S. Sheu. 2006. Thevoltage-dependent anion channel (VDAC): function in intracellular signal-ing, cell life and cell death. Curr. Pharm. Des. 12:2249–2270.

42. Simpson, A. G., J. R. Stevens, and J. Lukes. 2006. The evolution and diver-sity of kinetoplastid flagellates. Trends Parasitol. 22:168–174.

43. Singha, U. K., E. Peprah, S. Williams, R. Walker, L. Saha, and M.Chaudhuri. 2008. Characterization of the mitochondrial inner membranetranslocator Tim17 from Trypanosoma brucei. Mol. Biochem. Parasitol. 159:30–43.

44. Smith, S. G., V. Mahon, M. A. Lambert, and R. P. Fagan. 2007. A molecularswiss army knife: OmpA structure, function and expression. FEMS Micro-biol. Lett. 273:1–11.

45. Stuart, K. D., A. Schnaufer, N. L. Ernst, and A. K. Panigrahi. 2005. Complexmanagement: RNA editing in trypanosomes. Trends Biochem. 30:97–105.

46. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedures andsome application. Proc. Natl. Acad. Sci. USA 76:4350–4354.

47. Tsujimoto, Y., and S. Shimizu. 2007. Role of the mitochondrial membranepermeability transition in cell death. Apoptosis 12:835–840.

48. Vercesi, A. E., R. Docampo, and S. N. Moreno. 1992. Energization-depen-dent Ca2� accumulation in Trypanosoma brucei bloodstream and procyclictrypomastigotes mitochondria. Mol. Biochem. Parasitol. 56:251–257.

49. Walker, R., L. Saha, G. C. Hill, and M. Chaudhuri. 2005. The effect ofover-expression of the alternative oxidase in the procyclic forms of Trypano-soma brucei. Mol. Biochem. Parasitol. 139:153–162.

50. Wickstead, B., K. Ersfeld, and K. Gull. 2002. Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomesof Trypanosoma brucei. Mol. Biochem. Parasitol. 125:211–216.

51. Wittig, I., H. P. Braun, and H. Schagger. 2006. Blue native PAGE. Nat.Protoc. 1:418–428.

52. Woods, A., T. Sherwin, R. Sasse, T. H. MacRae, A. J. Baines, and K. Gull.1989. Definition of individual components within the cytoskeleton ofTrypanosoma brucei by a library of monoclonal antibodies. J. Cell Sci. 93:491–500.

53. World Health Organization. 2009. Report on infectious diseases. World HealthOrganization, Geneva, Switzerland. www.who.int/tropics/trypanosomiasis_african/en/.

54. Yehezkel, G., S. Abu-Hamad, and V. Shoshan-Barmatz. 2007. An N-terminalnucleotide-binding site in VDAC1: Involvement in regulating mitochondrialfunction. J. Cell. Physiol. 212:551–561.

55. Zalk, R., A. Israelson, E. S. Garty, H. Azoulay-Zohar, and V. Shoshan-Barmatz. 2005. Oligomeric states of the voltage-dependent anion channeland cytochrome c release from mitochondria. Biochem. J. 386:73–83.

56. Zíkova, A., E. Horakova, M. Jirku, P. Dunajcikova, and J. Lukes. 2006. Theeffect of down-regulation of mitochondrial RNA-binding proteins MRP1and MRP2 on respiratory complex in procyclic Trypanosoma brucei. Mol.Biochem. Parasitol. 149:65–73.



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