Functional Complementation Analyses Reveal that the Single PRATFamily Protein of Trypanosoma brucei Is a Divergent Homolog ofTim17 in Saccharomyces cerevisiae

Ebony Weems,a Ujjal K. Singha,a VaNae Hamilton,a Joseph T. Smith,a Karin Waegemann,b Dejana Mokranjac,b Minu Chaudhuria

Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee, USAa; Adolf Butenandt Institute, Physiological Chemistry, University ofMunich, Munich, Germanyb

Trypanosoma brucei, a parasitic protozoan that causes African trypanosomiasis, possesses a single member of the presequenceand amino acid transporter (PRAT) protein family, which is referred to as TbTim17. In contrast, three homologous proteins,ScTim23, ScTim17, and ScTim22, are found in Saccharomyces cerevisiae and higher eukaryotes. Here, we show that TbTim17cannot rescue Tim17, Tim23, or Tim22 mutants of S. cerevisiae. We expressed S. cerevisiae Tim23, Tim17, and Tim22 in T. bru-cei. These heterologous proteins were properly imported into mitochondria in the parasite. Further analysis revealed that al-though ScTim23 and ScTim17 were integrated into the mitochondrial inner membrane and assembled into a protein complexsimilar in size to TbTim17, only ScTim17 was stably associated with TbTim17. In contrast, ScTim22 existed as a protease-sensi-tive soluble protein in the T. brucei mitochondrion. In addition, the growth defect caused by TbTim17 knockdown in T. bruceiwas partially restored by the expression of ScTim17 but not by the expression of either ScTim23 or ScTim22, whereas the expres-sion of TbTim17 fully complemented the growth defect caused by TbTim17 knockdown, as anticipated. Similar to the findingsfor cell growth, the defect in the import of mitochondrial proteins due to depletion of TbTim17 was in part restored by the ex-pression of ScTim17 but was not complemented by the expression of either ScTim23 or ScTim22. Together, these results suggestthat TbTim17 is divergent compared to ScTim23 but that its function is closer to that of ScTim17. In addition, ScTim22 couldnot be sorted properly in the T. brucei mitochondrion and thus failed to complement the function of TbTim17.

Amajority of proteins in the mitochondria are encoded by nuclearDNA. These proteins are imported by the translocase of the mi-

tochondrial outer membrane (TOM) and the translocase of the mi-tochondrial inner membrane (TIM) (1, 2). The TOMs and TIMs aremultiprotein complexes whose structure and function have been ex-tensively characterized in fungi and recently in humans and plants.The TOM complex serves as the entry gate for virtually all mitochon-drial proteins (3). There are two TIM complexes, TIM23 and TIM22,in the majority of eukaryotes analyzed so far. Unlike TOM, the TIMcomplexes have substrate specificities. The TIM23 complex importsproteins that contain an N-terminal targeting signal (MTS) into themitochondrial matrix and, if they contain an additional sorting sig-nal, into the inner membrane (4, 5). Tim23 and Tim17, together withthe receptor Tim50, form the core of the TIM23 complex. This corecomplex seems to be sufficient for transport of proteins into the mi-tochondrial inner membrane by a stop-transfer pathway. For trans-location of proteins to the mitochondrial matrix, the ATP-dependentaction of the import motor of the TIM23 complex is additionallyrequired (4–6). The TIM22 complex, on the other hand, is involvedin the translocation and insertion of a special class of mitochondrialinner membrane proteins. These proteins have multiple internal tar-geting signals, such as mitochondrial metabolite carrier proteins(MCPs) (7, 8). The membrane-embedded part of TIM22 consists ofTim22, Tim54, and Tim18. Tim12 is an additional component of thiscomplex and aids with the docking of MCPs carried by the small Timproteins (Tim9 and Tim10) in the intermembrane space (IMS) (9).

Tim17, Tim23, and Tim22 all belong to the presequence andamino acid transporter (PRAT) protein family (10, 11). Theseproteins have 4 transmembrane domains located at the center andpossess a PRAT signature motif [(G/A)X2(F/Y)X10RX3DX6(G/A/S)GX3G]. In spite of the similarity of their secondary structure,

these proteins perform distinct functions. Saccharomyces cerevi-siae Tim23 (ScTim23) and ScTim22 form a protein import chan-nel in the TIM23 and TIM22 complexes, respectively (12, 13). Thefunction of ScTim17 is less clear; it seems to act as the structuralcomponent that is involved in gating the Tim23 channel (14).Homologs of these Tim proteins are also present in human andplants (15–18).

Trypanosoma brucei belongs to a group of ancient and diver-gent eukaryotes. It possesses a single mitochondrion with manyunique and essential activities (19–21). Similar to other eu-karyotes, hundreds of nuclear DNA-encoded proteins are im-ported into the T. brucei mitochondrion. However, T. brucei pos-sesses relict import machinery for mitochondrial proteins (22).There is no canonical TOM complex in this parasite. Instead, anarchaic protein termed ATOM imports proteins across the outermitochondrial membrane (23, 24). T. brucei possesses a single

Received 25 August 2014 Accepted 7 January 2015

Accepted manuscript posted online 9 January 2015

Citation Weems E, Singha UK, Hamilton V, Smith JT, Waegemann K, Mokranjac D,Chaudhuri M. 2015. Functional complementation analyses reveal that the singlePRAT family protein of Trypanosoma brucei is a divergent homolog of Tim17 inSaccharomyces cerevisiae. Eukaryot Cell 14:286 –296. doi:10.1128/EC.00203-14.

Address correspondence to Minu Chaudhuri, mchaudhuri@mmc.edu.

E.W. and U.K.S. contributed equally to this article.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/EC.00203-14.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/EC.00203-14

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PRAT family protein named T. brucei Tim17 (TbTim17) (25, 26)and also has a homolog of Tim50 (27). Three small Tim proteinshave been identified in T. brucei; however, their functions have notbeen characterized as yet. TbTim17 is localized in the mitochon-drial inner membrane. It is essential for the import of proteins intothe mitochondrion and for cell survival (25, 26). We recentlyshowed that TbTim17 is present in a larger protein complex of1,100 kDa (28). TbTim17 is also found to be associated with sev-eral trypanosome-specific proteins (TbTim62, TbTim54) that areinvolved in mitochondrial protein import (28). In addition, re-cent evidence indicates that TbTim17 is involved in the import oftRNAs into the T. brucei mitochondrion (29). Although T. bruceipossesses a large set of mitochondrial metabolite carrier proteins,the homologs of any of the components of the mitochondrial car-rier translocase TIM22 have not been detected in trypanosomegenome databases. However, it is speculated that TbTim17 alongwith small TbTim proteins perform this function (22).

TbTim17 possesses 4 predicted transmembrane domains andhas an �20 to 30% primary sequence similarity with fungalTim23, Tim17, and Tim22 (25). However, the functional equiva-lent of TbTim17 among these three proteins has not been deter-mined. Here, we performed a functional complementation anal-ysis of TbTim17 with Saccharomyces cerevisiae Tim17, Tim23, andTim22 and vice versa. We show that the function of TbTim17 ispartially complemented by the expression of ScTim17 but not by theexpression of ScTim23 or ScTim22. All three proteins were targetedto the mitochondrion in T. brucei. ScTim23 and ScTim17, but notScTim22, were integrated into the mitochondrial inner mem-brane. ScTim23 and ScTim17 also assembled into a protein com-plex similar in size to one of the TbTim17 protein complexes;however, only ScTim17 was stably associated with TbTim17 andparticipated in mitochondrial protein import. Therefore, theseresults indicate that TbTim17 is functionally closer to ScTim17.

MATERIALS AND METHODST. brucei strains, media, and cell growth. The procyclic form of cells ofthe T. brucei 427 double-resistant 29-13 cell line expressing a tetracyclinerepressor gene and a T7 RNA polymerase was grown in Schneider’s Dro-sophila medium supplemented with 10% fetal bovine serum and antibi-otics (50 �g/ml hygromycin and 15 �g/ml G418) (30). To measure cellgrowth, cells were seeded at a density of 2 � 106 cells/ml in fresh mediumcontaining the appropriate antibiotics. Cells were harvested at differenttime points (0 to 9 days), and cell numbers were counted using a Neu-bauer hemocytometer. The log of the cumulative cell numbers versus thetime (days) of incubation was plotted.

Generation of plasmid constructs and transfection. The open read-ing frames (ORFs) for ScTim17, ScTim23, and ScTim22 were PCR ampli-fied using the corresponding cDNA clones as the templates and sequence-specific primers. The forward and reverse primers were designed to addrestriction sites HindIII and BamHI, respectively, at the 5= ends (see TableS1 in the supplemental material). The PCR products were cloned into apHD1344 trypanosome expression vector (a generous gift from J. Carnes)within the HindIII and BamHI restriction sites. Plasmid DNAs were lin-earized by NotI and transfected into cells of a previously developedTbTim17 RNA interference (RNAi) cell line (25). Transfected cells wereselected by puromycin (1 �g/ml). The inserted gene is constitutively ex-pressed from this vector by a read-through transcription from the tubulinlocus. ORFs for ScTim proteins were also cloned into the pLew100-Bsdvector (a generous gift from George Cross) within the HindIII and BamHIsites. The nucleotide sequence-encoded 2� myc epitope (EQKLISEEDL)was added to the 5= end of the reverse primers (see Table S1 in the sup-plemental material) for inducible expression of ScTim17, ScTim23, and

ScTim22 with a 2� myc tag at the C-terminal end. For generation ofTbTim17 double-stranded RNA targeted to the 3= untranslated region(UTR) of the TbTim17 transcript, the 3= UTR of TbTim17 was PCR am-plified from T. brucei genomic DNA using the forward and reverse prim-ers containing the restriction sites BamHI and HindIII, respectively, at the5= ends (see Table S1 in the supplemental material). The 476-bp ampliconwas subcloned into the multiple-cloning site of the tetracycline-inducible(Ti) T7 double-headed promoter plasmid vector p2T7Ti-177 (31) be-tween the BamHI and HindIII restriction enzyme sites. After transfection,cells were selected by phleomycin (2.5 �g/ml). A wild-type TbTim17 con-struct was also generated by subcloning the open reading frame ofTbTim17 into the pHD-1344 vector as described above for the S. cerevisiaeyeast proteins. Plasmid DNAs were linearized by NotI and transfected intocells of the TbTim17 3=UTR RNAi cell line, and the transfected cells wereselected by puromycin (1.0 �g/ml).

Subcellular fractionation. Fractionation of T. brucei procyclic cellswas performed as described previously (25, 27). Briefly, 2 � 108 cells wereresuspended in 500 �l of SMEP buffer (250 mM sucrose, 20 mM MOPS[morpholinepropanesulfonic acid]-KOH, pH 7.4, 2 mM EDTA, 1 mMphenylmethylsulfonyl fluoride [PMSF]) containing 0.03% digitonin andincubated on ice for 5 min. The cell suspension was then centrifuged for 5min at 6,800 � g and 4°C. The resultant pellet was considered a crudemitochondrial fraction, and the supernatant contained soluble cytosolicproteins.

Isolation and postisolation treatments of mitochondria. Mitochon-dria were also isolated from the parasite after lysis via nitrogen cavitationin isotonic buffer as described previously (25, 27). The isolated mitochon-dria were stored at a protein concentration of 10 mg/ml in SME buffer(250 mM sucrose, 20 mM MOPS-KOH, pH 7.4, 2 mM EDTA) containing50% glycerol at �70°C. For limited proteinase K (PK) digestion, mito-chondria in SME buffer (1 mg/ml) were treated with various concentra-tion of PK (0 to 150 �g/ml) for 30 min on ice. After incubation, the PK wasinhibited by PMSF (2 mM) and the mitochondria were reisolated by cen-trifugation at 10,000 � g and 4°C for 10 min. For alkali extraction, mito-chondria (100 �g) isolated from T. brucei were treated with 100 mMNa2CO3 (100 �l) at pH 11.5 for 30 min on ice (25, 27). The supernatantand pellet fractions were collected after centrifugation and analyzed bySDS-PAGE and immunoblotting.

Mitochondrial protein import assay in vivo. Cells with TbTim17RNAi containing a genome-integrated copy of the mitochondrial RNA-binding protein MRP2 with a 2� myc tag (MRP2-2� myc) at the Cterminus were previously developed in our laboratory to study the effectof TbTim17 RNAi on in vivo targeting of MRP2 (28). Cells of this cell line(MRP2-2� myc/Tim17 RNAi) were further transfected with either thepHD1344-ScTim17, pHD1344-ScTim23, or pHD1344-ScTim22 con-struct, and cells were selected by puromycin as described above. Thesestably transfected cells were cultured in the presence of hygromycin (50�g/ml), G418 (15 �g/ml), phleomycin (2.5 �g/ml), blasticidin (10 �g/ml), and puromycin (1.0 �g/ml) to maintain the different constructsintegrated into the parasite genome. Cells were then induced with doxy-cycline for expression of MRP2-2� myc as well as TbTim17 double-stranded RNA. T. brucei cells transfected only with pLew100-Bsd-MRP2-2� myc were used as controls. At different time points afterinduction with doxycycline, cells were lysed and the mitochondrial andcytosolic fractions were separated. Equal amounts of proteins from themitochondrial fractions were analyzed by SDS-PAGE and immunoblot-ting to assess the level of MRP2-2� myc in the mitochondria.

Yeast complementation. T. brucei Tim17 was cloned in yeast expres-sion vector pVT-U, which enabled the expression of cloned genes underthe control of the constitutive alcohol dehydrogenase promoter (32). Totest complementation in yeast, plasmids were transformed into the yeaststrains GAL-Tim23, GAL-Tim17, and GAL-Tim22 using a lithium acetatemethod (33, 34). Empty plasmid and plasmids containing ScTim23,ScTim17, and ScTim22 were transformed as controls. The yeast strainsGAL-Tim17, GAL-Tim23, and GAL-Tim22, expressing ScTim17,

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ScTim23, and ScTim22, respectively, under the control of the GAL pro-moter, have been previously described (35). Since these Tim proteins areessential, all of these strains require the presence of galactose (Gal) in thegrowth medium. The ability of T. brucei Tim17 to substitute for ScTim23,ScTim17, or ScTim22 was therefore assessed using media lacking or con-taining 0.5% (wt/vol) Gal. Two independent transformants were analyzedfor each transformation.

SDS-PAGE and Western blot analysis. Proteins from whole cells orisolated mitochondria were separated on an SDS-polyacrylamide gel andimmunoblotted with polyclonal antibodies for the myc epitope (Abcam),TbTim17 (25), T. brucei voltage-dependent anion channel protein(VDAC) (36), T. brucei serine/threonine protein phosphatase 5 (TbPP5)(37), T. brucei mitochondrial RNA-binding protein RBP16 (38), and T.brucei mitochondrial heat shock protein 70 (mHsp70) (39) or monoclo-nal antibodies for trypanosome alternative oxidase (TAO) (40) and T.brucei �-tubulin (41). Blots were developed with appropriate secondaryantibodies and an enhanced chemiluminescence (ECL) kit (Pierce).

Blue native (BN) PAGE analysis. Mitochondrial proteins (100 �g)were solubilized in 50 �l of the ice-cold native buffer (50 mM bis-Tris, pH7.2, 50 mM NaCl, 10% [wt/vol] glycerol, 1 mM PMSF, 1 �g/ml leupeptin,1% digitonin). The solubilized mitochondrial proteins were clarified bycentrifugation at 100,000 � g for 30 min at 4°C. The supernatants weremixed with 2.5 �l of native PAGE G250 sample additive (Invitrogen) andwere electrophoresed on a precast (4 to 16%) bis-Tris polyacrylamide gel(Invitrogen) according to the manufacturer’s protocol. Protein com-plexes were detected by immunoblot analysis. Molecular size marker pro-teins apoferritin dimer (886 kDa), apoferritin monomer (443 kDa),�-amylase (200 kDa), alcohol dehydrogenase (150 kDa), and bovine se-rum albumin (66 kDa) were electrophoresed on the same gel and visual-ized by Coomassie staining.

Coimmunoprecipitation (co-IP). Mitochondrial proteins (200 �g)isolated from T. brucei wild type and those expressing ScTim23-2� myc,

ScTim17-2� myc, and TbTim17-2� myc were solubilized with nativebuffer (200 �l). Solubilized proteins were then subjected to immunopre-cipitation with anti-myc-conjugated Sepharose beads at 4°C overnight.The beads were washed sequentially with the same buffer, and specificallybound proteins were eluted with 2� Laemmli buffer and analyzed bySDS-PAGE and immunoblotting.

RESULTSComparison of primary and secondary structures of TbTim17with its homologs in yeast. TbTim17 possesses a PRAT motifwithin amino acid residues 83 to 113 (Fig. 1A). Multiple-sequencealignment of TbTim17 with ScTim23, ScTim17, and ScTim22 us-ing the Clustal W program (42) revealed that the PRAT motif islocated within the C-terminal half of each of these proteins and isin a largely conserved region (Fig. 1A). In contrast, the N-terminalregions are more divergent. A dendrogram analysis showed thatTbTim17 is closer to ScTim22 and ScTim17 than to ScTim23 (seeFig. S1A in the supplemental material). The primary sequence ofTbTim17 showed 21.8, 29.7, and 31.2% similarity and 7.1, 18, and16.8% identity with the primary sequences of ScTim23, ScTim17,and ScTim22, respectively (see Fig. S1B in the supplemental ma-terial). The secondary structure predicted by the TMPred server(43) indicated that TbTim17 possesses 4 transmembrane domains(TMs; TM1 to TM4), similar to the findings for its homologs inyeast (Fig. 1B). The positions of the TMs and the lengths of theloop regions (L1 to L3) of TbTim17 are more similar to those ofScTim23 and ScTim17 than to those of ScTim22. The N-terminalhydrophilic region of TbTim17 (1 to 29 amino acid residues) ismuch shorter than the corresponding region in ScTim23 andslightly longer than that seen in ScTim17. In contrast, the C-ter-

FIG 1 Primary and the secondary structure analysis of TbTim17 and S. cerevisiae Tim23, Tim17, and Tim22. (A) Multiple-sequence alignment of TbTim17 andthe ScTim23, ScTim17, and ScTim22 proteins. Similar amino acid residues are outlined. Identical residues are indicated by asterisks at the bottom of thesequence. The PRAT domain is shown by a red line on top of the sequence. (B) Schematics of the predicted secondary structures of TbTim17, ScTim23, ScTim17,and ScTim22. Transmembrane domains (TMs) are identified in boxes, and the position numbers of the amino acid (AA) residues within each protein are shown.

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minal hydrophilic region of TbTim17 is relatively longer than thatseen in ScTim23 but shorter than that in ScTim17. Therefore, bybioinformatics analysis alone, it is difficult to assess which of thesethree fungal Tim proteins is functionally closer to TbTim17.

Conditional mutants of ScTim17, ScTim23, and ScTim22cannot be rescued by TbTim17. Next, we evaluated if TbTim17could complement the growth of yeast strains depleted of eitherTim23, Tim17, or Tim22. Since these ScTim proteins are essential,the GAL-Tim17, GAL-Tim23, and GAL-Tim22 yeast strains wereused for the complementation analysis. In these cells the endoge-nous copy of the respective gene was placed under the control ofthe GAL promoter so that yeast cells could survive only in thepresence of galactose. Transformation with an expression vectorcontaining TbTim17 or the empty vector did not allow these cellsto grow in a galactose-free medium (Fig. 2A). However, transfor-mation with the same vector containing the respective genes foryeast Tim proteins clearly rescued the growth in the galactose-freemedium. These results indicate that TbTim17 is unable to func-tionally complement either ScTim23, ScTim17, or ScTim22. Toanalyze the expression of TbTim17 in yeast, the yeast strains de-picted in Fig. 2 were grown in the presence of galactose, and themitochondrial and cytosolic fractions were isolated as describedpreviously (44). Analysis of proteins from these fractions revealedthat TbTim17 was expressed at comparable levels and was prop-erly targeted to the mitochondria in all yeast strains (Fig. 2B). Theyeast mitochondrial escape 1 (Yme1) protein was used as a markerfor the mitochondrial fractions. These results demonstrate thatTbTim17 can be stably expressed in yeast mitochondria; however,it likely cannot properly associate with other components of theTIM23 and TIM22 complexes.

Ectopically expressed ScTim17, ScTim23, and ScTim22 aretargeted to T. brucei mitochondria. To assess if S. cerevisiae

Tim17, Tim23, and Tim22 can be expressed and properly targetedto mitochondria in T. brucei, we transfected the procyclic form ofT. brucei with the C-terminally 2� myc-tagged constructs of theScTim proteins. Analysis of total cellular proteins by Western blotanalysis using an anti-myc antibody showed that the S. cerevisiaeproteins were expressed with the anticipated sizes upon induction

FIG 2 Analysis of S. cerevisiae mutants for complementation of Tim17, Tim23, and Tim22 by TbTim17. (A) Growth phenotype of yeast strains GAL-TIM23,GAL-TIM17, and GAL-TIM22 grown in the absence (� galactose) or presence (� galactose) of galactose. The yeast strains were transformed with an expressionplasmid containing TbTim17. Empty plasmids and plasmids containing wild-type copies of ScTim23, ScTim17, and ScTim22 were transfected into theirrespective yeast cells as the negative and positive controls, respectively. Agar plate schematics show the positions of cells transformed with different plasmidconstructs. Tb-Tim17#1 and Tb-Tim17#2 refer to two independent yeast transformants. (B) The depicted yeast strains were grown in a liquid mediumcontaining galactose. Mitochondrial (lanes M) and cytosolic (lanes C) fractions were isolated and analyzed by SDS-PAGE, followed by Western blotting usingantibodies specific for TbTim17 and Yme1, which was used as a control for the mitochondrial fractions.

FIG 3 Expression and subcellular localization of ScTim17, ScTim23, andScTim22 in T. brucei. (A) The T. brucei procyclic-form cells transfected withthe expression constructs for either ScTim23-2� myc, ScTim17-2� myc, orScTim22-2� myc were grown in the absence (lanes �) and presence (lanes �)of doxycycline (Dox). Cells were harvested at 48 h postinduction, and proteinswere analyzed by immunoblotting using antibodies for the myc epitope.VDAC was used as the loading control. Arrows, positions of ScTim23,ScTim17, and ScTim22 on the respective blots; *, nonspecific interactions withanti-myc antibody. (B) Analysis of subcellular fractions, i.e., the total (lanes T),cytosolic (lanes C), and mitochondrial (lanes M) fractions, from ScTim23-2�myc-, ScTim17-2� myc-, and ScTim22-2� myc-expressing T. brucei by im-munoblot analysis using antibodies for the myc epitope, T. brucei VDAC, andTbPP5. Ten micrograms of protein was loaded per lane.

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with doxycycline (Fig. 3A). We did not observe any changes ingrowth pattern due to expression of these heterologous proteins inT. brucei (see Fig. S2A in the supplemental material). In addition,the expression levels of these ScTim proteins were consistent for atleast 6 days postinduction (see Fig. S2B in the supplemental ma-terial). To determine the subcellular location of the heterologousproteins in T. brucei, we separated the cytosolic and mitochondrialfractions from the cell lysates. Analysis of the fractions revealedthat ScTim23, ScTim17, and ScTim22 were enriched in the mito-chondrial fractions of T. brucei (Fig. 3B). A mitochondrial protein,VDAC, and a cytosolic protein, T. brucei PP5 (TbPP5), were usedas markers for the corresponding fractions.

ScTim17 and ScTim23 are integrated into the mitochondrialinner membrane in T. brucei. Limited protease digestion of theisolated mitochondria was performed to further analyze the sub-mitochondrial location of the heterologous proteins in T. brucei.Analysis of mitochondrial proteins after protease digestion re-vealed that ScTim23 and ScTim17 were protected even aftertreatment with 150 �g/ml of PK (Fig. 4A and B). However, bothScTim23 and ScTim17 were completely digested when mitochon-drial membranes were solubilized by Triton X-100, showing thatthese proteins are not per se resistant to PK. A T. brucei mitochon-drial inner membrane protein (TbTim17), an outer membraneprotein (VDAC), and a matrix protein (mHsp70) were used ascontrols. TbTim17 and mHsp70 were protected from PK diges-tion in all mitochondrial samples, as expected. Although VDAC isan outer membrane protein, this �-barrel protein is mostly em-bedded in the membrane and is thus protected upon PK treat-ment. Furthermore, tubulin is not a mitochondrial protein. It ispresent in the T. brucei mitochondrial preparation as a peripher-

ally associated protein. Due to the unavailability of a suitablemarker protein that is exposed to the cytosolic face of the mito-chondrial outer membrane in T. brucei, we used an antitubulinantibody to test the level of this protein in our samples before andafter PK treatment. We found that the level of tubulin was reducedafter treatment with PK even at 50 �g/ml, indicating that proteasetreatment digested only the peripherally associated outer mem-brane proteins. Taking the findings altogether, we concluded thatScTim23 and ScTim17 are located in the mitochondrial innermembrane in T. brucei. In contrast to ScTim23 and ScTim17,ScTim22 was sensitive to PK even at a very low concentration(such as 25 �g/ml) (Fig. 4C), whereas VDAC, TbTim17, andmHsp70 were protected from PK treatment. Therefore, these re-sults suggest that although ScTim22 is targeted to T. brucei mito-chondria, it is not localized in the mitochondrial inner membrane.

Next, we investigated if ScTim23, ScTim22, and ScTim17 wereintegrated into mitochondrial membranes in T. brucei. Mitochon-dria isolated from T. brucei expressing the 2� myc-tagged copiesof ScTim23, ScTim22, and ScTim17 were extracted with Na2CO3,and the proteins in the soluble and pelleted fractions were ana-lyzed. ScTim23 and ScTim17 were mostly found in the pellet frac-tions, as was the endogenous mitochondrial membrane proteinVDAC (Fig. 4D). RBP16, a soluble matrix protein in T. bruceimitochondria, was found in the soluble fraction, as expected. In-terestingly, ScTim22 was mostly fractionated as a soluble protein,suggesting that ScTim22 failed to be integrated into the mitochon-drial membrane of T. brucei.

ScTim23 and ScTim17 are assembled into protein complexessimilar in size to TbTim17 in T. brucei. To investigate if ScTim23and ScTim17 were assembled in the TbTim17 protein complex in

FIG 4 Intramitochondrial location of ScTim23-2� myc, ScTim17-2� myc, and ScTim22-2� myc in T. brucei. (A to C) Limited protease digestion ofmitochondria (100 �g) isolated from T. brucei expressing ScTim23, ScTim17, or ScTim22. Mitochondria were treated with various concentrations (0 to 150�g/ml) of proteinase K (Prot. K), as described in Materials and Methods. Proteins were analyzed by immunoblotting using anti-myc antibodies. Antibodies forTbTim17, VDAC, mHsp70, and tubulin were used as controls. As indicated, some samples were also treated with Triton X-100 (1%) along with proteinase K. (D)Alkali extraction of mitochondria isolated from T. brucei expressing ScTim23-2� myc, ScTim17-2� myc, or ScTim22-2� myc. Proteins from equal volumes ofthe supernatant (lanes S) and pelleted (lanes P) fractions were analyzed by immunoblotting using anti-myc, anti-RBP16, and anti-VDAC antibodies.

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T. brucei, BN-PAGE analysis was performed. Mitochondria iso-lated from T. brucei expressing ScTim23-2� myc, ScTim17-2�myc, or ScTim22-2� myc were solubilized with 1% digitonin.Mitochondria from the wild-type and the TbTim17-knockdown(KD) T. brucei strains were used in parallel as a positive controland a negative control, respectively. After analysis of the mito-chondrial extract on a BN-polyacrylamide gel, TbTim17 proteincomplexes were detected by immunoblot analysis using anti-TbTim17 antibody. A duplicate blot was probed with anti-mycantibody for detection of potential ScTim23 and ScTim17 proteincomplexes. Anti-TbTim17 antibody detected two protein com-plexes from mitochondrial extracts obtained from the T. bruceiwild type within a molecular size range of from 300 to 1,100 kDa(Fig. 5A). The lower protein band had a relatively broader molec-ular size (300 to 400 kDa) than the larger complex (�1,100 kDa).Similar complexes were also observed in the mitochondrial ex-tracts from those cells expressing ScTim23-2� myc, ScTim17-2�myc, and ScTim22-2� myc. Furthermore, both of these proteincomplexes were significantly reduced in TbTim17-KD mitochon-dria, indicating the presence of TbTim17 in these complexes. Wereported previously that TbTim17 is present in a megadalton-sizecomplex (28). Using a higher concentration of the anti-TbTim17antibody and a standardized precast gel, we have now identified 2distinct complexes for TbTim17. The anti-myc antibody specifi-cally detected an �300-kDa protein complex in the mitochondrialextract obtained from T. brucei expressing ScTim23-2� myc andScTim17-2� myc but not from the wild-type mitochondria ormitochondria from which TbTim17 was depleted (Fig. 5A), indi-cating that these heterologous proteins are assembled in this com-plex, which is similar in size to that of one of the complexes formedby TbTim17. Interestingly, this complex was not detected by theanti-myc antibody in the mitochondrial extract obtained from T.brucei expressing ScTim22-2� myc, which supports our previousobservation that although ScTim22 is targeted to mitochondria, itis not integrated into the mitochondrial membrane and it is notassembled into a membrane complex similar to ScTim23 and Sc-Tim17. The anti-myc antibody did show some cross-reactivitywith other protein complexes (�600 kDa and larger). Since thesecomplexes were also detected at similar intensities in wild-typemitochondria using the anti-myc antibody, we did not considerthem to be specific complexes formed by ScTim proteins. Al-though the intensity of these nonspecifically interacting proteinbands was much weaker in TbTim17-KD mitochondrial samples,this could be due to a decrease in the nonspecifically interactingproteins in these mitochondria. In contrast, VDAC protein com-plexes were detected in all samples showing equal loading.

To further analyze the possible association of ScTim23, ScTim17,and ScTim22 with TbTim17 in T. brucei, co-IP experiments wereperformed. Anti-myc antibody pulled down ScTim23, ScTim17,and ScTim22 from the respective mitochondrial extracts (Fig. 5B).This antibody did not detect any protein in the immunoprecipi-tate from wild-type mitochondrial extracts. Probing of this blotwith anti-TbTim17 antibody showed that TbTim17 was coimmu-noprecipitated with ScTim17 but not with ScTim23 from the re-spective mitochondrial extract. We found that the TbTim17 levelin the input lane for the wild-type control was comparable to thatin other samples except for those containing ScTim23-myc mito-chondria. We observed that the TbTim17 level was upregulated inthese cells. Although the reason for this upregulation is not clear atpresent, we speculate that expression of ScTim23 is inhibitory

for TbTim17 function and, to compensate for this effect, theTbTim17 levels were increased. In spite of the presence of a largeamount of TbTim17 in the mitochondrial extract, this protein wasnot found in the immunoprecipitate, showing that ScTim23 is notstably associated with TbTim17. On the other hand, a large pro-portion of TbTim17 was found to be associated with ScTim17,indicating that these two proteins interact in a stable manner in T.brucei. A trace amount of TbTim17 was found in the immunopre-cipitate from the wild-type and ScTim22 mitochondrial extracts;however, this could have been due to a nonspecific interaction ofthis protein with the agarose beads or the anti-myc antibody. Wealso used mitochondria from T. brucei expressing TbTim17-2�

FIG 5 Analysis of T. brucei mitochondrial membrane protein complexes andcoimmunoprecipitation analysis. (A) BN-PAGE analysis of the mitochondrialprotein complexes. Mitochondria were isolated from T. brucei cells at 4 dayspostinduction. Samples of mitochondria (100 �g) from T. brucei cells express-ing ScTim23-2� myc, ScTim17-2� myc, and ScTim22-2� myc proteins aswell as from the wild-type (Wt) and the TbTim17-KD T. brucei strains weresolubilized with digitonin (1.0%). The solubilized supernatants were clarifiedby centrifugation at 100,000 � g and analyzed by BN-PAGE. Protein com-plexes were detected by immunoblot analysis using antibodies to TbTim17,the myc epitope, and VDAC. Molecular size marker proteins apoferritin dimer(886 kDa), apoferritin monomer (443 kDa), �-amylase (200 kDa), alcoholdehydrogenase (150 kDa), and bovine serum albumin (66 kDa) were run onthe gel and visualized by Coomassie blue staining. (B) Coimmunoprecipita-tion by anti-myc antibodies. Mitochondrial proteins (200 �g) were solubilizedwith digitonin (1.0%) at a protein concentration of 1 mg/ml. The mitochon-drial lysates from the T. brucei wild type and cells expressing ScTim23-2� myc,ScTim17-2� myc, ScTim22-2� myc, and TbTim17-2� myc were subjectedto immunoprecipitation with anti-myc-Sepharose beads. Ten percent of thetotal soluble protein input (lanes In) and 50% of the immunoprecipitated(lanes ppt) proteins were analyzed by SDS-PAGE and immunoblotted withanti-myc, anti-TbTim17, and anti-TAO antibodies.

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myc as the positive control. Anti-myc antibody pulled down theendogenous TbTim17 along with the ectopically expressedTbTim17-2� myc, showing that they were associated in vivo. Ananti-TAO antibody was used as a negative control; no band wasdetected from the samples immunoprecipitated with this anti-body, indicating the specificity of the interactions (Fig. 5B). Theseresults show that ScTim17 is associated with TbTim17 in vivo.Although ScTim23 is present in protein complexes similar to Sc-Tim17 and TbTim17 on BN-polyacrylamide gels, it is apparentlymore weakly associated with TbTim17 than it is with ScTim17.

ScTim17 partially complements the growth defect caused byTbTim17 RNAi. To assess if S. cerevisiae Tim17, Tim23, andTim22 could complement the growth defect in T. brucei causedby TbTim17 RNAi, we induced cells of the doubly transfectedcell lines ScTim17/TbTim17 KD, ScTim23/TbTim17 KD, andScTim22/TbTim17 KD using doxycycline. The growth of cells ofeach cell line in the presence and absence of doxycycline was com-pared with that of TbTim17-KD cells under the same conditions.Induction of TbTim17 KD inhibited cell growth after 4 days (Fig.6A), as previously shown (25, 28). Doxycycline treatment alsodrastically reduced the growth of the ScTim23/TbTim17-KD cells,and cell growth ceased after 4 days (Fig. 6B). Interestingly, whenScTim17 was expressed in TbTim17-KD cells, we found that cellgrowth was not fully inhibited even within 8 days, although thegrowth rate was slightly lower over the entire period of analysis(Fig. 6C). These results show that ScTim17 at least partially com-plemented the growth defect caused by TbTim17 KD. Comparingthe growth of ScTim22/TbTim17-KD cells in the presence andabsence of doxycycline revealed that expression of ScTim22 failedto restore the growth of T. brucei, when TbTim17 was reduced byRNAi (Fig. 6D). Induced cells grew at a lower rate from the begin-ning, and growth ceased after day 6. These results suggest thatneither the yeast Tim23 nor the yeast Tim22 is capable of comple-menting the TbTim17 function in T. brucei. Immunoblot analysisof proteins from isolated mitochondria at day 4 after inductionwith doxycycline showed that ScTim proteins were present in themitochondria, while the endogenous levels of TbTim17 were re-duced, as expected (Fig. 6E). However, the levels of ScTim pro-teins were not equivalent. Interestingly, the level of ScTim17 wassignificantly lower than that of ScTim23 and ScTim22 inTbTim17-KD cells. As mentioned earlier, a similar observationwas not seen when we expressed ScTim proteins in the parental T.brucei line (Fig. 3A; see also Fig. S2B in the supplemental mate-rial). Therefore, this indicates that TbTim17 is possibly requiredfor the import/stability of ScTim17 in T. brucei. In spite of thelower level of ScTim17 seen in mitochondria, expression of thisprotein showed growth complementation for TbTim17 KD.These results thus support the notion that TbTim17 is function-ally most similar to ScTim17.

TbTim17 expression fully complements the growth defectcaused by TbTim17 RNAi. To assess if expression of TbTim17 canreverse the growth defect in T. brucei by TbTim17 RNAi, we de-veloped an RNAi cell line in which the double-stranded RNA forthe 3=UTR of the TbTim17 transcript was produced and targetedonly the endogenous copy. Thus, the ectopically expressed copy ofTbTim17 was unharmed. For this purpose, we first establishedthat this 3= UTR RNAi could effectively knock down TbTim17.We determined that the TbTim17 protein level was reduced 60 to70% within 6 days due to induction of RNAi targeted to the 3=UTR of the TbTim17 transcript (see Fig. S3A and B in the supple-

mental material). In comparison to the findings for the RNAi cellline that was previously generated by targeting the coding regionof this transcript, we found that this new cell line was relatively lessrobust (see Fig. S3A and B in the supplemental material). Thiscould be because the 3=UTR of the TbTim17 transcript could havefolded secondary structures, which may inhibit the annealing ofsome interfering RNAs.

Analysis of the growth kinetics clearly showed that doxycyclinesignificantly reduced cell growth upon induction of the expressionof the double-stranded RNA (Fig. 7A). Next, we monitored thegrowth kinetics of cells of the T. brucei cell line that expressed awild-type copy of TbTim17, in addition to the double-strandedRNA for the 3= UTR of the TbTim17 transcript. The resultsshowed that upon induction with doxycycline, the cell growthdefect caused by TbTim17 KD (by the use of RNAi targeted to the3= UTR) was fully complemented by the expression of wild-typeTbTim17 (Fig. 7B).

To investigate if the ectopic copy of TbTim17 was expressed inT. brucei, cells were harvested at 48 h postinduction and proteins

FIG 6 Growth phenotype of TbTim17-KD cells transfected with expressionconstructs for ScTim23, ScTim17, or ScTim22. TbTim17-KD cells (A) andTbTim17-KD cells transfected with ScTim23 (B), ScTim17 (C), or ScTim22(D) expression constructs were allowed to grow in the absence (uninduced)and presence (induced) of doxycycline. Cell numbers were counted at differ-ent time points after induction with doxycycline. The log cumulative cell num-bers were plotted versus the times postinduction (in days). The results arerepresentative of those from three independent experiments for each cell type.(E) Levels of ScTim23, ScTim22, and ScTim17 expression in TbTim17-KDcells. Mitochondria were isolated from T. brucei parental (wild-type), Sc-Tim23-myc/TbTim17-KD, ScTim22-myc/TbTim17-KD, and ScTim17-myc/TbTim17-KD cells at day 4 after induction with doxycycline. Mitochondrialproteins (100 and 50 �g) were analyzed by immunoblotting using antibodiesfor the myc epitope, TbTim17, and VDAC as probes.

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were analyzed by SDS-PAGE and Western blotting. The parentalT. brucei strain (wild type) was grown under the same conditionsand used as a control. As expected, there was no change inTbTim17 protein levels in wild-type cells that were either treatedor untreated with doxycycline (Fig. 7C). However, two distinctprotein bands were seen in TbTim17/TbTim17-KD cells. Thelower band (�19 kDa) was the endogenous TbTim17, which wasreduced after induction of RNAi with doxycycline (Fig. 7C). Theupper band, which is the ectopically expressed TbTim17 with amyc epitope tag at the C terminus, was expressed at the same levelin cells grown in the presence or absence of doxycycline. As ex-pected, the TbTim17 protein level was reduced in TbTim17-KDcells grown in the presence of doxycycline.

To confirm the localization of the ectopically expressedTbTim17 in mitochondria, a subcellular fractionation analysiswas performed after harvesting TbTim17/TbTim17-KD cellsgrown in the presence of doxycycline for 48 h (Fig. 7D). T. brucei

VDAC and TbPP5 were used as mitochondrial and cytosolic markerproteins, respectively. The ectopically expressed TbTim17-myc wasfound exclusively in the mitochondrial fraction, similar to the find-ings for endogenous TbTim17. Together, these results demon-strate that RNAi targeted to the 3= UTR of the TbTim17 tran-script did not affect the ectopic expression of TbTim17-myc. Inaddition, expression of TbTim17-myc was capable of rescuingcells after TbTim17 KD.

ScTim17 partially complements the defect in mitochondrialprotein import caused by depletion of TbTim17. Next, we per-formed mitochondrial protein import assays to assess if ScTimproteins were functional in T. brucei. For this purpose, we ex-pressed ScTim23, ScTim17, and ScTim22 individually in cells ofthe MRP2-2� myc/TbTim17-KD cell line. In these cells, bothMRP2-2� myc and the double-stranded TbTim17 transcript wereexpressed after induction with doxycycline. At different timepoints (24 to 80 h) during induction, the level of MRP2-2� mycin mitochondria was assessed by immunoblot analysis. TheMRP2-2� myc protein was expressed in cells of the MRP2-2�myc-transfected T. brucei cell line and was found in the mitochon-drial fraction at all time points (Fig. 8A). However, in doublytransfected cells where both TbTim17 double-stranded RNA andMRP2-2� myc were induced simultaneously by doxycycline, wefound that the level of this protein was drastically reduced after 48h (Fig. 8B), showing that reduction of the TbTim17 level by RNAihampered the import of this nuclear DNA-encoded mitochon-drial protein. Next, we investigated whether the expression of anyof ScTim proteins aided the import of MRP2-2� myc into mito-chondria when TbTim17 was depleted by RNAi. The expression ofScTim23 (Fig. 8C) and ScTim22 (Fig. 8E) failed to help regain thelevel of MRP2-2� myc in mitochondria. However, expression ofScTim17 partially restored this level in mitochondria whenTbTim17 was depleted (Fig. 8D). To assess the relative levels ofMRP2 in different mitochondrial samples, the intensity of theMRP2 protein band was quantitated for each sample at 72 hpostinduction, its intensity was normalized to the intensity of thecorresponding tubulin band, and the results were plotted aftersetting the level of MRP2 in singly transfected T. brucei cells (T.brucei 29-13 MRP2-2� myc) equal to 100 (Fig. 8F). The resultsshowed that the reduction of MRP2 levels in both ScTim23/TbTim17-KD and ScTim22/TbTim17-KD cells (50 to 60%) wassimilar to that in cells with TbTim17 KD. However, in ScTim17/TbTim17-KD mitochondria the level of MRP2 was retained at alevel �80% of that for the controls. Together, these results showthat TbTim17 is functionally closer to ScTim17 than ScTim23.Due to improper sorting, ScTim22 was also not functional in T.brucei.

DISCUSSION

We analyzed the functional similarity of TbTim17 with its ho-mologs in S. cerevisiae by complementation analyses. Our resultsshow that, despite the similarities in their primary and secondarystructures, TbTim17 is functionally divergent from its yeast ho-mologs. TbTim17 could not complement the function of any ofthe three yeast PRAT family members, ScTim17, ScTim23, andScTim22. Although ScTim23 was properly integrated and assem-bled into protein complexes like endogenous TbTim17 was, it wasnonfunctional in T. brucei. Only ScTim17 partly complementedthe deficit of TbTim17 in T. brucei. Although ScTim22 was ex-pressed and targeted to T. brucei mitochondria, it was not inte-

FIG 7 Ectopically expressed TbTim17 complements the RNAi growth defect.(A and B) TbTim17-KD (in which TbTim17 was knocked down by the use ofRNAi targeted to the 3= UTR) (A) and TbTim17/TbTim17-KD (B) cells weregrown in the presence of doxycycline (induced) or left uninduced. Cell num-bers were counted at different time points of growth, and the log cumula-tive cell numbers were plotted versus the times postinduction (in days).Data represent the log of the mean values from three independent experi-ments. (C) TbTim17 protein levels in T. brucei 427 29-13 (wild-type) cells,cells with TbTim17 RNAi transfected with a wild-type copy of TbTim17(TbTim17/TbTim17 KD), and TbTim17-KD cells grown in the absence (�)and presence of (�) doxycycline (Dox). The anti-TbTim17 antibodies wereused to detect protein in the immunoblot. TbTim17-myc and endogenousTbTim17 are indicated. Tubulin was used as the loading control. (D) Analysisof the total (lanes T), cytosolic (lanes C), and mitochondrial (lanes M) frac-tions from TbTim17-KD/TbTim17 cells by immunoblot analysis using anti-TbTim17, anti-TbPP5 and anti-VDAC antibodies. Ten micrograms of proteinwas loaded per lane.

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grated into the mitochondrial inner membrane, explaining its in-ability to support the function of TbTim17. Taken together, thesedata suggest that TbTim17 is the closest to ScTim17.

In yeast, the TIM23 complex contains at least 11 different sub-units which coordinate the recognition, translocation, and inser-tion of presequence-containing precursor proteins. ScTim17forms a stable subcomplex with ScTim23 and also seems to di-rectly interact with Tim14 and Pam17 (5, 6). In T. brucei, we foundthat TbTim17 interacts with TbTim50 (27) and with a few othertrypanosome-specific proteins (28). Together they possibly formthe core translocase of �300 kDa, as observed by BN-PAGE anal-ysis. The core complex likely further associates with other factorsto form a larger complex of 1,100 kDa. Data presented here showthat ScTim17 can associate with TbTim17 in T. brucei and mostlikely assembles in the core complex. Thus, it could at least partlycomplement the downregulation of TbTim17. ScTim23, on theother hand, possesses a longer hydrophilic region at its N termi-nus, which may hamper its stable association with TbTim17and/or other components of the 300-kDa TbTim17 protein com-plex. Further analysis with an N-terminally truncated version ofScTim23 may prove this hypothesis.

Tim23 and Tim17 in yeast are imported into the mitochondrialinner membrane via internal targeting signals located within thetransmembrane domains of these proteins (44, 45). ScTim23 andScTim17 were shown to be recognized by the Tom70 receptor,located on the cytosolic face of the outer mitochondrial mem-brane (44). Then these proteins cross the outer mitochondrialmembrane through the Tom40 channel and are finally insertedinto the inner membrane via the TIM22 complex with the assis-tance of small Tim chaperones in the inter membrane space (IMS)(44). A homolog of Tom70 has not been identified in T. brucei.Despite these deficiencies, the T. brucei mitochondrion is capable

of recognizing the targeting signals of ScTim17 and ScTim23. Thisis because TbTim17 most likely possesses an internal targetingsignal similar to the targeting signals of ScTim17 and ScTim23.Besides, we have recently characterized a presequence-like inter-nal targeting signal in the mitochondrial protein TAO (46).Therefore, T. brucei must possess unidentified receptors for theimport of such proteins into mitochondria.

ScTim22, however, takes a slightly different route to reach itsdestination in yeast mitochondria. Although the targeting signalfor ScTim22 has not been well characterized, it has been shownthat this protein requires the Tom20 receptor, and after translo-cation through the Tom40 channel, it is also inserted into themitochondrial inner membrane via the TIM22 complex (47, 48).Recent reports show that the import and assembly of Tim22 infungi depend on Tim18, a component of the TIM22 translocase,as well as Mia40, an IMS protein (48, 49). Oxidation of a pair ofintramolecular sulfhydryl groups in Tim22 is critical for the im-port and assembly of this protein into the mitochondrial innermembrane (48, 49). Trypanosomatids do not have a homolog ofeither Tim18 or Mia40, which could potentially explain whyScTim22 was unable to assemble into the T. brucei mitochondrialinner membrane.

T. brucei also lacks a protein complex similar to TIM22, whichin fungi is essential for insertion of Tim23, Tim17, and Tim22.However, the parasite is fully capable of inserting heterologousproteins (Tim23 and Tim17) as well as TbTim17 and many otherendogenous internal signal-containing proteins. It can be specu-lated that TbTim17 plays a role in the import of these proteins.Experiments are needed to establish the role of TbTim17 in theimport of MCPs and other internal signal-containing proteins likeTbTim17 itself.

Like trypanosomatids, some microsporidian species also pos-

FIG 8 Effect of expression of ScTim17, ScTim23, and ScTim22 on mitochondrial localization of MRP2 in TbTim17-knockdown cells. T. brucei cells containingthe double constructs MRP2-2� myc and TbTim17 KD were further transfected with the expression cassettes for either ScTim23, ScTim17, or ScTim22, andstable cell lines were generated. T. brucei cells singly transfected with MRP2-2� myc (A) and doubly transfected with the MRP2-2� myc and TbTim17-KDconstructs (B), as well as cells triply transfected with MRP2-2� myc/ScTim23/TbTim17 KD (C), MRP2-2� myc/ScTim17/TbTim17 KD (D), and MRP2-2�myc/ScTim22/TbTim17 KD (E), were induced with doxycycline. Cells were harvested at different time points (24 to 80 h) after induction. Proteins from themitochondrial fractions were analyzed by SDS-PAGE and immunoblotting using anti-myc, -VDAC, and -tubulin antibodies as probes. (F) The intensities of theMRP2-2� myc protein bands from different samples were measured at 72 h postinduction and normalized to the intensity of the corresponding tubulin band.The percent reduction of the MRP2 protein levels was quantitated after setting the values for wild-type (Wt) cells equal to 100. Bars indicate standard errors,which were calculated from three independent experiments.

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sess a single PRAT protein (50). Microsporidians possess mito-somes, the relict form of mitochondria, and have been classified inthe group of fungi (50, 51). Mitosomes are double-membrane-bound organelles which do not have any electron transport systemor an organelle genome. However, mitosomes possess an Fe-Scluster assembly system, which is the only conserved essentialfunction among all mitochondria and mitochondrion-like organ-elles. Mitosomes import proteins from the cytosol, and many ofthese proteins possess an N-terminal targeting signal with charac-teristics similar to those of the N-terminal targeting signals ofmitochondrial proteins. Thus, it is likely that the microsporidianPRAT protein will also be more similar to Tim17 than to Tim23 infungi. Parasitic protozoa, such as Giardia and Entamoeba, alsopossess mitosomes (52, 53). Surprisingly, no member of the PRATfamily of proteins has been identified in the genomes of theseprotozoa (52, 53). Therefore, it is not clear at this time how mito-somal matrix proteins cross the inner membrane in this group ofparasites.

In contrast to these organisms, trypanosomatids possess afully developed mitochondrion with an electron transport sys-tem and a mitochondrial membrane potential. Moreover, theirmitochondrial proteins seem to be targeted to the organelle byeither N-terminal or internal signals. Still, a single protein,TbTim17, appears to manage the import of both these classes ofproteins. This likely explains the divergent nature of this T.brucei protein from its yeast counterparts. It is not yet clear howTbTim17 performs the overall protein import process. Functionalcharacterization of the T. brucei homologs of small Tim proteins,TbTim17-associated trypanosome-specific Tim proteins, mito-chondrial Hsp70, and TbTim17 together is necessary to under-stand this important cellular process in a divergent eukaryotelike T. brucei. In any case, our studies show that TbTim17 isstructurally and functionally closer to Tim17 in fungi, suggestingthat Tim17 is an ancient member of this translocator proteinfamily.

ACKNOWLEDGMENTS

We thank George Cross for the pLew100 vectors and the T. brucei 42729-13 cell line, Jason Caenes for the pHD1344 vector, Laurie Read for theRBP16 antibodies, and Paul Englund for mHsp70 antibodies.

This work was supported by grant 2SC1GM081146 from the NationalInstitutes of Health to M.C. and by a grant from the Deutsche Forschungs-gemeinschaft to D.M. E.W., J.T.S., and V.H. were supported by traininggrants T32HL007737, T32AI007281, and 2R25GM059994. The Molecu-lar Biology Core Facility at Meharry Medical College is supported by NIHgrant U54RR026140/U54MD007593.

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