Trypanosoma brucei Cytochrome c1 Is Imported into Mitochondria Along an Unusual Pathway*

Jeffrey W. PriestDagger and Stephen L. Hajduk§

From the Departments of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received for publication, December 19, 2002, and in revised form, February 3, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In most eukaryotic organisms, cytochrome c1 is encoded in the nucleus, translated on cytosolic ribosomes, and directed to its final destination in the mitochondrial inner membrane by a bipartite, cleaved, amino-terminal presequence. However, in the kinetoplastids and euglenoids, the cytochrome c1 protein has been shown to lack a cleaved presequence; a single methionine is removed from the amino terminus upon maturation, and the sequence upstream of the heme-binding site is generally shorter than that of the other eukaryotic homologs. We have used a newly developed mitochondrial protein import assay system from Trypanosoma brucei to demonstrate that the T. brucei cytochrome c1 protein is imported along a non-conservative pathway similar to that described for the inner membrane carrier proteins of other organisms. This pathway requires external ATP and an external protein receptor but is not absolutely dependent on a membrane potential or on ATP hydrolysis in the mitochondrial matrix. We propose the cytochrome c1 import in T. brucei is a two-step process first involving a membrane potential independent translocation across the outer mitochondrial membrane followed by heme attachment and a membrane potential-dependent insertion into the inner membrane.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Most of the mitochondrial proteins are encoded within the nucleus, synthesized on cytosolic ribosomes, and directed to their final mitochondrial destination by cleavable amino-terminal presequences (reviewed in Refs. 1 and 2). In many cases, mitochondrial protein import is receptor-mediated, is dependent upon an inner mitochondrial membrane potential, and requires the hydrolysis of ATP both in the cytosol and in the mitochondrial matrix. Constellations of proteins in the inner and outer mitochondrial membranes and in the intermembrane space (IMS)1 have been implicated in the recognition, transfer, and integration of proteins from the cytosol (reviewed in Ref. 3). Many of the imported mitochondrial proteins (notably the Rieske iron-sulfur protein (ISP) subunit of the cytochrome c reductase complex) have a bipartite, amino-terminal presequence that directs the proteins along a conservative sorting pathway; the first part of the presequence directs the protein into the mitochondrial matrix, and the second part of the signal causes the protein to be sorted into or through the inner mitochondrial membrane to its final destination (4).

The cytochrome c1 subunit of the cytochrome c reductase complex of most eukaryotic organisms is synthesized as a preprotein with a long (60-80 amino acids) amino-terminal extension that has been shown to direct the import of the protein into the mitochondrion in the presence of a membrane potential (5-8). Although the cytochrome c1 presequence does possess the characteristics of a typical conservative sorting signal and is processed in two distinct steps on opposite sides of the inner mitochondrial membrane, the precise pathway used for the import of this protein has remained controversial (9, 10). In contrast to the typical conservatively sorted preprotein, the cytochrome c1 preprotein does not appear to require the presence of matrix ATP for import and sorting (11-13). To explain this observation, some researchers (14) have suggested that the preprotein may bypass the ATP-requiring matrix chaperones by "looping" through the matrix on its way to the inner membrane along the conservative sorting pathway. Others (15-17) have suggested that a stretch of hydrophobic residues located within the amino-terminal presequence may act to arrest the transfer of the preprotein within the inner mitochondrial membrane (stop-transfer pathway), thus preventing the mature part of the protein from having any contact with the matrix. Regardless of the exact pathway, there is general agreement that cytochrome c1 protein import requires a membrane potential and external ATP (but not matrix ATP) and that the preprotein is processed first by the matrix processing protease and then by the Imp2 protease on the outer surface of the inner mitochondrial membrane (9, 12, 18-21). In addition, cytochrome c1 heme lyase, an enzymatic activity that is localized in the IMS, is required to form two covalent thioester bonds between the vinyl groups of heme b and the cysteines in the CXXCH motif of the apoprotein (9, 22) before the protein can be assembled into the reductase complex. Recent work has shown that the carboxyl-terminal region of the yeast cytochrome c1 protein contains a previously unrecognized targeting signal that acts in a membrane potential-dependent fashion to insert the alpha -helical membrane anchor of the protein into the inner membrane so that the mature protein has the proper Nout-Cin orientation (23). Apparently, both the amino- and the carboxyl-terminal targeting sequences are required by the yeast system for proper membrane insertion and orientation because the carboxyl-terminal signal, by itself, could direct the import to the intermembrane space of only a limited fragment of the yeast cytochrome c1 protein.

Our previous analysis of the kinetoplastid c1 cytochromes has shown that an essential element that is required by both the stop-transfer pathway and the conservative sorting pathway models, namely a cleaved amino-terminal presequence, is totally absent; a single methionine is removed from the amino terminus of the kinetoplastid c1 cytochromes to form the mature protein (24). The absence of a cleaved presequence on these c1 cytochromes suggested that they might be imported into the mitochondrion along an alternative type of pathway that bypasses the mitochondrial matrix altogether.

In Neurospora crassa, several types of proteins are imported into the mitochondrion in the absence of a cleavable presequence and along alternative pathways. The cytochrome c import pathway into the IMS does not utilize a receptor nor does it require a membrane potential or the hydrolysis of ATP (reviewed in Ref. 25). Import, which has been shown to require the presence of heme, a source of reducing equivalents, and a functional cytochrome c heme lyase, is thought to be driven by a conformational change that results from holoprotein formation (26-28). Cytochrome c heme lyase is itself imported into the IMS in the absence of a cleaved presequence. Import requires a membrane receptor but is not dependent upon either ATP hydrolysis or a membrane potential (29, 30). The enzyme is thought to be associated with the mitochondrial membrane. The ATP/ADP carrier protein (AAC) and other inner membrane metabolite carrier molecules are imported along a receptor-mediated pathway to the outer surface of the inner mitochondrial membrane in the absence of a membrane potential and without matrix ATP hydrolysis (31-35). External ATP is required to maintain import competence (36). However, in contrast to the other pathways, a membrane potential is required for the assembly of the AAC dimer and insertion of the functional complex into the inner membrane (33, 34). Because the kinetoplastid cytochrome c1 must be inserted into the inner membrane, the AAC-type pathway seemed the most likely candidate of the alternative pathways that have been described so far.

In this work we demonstrate that the Trypanosoma brucei cytochrome c1 is indeed imported along an alternative pathway. The import, membrane insertion, and proper orientation of the mature protein in the membrane are likely directed by a carboxyl-terminal signal sequence similar to the one described in the yeast system. Given that the T. brucei Rieske ISP subunit of the cytochrome c reductase complex is processed differently in these organisms than in other eukaryotes (37), we suggest that the assembly of the reductase complex may be different in the trypanosomes.

    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Construction of Clones, Transcription, and Translation-- A PCR was used to generate a coding sequence clone of the T. brucei cytochrome c1 using a TaqI genomic clone of the gene as target.2 Deoxyoligonucleotides XD-1 (5'-ATGGCAGGTAAGAAAGCTCAC-3') and OR-21 (5'-CTCTCGCTCCACATCATACAGGC-3') were used for hot start PCR with AmpliTaq DNA polymerase as directed by the manufacturer (PerkinElmer Life Sciences). The resulting 816-bp product (GenBankTM accession number AF102980; Fig. 1A) was cloned into the SmaI restriction site of Bluescript SK+ plasmid (Stratagene). Cleavage of the clone with BamHI generated a template for RNA synthesis with T7 RNA polymerase.

A cDNA clone (pPNE-6) of the mitochondrial matrix HSP70 from Crithidia fasciculata in Bluescript SK- (GenBankTM accession number M95682) was kindly provided by Dr. Paul Englund (The Johns Hopkins University, Baltimore) (38). Because the 1944-bp coding sequence did not transcribe and translate efficiently in vitro, the clone was shortened by excision of an EcoRV fragment from the 3' end. The open reading frame of the resulting clone included the first 678 bp of the HSP70 coding sequence and terminated at an in-frame stop codon 54 bp downstream of the EcoRV restriction site in the Bluescript plasmid sequence. Thus the 26-kDa truncated HSP70 translation product (HSP70Delta RV) contained the 20-amino acid, cleaved, amino-terminal presequence, 206 amino acids of the mature HSP70 sequence, and 18 amino acids of unrelated sequence from the plasmid. The clone was cleaved with HinfI to generate a template for RNA synthesis with T3 RNA polymerase. The clone for the T. brucei Rieske ISP of the cytochrome bc1 complex was cleaved and transcribed as described previously (37).

5'-7-Methyl-GpppG capped mRNA was synthesized from the plasmid templates using either the mMessage mMachine in vitro transcription kit (Ambion) or the method described previously (37). If the latter method was used, the cap was often deleted from the cytochrome c1 reaction because this was not found to be necessary for efficient translation. In vitro translations were performed using a rabbit reticulocyte translation system (Promega) with [35S]methionine (PerkinElmer Life Sciences) as described previously (37).

Standard Mitochondrial Protein Import and Analysis-- T. brucei (TREU 667) procyclic cells were grown in shaking cultures, collected by centrifugation, and lysed in a nitrogen cavitation bomb as described previously (37). A crude mitochondrial fraction was isolated by differential centrifugation and stored in 50% glycerol storage buffer (37). A modified lysis, storage, and dilution buffer (0.25 M sucrose, 20 mM MOPS/KOH (MOPS/KOH, MOPS buffer adjusted to pH 7.2 with KOH), pH 7.2, and 2 mM EDTA) (SME-20) was used to improve the reproducibility of the import reactions (39). Protein import reactions were initiated by the addition of 5 µl of rabbit reticulocyte translation mixture to 20 µl of mitochondria (about 100 µg of protein from 1.5 × 108 cells) in basal import buffer composed of 0.25 M sucrose, 20 mM MOPS/KOH, pH 7.2, 80 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol, and 1 mg/ml fatty acid-free bovine serum albumin (final concentrations). The "complete" import buffer used in some reactions was made by supplementing basal import with an ATP-regenerating system (10 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase, and 2 mM ATP) and an energizing mixture (8 mM potassium ascorbate, 0.2 mM N,N,N',N'-tetramethylphenylenediamine, and 5 mM NADH). Reactions were terminated by transfer to 4 °C, and the mitochondria were collected by centrifugation at 14,000 × g for 10 min at 4 °C. Unless otherwise indicated, pellets were resuspended in 50 µl of SME-20 buffer containing 30 µg/ml proteinase K (Roche Molecular Biochemicals) and digested at room temperature for 15 min. Antimycin A (15 µM) and oligomycin (30 µM) or carbonylcyanide m-chlorophenylhydrazone (CCCP) (50 µM) and valinomycin (0.5 µM) were included in the indicated reactions to dissipate the membrane potential and thereby decrease the possibility of continued import during the protease digestion step. Following the addition of 1 volume of ice-cold SME-20 containing 4 mM phenylmethylsulfonyl fluoride (PMSF), the mitochondria were again collected by centrifugation. Labeled proteins were resolved on 12% SDS-polyacrylamide gels, visualized by fluorography, and quantitated by storage phosphor autoradiography (Amersham Biosciences model 400E PhosphorImager) as described previously (37). The analysis of imported proteins in the presence of proteinase K and the detergent CHAPS was performed by acetone precipitation as described previously (37).

Import Followed by Sucrose Gradient and Western Blot Analysis-- Scaled up import reactions (500-µl final volume containing 100 µl of reticulocyte lysate and the mitochondria from ~3.0 × 109 cells) were performed for ISP, cytochrome c1, and cytochrome c1 in the presence of 100 µM CCCP using basal import buffer supplemented with the ATP-regenerating system described above. Following a 30-min incubation at room temperature, the mitochondria were collected by centrifugation and resuspended in 500 µl of SME-20 buffer containing 30 µg/ml of proteinase K, 30 µM oligomycin, and 15 µM antimycin A. After a 15-min digestion at room temperature, 500 µl of SME-20 buffer containing 4 mM PMSF were added, and the mitochondria were again collected by centrifugation. The mitochondria from each import reaction were resuspended in 500 µl of SME-20 containing 1 mM PMSF and loaded onto the top of linear sucrose gradients as described by Opperdoes et al. (40) (1.18-2.0 M sucrose with 25 mM Tris, pH 7.4, and 1 mM EDTA on top of a cushion of 0.8 ml of 2.5 M sucrose in the same buffer). The gradients were centrifuged for 17 h at 30,000 rpm (60,000 × g at the sample zone) at 6 °C in a SW41Ti rotor (Beckman Instruments). Gradients were fractionated (12 times, 1 ml) from the bottom of the centrifuge tubes using a pump and a thin capillary tube to minimize mixing. The fractions were numbered in reverse order so that fraction 1 represented the top (least dense) fraction of the gradient. Each sucrose gradient fraction was slowly diluted with 9 ml of 20 mM MOPS, pH 7.2, with gentle vortexing and then centrifuged for 15 min at 15,700 × g at 4 °C in a swinging bucket rotor (10,000 rpm; Beckman Instruments JS 13.1 rotor). Mitochondrial pellets were dissolved in 50 µl of SDS sample loading buffer with 2 mM PMSF.

The proteins from each fraction (20 µl/lane) were resolved on 12% polyacrylamide minigels and electrotransfered onto polyvinylidene difluoride (PVDF) membranes (Immobilon P, Millipore). The blots were probed with a rabbit polyclonal antibody raised against the T. brucei cytochrome c1.3 The rabbit serum was diluted 1:200 in phosphate-buffered saline (0.85% NaCl and 10 mM Na2HPO4, pH 7.2) (PBS) containing 0.3% Tween 20 (Tween/PBS). Bound antibodies were detected with a biotin-labeled rat anti-rabbit IgG monoclonal antibody (1:1000 in Tween/PBS) (Zymed Laboratories Inc., S. San Francisco, CA) and alkaline phosphatase-labeled streptavidin (1:1000 in Tween/PBS) (Invitrogen). Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate were used to visualize the bound antibodies. As an additional confirmation that the anti-cytochrome c1 antibody was indicating the correct location of the mitochondria on the sucrose gradient, one of the blots was probed with a mouse monoclonal antibody against the Rieske ISP of the cytochrome bc1 complex (III.30.21.3 ascites diluted 1:125 in Tween/PBS) (41). A biotin-labeled rat anti-mouse IgG monoclonal antibody was used as the secondary antibody for this blot (1:1000 in Tween/PBS; Zymed Laboratories Inc.). The Western blots were sprayed with En3Hance and exposed to x-ray film as directed by the manufacturer (PerkinElmer Life Sciences).

Two additional 12% polyacrylamide gels (10 µl/lane from the cytochrome c1 import reaction gradient) were run to examine the protein profiles of the sucrose gradient fractions. One gel was transferred to a PVDF membrane, which was stained with AuroDye Forte as directed by the manufacturer (Amersham Biosciences). For a more accurate indication of the relative protein contents of each gradient fraction, the second gel was stained with Coomassie Brilliant Blue R-250 (42).

Pretreatment of Mitochondria with Proteinase K-- Mitochondria from ~3 × 108 cells (containing about 200 µg of protein) were resuspended in 100 µl of SME-20 buffer with proteinase K at various concentrations (0.1, 0.5, 1.5, 4.5, and 15 µg/ml). After a 15-min incubation at 4 °C, the samples were diluted with 9 volumes of cold SME-20 containing 1 mM PMSF, and the mitochondria were collected by centrifugation for 10 min at 4 °C. Control reactions (no addition during the initial incubation step at 4 °C) were diluted with 900 µl of either SME-20 buffer alone, SME-20 buffer with 1 mM PMSF, or SME-20 buffer with 1 mM PMSF and 1.5 µg of proteinase K (equivalent to the amount of protease found in the 15 µg/ml digestion). Mitochondrial pellets were washed once (without resuspension) with 150 µl of SME-20 with 1 mM PMSF. The mitochondrial pellets were then resuspended in 40 µl of import buffer with an ATP-regenerating system and the energizing mixture and were incubated with 10 µl of labeled rabbit reticulocyte lysate for 30 min at room temperature. Mitochondria were collected by centrifugation at 4 °C and resuspended in 100 µl of SME-20 buffer. One-half of each sample was collected by centrifugation, run on a 12% SDS-polyacrylamide gel, and stained with Coomassie Blue. The other half of each sample was treated with 30 µg/ml proteinase K and analyzed as described for the standard import reaction.

Sequence Analysis-- Sequences were aligned using the ClustalW program of Thompson et al. (43). The trypanosome protein sequence was analyzed for transmembrane alpha -helical domains using the PHDhtm program (44).

Miscellaneous Reagents-- All reagents were purchased from Sigma unless otherwise stated. ATP and ADP were made as 100 mM concentrated stocks at pH 7.2 and kept at -80 °C until use. A concentrated stock (1000 units/ml) of potato apyrase (grade VIII) was made in 20 mM MOPS/KOH, pH 7.2. Atractyloside (potassium salt) was dissolved in water at a concentration of 10 mM. An inhibitory concentration of 400 µM atractyloside was chosen for the assays based upon studies done in Euglena gracilis by Datta and Kahn (45). Valinomycin, CCCP, oligomycin, antimycin-A (mixture), and PMSF were made as 100-fold concentrated stocks in ethanol.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

T. brucei Cytochrome c1 Lacks a Presequence-- Fig. 1A shows the sequence of the T. brucei cytochrome c1 in alignment with the Saccharomyces cerevisiae and E. gracilis homologs (GenBankTM accession numbers CAA25375 and JQ0021, respectively) (5, 46). The yeast protein has a 61-amino acid presequence that is required for mitochondrial protein import (5). As with the E. gracilis cytochrome c1 and the other kinetoplastid c1 cytochromes that have been cloned to date (24), the T. brucei sequence does not have an amino-terminal extension that might act as a signal for protein import into the mitochondrion; the initial methionine (Met-1) of the protein aligns in close proximity with the mature amino termini of the yeast and E. gracilis proteins (Met-62 and Gly-2, respectively) (indicated in green). If the T. brucei cytochrome c1 is processed in the same manner as the C. fasciculata protein (24, 47), the mature amino terminus is formed by the removal of a single methionine residue to reveal Ala-2 (green arrowhead).


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Fig. 1.   Amino acid sequence comparison of T. brucei and N. crassa c1 cytochromes. A, the translated amino acid sequence of T. brucei (T.b.) cytochrome c1 from DNA sequence AF102980 was aligned with the S. cerevisiae (S.c.) cytochrome c1 protein sequence (GenBankTM accession number CAA25375) (5) and with the E. gracilis (E.g.) protein sequence (JQ0021) (46) by using the ClustalW program (43). Amino- and carboxyl-terminal segments were optimized manually. Residues that are identical between the three sequences are indicated in yellow, and conservative substitutions are indicated in blue. The mature amino termini of the yeast and E. gracilis proteins are shaded green, and the predicted mature amino terminus of the trypanosome protein is indicated by a green arrowhead. Two clusters of positively charged residues near the amino terminus of the trypanosome protein sequence are indicated by italics and are shaded in red. Amino acids in the yeast (23) and trypanosome sequences that are predicted to span the lipid bilayer are indicated by an underline, and sequences that may form an amphipathic alpha -helix are indicated in boldface. B, alpha -helical plot of residues 239-256 of trypanosome cytochrome c1. Positively charged residues are indicated by + and hydrophobic residues are circled.

Given that the kinetoplastid and euglenoid c1 cytochromes lack the amino-terminal targeting sequence found in other eukaryotes, what sequence could act in its place? The trypanosome and other kinetoplastid cytochromes c1 (but not the E. gracilis protein) do contain a cluster of positively charged residues at the amino terminus (Fig. 1A, italics) that might play a role in protein import (24). Although their precise role is unclear, concentrations of positively charged residues have been found at the amino terminus of a number of imported trypanosome mitochondrial proteins (37, 48, 49). The T. brucei protein also has a region near the carboxyl terminus that is predicted to form a membrane-spanning alpha -helix (Fig. 1A, amino acids 222-238 indicated by underlines). An alpha -helical plot of the 18 residues that follow the transmembrane domain (amino acids 239-256 indicated in boldface) shows that this region may form an amphipathic alpha -helix with the positively charged residues and the hydrophobic residues localized on opposite sides of the structure (Fig. 1B). The membrane spanning alpha -helix (Fig. 1A, underlines) and the amphipathic alpha -helix (boldface) identified in the yeast protein by Arnold et al. (23) align with the predicted helices in the trypanosome protein. In the yeast protein, these carboxyl-terminal regions have been implicated as a secondary signal for the membrane potential-dependent insertion of cytochrome c1 into the inner mitochondrial membrane.

Cytochrome c1 and Truncated HSP70 Are Imported into Isolated Mitochondria-- In earlier work, we demonstrated that a crude mitochondrial fraction isolated from T. brucei procyclic cells by nitrogen cavitation lysis and differential centrifugation was competent for the import of the ISP subunit of the cytochrome bc1 complex but did not import a non-mitochondrial protein, luciferase (37). Aliquots of the crude mitochondrial fraction were incubated with in vitro translated, [35S]methionine-labeled T. brucei cytochrome c1, and the amount of imported protein was determined following digestion with increasing amounts of proteinase K (Fig. 2, Cyt c1). In parallel reactions (Fig. 2, HSP70Delta RV), we used the truncated form of the C. fasciculata mitochondrial HSP70 protein as a control protein for mitochondrial import. Like the ISP (37), the HSP should be targeted to the mitochondrial matrix along an ATP-dependent and membrane potential-dependent import pathway, and it contains a cleavable, amino-terminal presequence that should be removed in a single digestion step inside the matrix. Based upon recovery of the [35S]methionine label, 42% of the input cytochrome c1 and 32% of the input HSP70Delta RV protein were found in association with the mitochondria following centrifugation and a SME-20 buffer wash step (Fig. 2, lane 1 and lane 5, respectively). Significant proportions of the bound cytochrome c1 and HSP70Delta RV proteins (7 and 50%, respectively) were resistant to digestion with 15 µg/ml proteinase K (Fig. 2, lane 2 and lane 6), and the recoveries did not change significantly even when the proteinase K concentration was increased to 60 µg/ml (5 and 43%; lanes 4 and 8, respectively). The observed level of cytochrome c1 import (5-7%) is similar to what has been reported for two other inner-membrane complex subunit proteins (ISP, 6%; NdhK, 6%) in this system (37, 39). The efficiency of import of the HSP70Delta RV (43-50%) was consistently and significantly higher than that of the cytochrome c1.


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Fig. 2.   T. brucei cytochrome c1 and C. fasciculata HSP70Delta RV proteins are imported into isolated trypanosome mitochondria. Standard import reactions (25-µl final volume) in complete import buffer as described under "Materials and Methods" were incubated for 30 min at room temperature. Mitochondria were collected by centrifugation and then resuspended in 50 µl of SME-20 buffer containing no protease (lane 1) or with 15 (lane 2), 30 (lane 3), or 60 (lane 4) µg/ml proteinase K (Prot. K). Reactions were incubated for 15 min at room temperature. Digestions were terminated by the addition of an equal volume of ice-cold SME-20 containing 4 mM PMSF, and the mitochondria were collected and analyzed as described under "Materials and Methods." Protease-resistant, labeled cytochrome c1 (lanes 1-4) and HSP70Delta RV (lanes 5-8) proteins were visualized by fluorography. The positions of molecular weight markers are indicated.

No processing products were noted for the cytochrome c1. The HSP70Delta RV protein did occasionally demonstrate a novel weak band at a slightly reduced molecular weight that may be the processed mature protein, but it was not consistent (see also Fig. 7). The mitochondrial preparation was known to be competent for processing because the ISP subunit of the cytochrome bc1 complex was cleaved to both the intermediate and mature forms in parallel experiments (data not shown). The apparent lack of efficient processing of the HSP70Delta RV protein might be the result of either the truncation of the protein used in our studies or the use of a Crithidia protein in a trypanosome import system.

Although we did not specifically test for the formation of holocytochrome c1, we believe that the imported protein is on the correct pathway for complex assembly. We observed an 18-32% stimulation in the level of cytochrome c1 import when NADH and flavin mononucleotide were included in the import buffer at 5 mM and 10 µM concentrations, respectively (data not shown). Reducing equivalents and the FMN cofactor have been reported as essential for the formation of holoprotein in other systems (9). No stimulation in the level of ISP or HSP70Delta RV import was observed in the presence of NADH and FMN (data not shown). In summary, the crude mitochondrial preparation was able to move both HSP70Delta RV and cytochrome c1 to a protease-protected location.

Cytochrome c1 Is Imported in the Absence of a Membrane Potential, but the Truncated HSP70 Is Not-- Insertion of proteins into or across the inner membrane has been shown to require the presence of an intact membrane potential (18). To assess the requirement of a membrane potential for import of the truncated HSP70 and the cytochrome c1 proteins, import reactions were run in the presence of various membrane potential inhibitors. As with the conservatively sorted ISP (37), CCCP (a protonophore that uncouples oxidative phosphorylation), valinomycin (a potassium ionophore), and a combination of oligomycin plus antimycin A (inhibitors of the mitochondrial F1F0-ATPase and of electron transport at the cytochrome b, respectively) were 94-98% effective at blocking import of the HSP70Delta RV protein (Fig. 3A, HSP70Delta RV, lanes 8-10). In contrast, import of the cytochrome c1 was decreased but was not completely eliminated by the presence of these membrane potential inhibitors (Fig. 3A, Cyt c1). Inclusion of CCCP (lane 3), valinomycin (lane 4), and antimycin A + oligomycin (lane 5) in the import reactions resulted in a 53, 81, and 72% inhibition, respectively, as compared with the control level of import.


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Fig. 3.   Cytochrome c1 is imported in the absence of a membrane potential. A, standard cytochrome c1 (upper panel) and HSP70Delta RV (lower panel) import reactions (25-µl final volume; 30-min incubations at room temperature) were conducted in basal import buffer supplemented with an ATP regeneration system (No inhib., lanes 1 and 6). The mitochondrial membrane potential was dissipated in the indicated reactions by preincubation (10 min at 4 °C) with 50 µM CCCP (lanes 3 and 8), 0.5 µM valinomycin (valin., lanes 4 and 9), or 15 µM antimycin A with 30 µM oligomycin (Anti. A + Oli., lanes 5 and 10). Reactions containing 2% ethanol (EtOH, lanes 2 and 7) were included to determined the effect of the solvent upon import. Post-import mitochondrial pellets were digested in SME-20 buffer with 30 µg/ml proteinase K for 15 min at room temperature and analyzed as in Fig. 2. B, standard 25-µl cytochrome c1 (lanes 1 and 2) and HSP70Delta RV (lanes 7 and 8) import reactions in complete import buffer, cytochrome c1 reactions in basal buffer with an ATP regeneration system (+ATP, lanes 3 and 4), and cytochrome c1 reactions in basal buffer with an ATP regeneration system and 50 µM CCCP (+ATP, +CCCP, lanes 5 and 6) were incubated for 30 min at room temperature. Post-import mitochondrial pellets were resuspended in SME-20 buffer with 100 mM KCl and 15 µg/ml proteinase K. CHAPS detergent (2%) was included in the indicated reactions (lanes 2, 4, 6, and 8). Digestions were incubated for 15 min at room temperature and then terminated by the addition of an equal volume of ice-cold SME-20 buffer with 4 mM PMSF. The proteins were precipitated with acetone (4 volumes of ice-cold acetone) at -20 °C overnight and collected by centrifugation (10 min at 4 °C). The resulting pellets were dried for 5 min under vacuum before solubilization in SDS loading buffer and analysis by SDS-PAGE and fluorography.

The observed resistance to protease digestion was not inherent to the translated protein; when CHAPS detergent was included in the SME-20 buffer following import, at least 97% of the cytochrome c1 (Fig. 3B, lanes 2, 4, and 6) and 100% of the HSP70Delta RV (Fig. 3B, lane 8) became sensitive to digestion at the lowest proteinase K concentration (15 µg/ml). While conducting these experiments, we noted that the levels of mitochondrial protein import did not decrease significantly when the respiratory substrates of the energizing mixture, NADH, N,N,N',N'-tetramethylphenylenediamine, and ascorbate, were deleted from the reaction mixture (compare lanes 1 and 3 in Fig. 3B). In general, these substrates were deleted from later assays.

A titration of CCCP was performed to determine the concentration necessary to inhibit import. Reactions were terminated after 5 min so as to be in the linear range of the import assay (see Fig. 5). As shown in Fig. 4, 50% of the import of the truncated HSP70 was inhibited by CCCP at a concentration of about 21 µM. This compares favorably with the 17 µM value for the Rieske ISP reported previously (37). In contrast, almost a 10-fold increase in the CCCP concentration to about 185 µM was required to achieve a similar level of inhibition of cytochrome c1 protein import. As shown in Fig. 3A, the ethanol used as solvent for the CCCP (lanes 2 and 7) had little effect on import by itself.


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Fig. 4.   Import of cytochrome c1 and HSP70Delta RV in the presence of different concentrations of CCCP. A, cytochrome c1 and HSP70Delta RV import reactions (25 µl) in basal import buffer supplemented with an ATP-regenerating system were conducted in the presence of various concentrations of CCCP as indicated (lanes 3-9 and lanes 12-18). The final concentration of the ethanol solvent in all of the CCCP reactions and in the control reactions in lanes 2 and 11 was 2%. In contrast to the reaction shown in Fig. 3, these reactions were incubated at room temperature for only 5 min so as to be in the linear range of the import assay (determined in Fig. 5). Mitochondria were collected by centrifugation at 4 °C and resuspended in 50 µl of SME-20 buffer containing 30 µg/ml proteinase K with 30 µM oligomycin and 15 µM antimycin A to dissipate the membrane potential. After 15 min at room temperature, the samples were treated and analyzed by fluorography as described previously. B, the amounts of the imported, labeled proteins in A were determined by storage phosphor autoradiography and expressed as a percentage of the control level of import with 2% ethanol (A, lanes 2 and 11).

Of the kinetoplastid proteins that have thus far been used in import assays (ISP, HSP70Delta RV, NdhK, and a dihydrolipoamide dehydrogenase construct), only the trypanosome cytochrome c1 has been shown to import in the absence of an inner mitochondrial membrane potential (37, 39, 49). We think it is not a coincidence that the cytochrome c1 is the only one of these proteins that lacks a cleaved amino-terminal presequence.

Import of Cytochrome c1 Is Time- and Temperature-dependent-- Fig. 5A shows that the accumulation of both the cytochrome c1 and the HSP70Delta RV proteins into the protease-inaccessible compartment was time-dependent and was essentially complete after 20-30 min. As demonstrated by the curves in Fig. 5B, the two proteins were imported at similar rates as follows: the t1/2 for cytochrome c1 was ~8.0 min and that for HSP70Delta RV was ~10.6 min. These times are slightly longer than the 6.5-min t1/2 for the mitochondrial ISP reported previously (37). The import of the cytochrome c1 protein was also found to be temperature-dependent. When the cytochrome c1 import reaction was maintained on ice for 30 min, the amount of protease-protected protein was decreased 9-fold relative to the standard import reaction at room temperature (data not shown). Like the standard cytochrome c1 import reaction, the import of cytochrome c1 in the presence of 200 µM CCCP (Fig. 4) was also shown to be time-dependent (t1/2 = 8.8 min) and to be temperature-sensitive (5-fold stimulation at room temperature versus 4 °C) (data not shown). The similar kinetic profiles observed for the import of HSP70Delta RV, cytochrome c1, and cytochrome c1 in the presence of CCCP as well as the marked reduction in import at low temperature would suggest that the import of these proteins is occurring along a receptor-mediated pathway.


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Fig. 5.   Import of cytochrome c1 and HSP70Delta RV are time-dependent. A, 200-µl mitochondrial import reactions (mitochondria from ~1.2 × 109 cells) in complete import buffer were conducted as described under "Materials and Methods." Cytochrome c1 reactions were run with (middle panel) and without (top panel) 200 µM CCCP. At the indicated time points 25-µl aliquots were withdrawn from the reaction and diluted with 75 µl of ice-cold SME-20 buffer containing sufficient CCCP and valinomycin for final concentrations of 50 and 0.5 µM, respectively. Mitochondria were collected by centrifugation at 4 °C and resuspended in 50 µl of SME-20 buffer containing 30 µg/ml proteinase K. After a 15-min incubation at room temperature, the samples were treated and analyzed by fluorography as described previously. B, the amounts of the imported, labeled proteins in A were determined by storage phosphor autoradiography and expressed as a percentage of the maximum level of import.

Import of Cytochrome c1 Requires an Exposed, Protease-sensitive Component-- With the notable exception of cytochrome c, the import of most proteins into the mitochondrion has been shown to require a protein receptor that is exposed on the exterior surface of the outer mitochondrial membrane (50). To confirm that a surface protein receptor was required for cytochrome c1 import, the mitochondrial fraction was incubated at 4 °C with low concentrations of proteinase K prior to the import reaction (Fig. 6A). Pretreatment of the crude mitochondrial fraction with proteinase K did not cause a drastic alteration in the overall protein profile; only two proteins present in the mitochondrial fraction were observed to be hypersensitive to protease digestion (proteins with apparent molecular masses of 70 and 17 kDa in the 4.5 and 15 µg/ml treatment lanes as indicated by arrows). In contrast, the ability of the mitochondria to import both cytochrome c1 and HSP70Delta RV proteins was affected by protease pretreatment (Fig. 6B). Import of both proteins was essentially eliminated by pretreatment of the mitochondria with 15 µg/ml proteinase K. Pretreatment of mitochondria with an equivalent concentration of PMSF-inactivated proteinase K (15*, lane 3) had no effect on the protein profile or on the ability to import (compare Fig. 6A, lanes 3 and 8 and the corresponding lanes in Fig. 6B). The level of protein import was also not affected by the presence of PMSF in the SME-20 wash buffer (lanes 1 and 2 in A and B). As shown by the curve in Fig. 6C, protease pretreatment had a similar effect on the import of both proteins; the IC50 for cytochrome c1 import was ~1.3 µg/ml whereas that for HSP70Delta RV import was ~1.0 µg/ml. We conclude that the import of both proteins requires a protein component that is exposed on the mitochondrial surface and that is hypersensitive to protease digestion.


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Fig. 6.   Import of both cytochrome c1 and HSP70Delta RV proteins requires an exposed, hypersensitive protein component on the mitochondria. A, prior to use in import reactions, aliquots of mitochondria from 3 × 108 cells were incubated in 100 µl of SME-20 buffer with the indicated concentrations of proteinase K (Prot. K) (lanes 4-8) for 15 min at 4 °C. The asterisk in lane 3 indicates that the proteinase K in this reaction was added at the same time as the PMSF dilution buffer (see below). Samples were then diluted with 900 µl of either SME-20 (lane 1), SME-20 with 1 mM PMSF and 1.5 mg of proteinase K (lane 3; equivalent to 15 µg/ml in the original incubation volume), or with SME-20 with 1 mM PMSF (lane 2 and lanes 4-8). The mitochondria were collected by centrifugation at 4 °C, washed once with SME-20 (lane 1) or SME-20 containing 1 mM PMSF (lanes 2-8), and used for 50-µl import reactions in complete buffer as described under "Materials and Methods." One-half of the post-import mitochondrial pellet was analyzed by SDS-PAGE and stained with Coomassie Blue. Arrows indicate the positions of two proteins at apparent molecular masses of 70 and 16 kDa that appear to be hypersensitive to digestion. (The 16-kDa protein is the upper band of a doublet.) Apparent molecular weights of the standards in lane M are given to the left of the panel. B, the other half of each reaction was digested with 30 µg/ml proteinase K for 15 min at room temperature and treated and analyzed as described previously. Cytochrome c1 import is shown in the top panel, and HSP70Delta RV import is shown in the bottom panel. C, the amounts of the imported, labeled proteins in lanes 3-8 in B were determined by storage phosphor autoradiography and expressed as a percentage of the level of import observed in the control lane 3 (PMSF-inactivated proteinase K).

Cytochrome c1 Is Imported into Mitochondria in the Absence of a Membrane Potential-- To rule out the possibility that the cytochrome c1 was imported to a non-mitochondrial compartment in the absence of a membrane potential, we scaled up the import reactions and then separated the membrane components by density on a linear sucrose gradient. Opperdoes et al. (40) demonstrated previously that mitochondria from isotonically lysed T. brucei procyclic cells can be cleanly resolved from the glycosomes and other microbodies by isopycnic centrifugation on a 1.18-2.0 M linear sucrose gradient (1.15-1.26 g/cm3). Mitochondria were shown to equilibrate over a broad density range centered at 1.17 g/cm3, whereas the glycosomes and other microbodies equilibrated as a sharp band at 1.23 g/cm3.

We imported both cytochrome c1 and the ISP in basal buffer supplemented with the ATP-regenerating system, and we also imported cytochrome c1 in the presence of the lowest CCCP concentration that completely inhibited HSP70Delta RV (100 µM; see Fig. 4B) and then resolved the imported products into 12 density fractions as shown in Fig. 7A. Fractions 7 and 8 (density range 1.207-1.227 g/cm3) contained a well demarcated, visible band that, under microscopic examination, was observed to contain large numbers of flagella and vesicles. No bands were obvious in the lower density range except in the tube containing the cytochrome c1 import in the presence of CCCP. In that tube, a faint yellow band was apparent near the top of the gradient. No 35S-labeled proteins were detected in the most dense fractions (density >1.227 g/cm3; fractions 9-12), and these fractions were not further analyzed.


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Fig. 7.   Most of the labeled cytochrome c1 protein is imported into mitochondria. To determine the density of the vesicles that contained the imported, labeled proteins, scaled-up reactions (500 µl final volume; mitochondria from 3 × 109 cells) were conducted for 30 min in basal import buffer with an ATP-regenerating system as described under "Materials and Methods." After treatment with protease, the vesicles were centrifuged on a linear sucrose gradient with the profile shown in A and fractionated as described under "Materials and Methods." No proteins or radioactive counts were detected in fractions 9-12, and these fractions were not further analyzed. A particulate pellet was obtained from each fraction and was dissolved SDS loading buffer. The proteins were resolved on a 12% polyacrylamide gel and transferred to a PVDF membrane. B, the top panel shows an AuroDye Forte-stained blot of the sucrose gradient fractions (10 µl/lane) from the cytochrome c1 import reaction. The numbers above the panel indicate the gradient fraction number from A. Apparent molecular weights of the standards run in lane M are indicated on the right side of the panel. The middle panel shows the locations of the native iron-sulfur protein (ISP) and cytochrome c1 (Cyt c1) proteins in the sucrose gradient (20 µl/lane) as determined by Western blot. The Western blot was then subjected to fluorography (bottom panel) to determine the location of the labeled, imported cytochrome c1 in the gradient. C shows the sucrose gradient fractionation of labeled protein from an ISP import reaction. The numbers above the panel indicate the gradient fraction number from A. D shows the sucrose gradient fractionation of labeled protein from a cytochrome c1 import reaction conducted in the presence 100 µM CCCP. The numbers above the panel indicate the gradient fraction number from A. The top panel shows the location of native cytochrome c1 protein (as determined by Western blot) in the sucrose gradient (20 µl/lane). The Western blot was then subjected to fluorography (bottom panel) to determine the location of the labeled, imported cytochrome c1 in the gradient.

Fig. 7B shows the protein profile (AuroDye), Western blot analysis, and 35S-labeled protein distribution in the cytochrome c1 sucrose gradient. As demonstrated by the AuroDye panel, the protein profile varied through the gradient; the lower density fractions were enriched for a different subset of proteins than the higher density fractions, and fractions 7 and 8 contained more total protein than the lower density fractions. From a Coomassie Blue-stained polyacrylamide gel of the gradient fractions, we estimate that 80-90% of the total protein recovered from the gradient was present in fractions 7 and 8 (data not shown). A Western blot analysis of the fractionated proteins indicated that, although cytochrome c1 was found throughout the gradient, it was enriched in the lower density fractions (fractions 2-5; density range 1.155-1.196 g/cm3). To be certain that the anti-cytochrome c1 antibody was giving an accurate representation of the distribution of the mitochondrial membranes in the gradient, the Western blot was also probed with a monoclonal antibody against the Rieske ISP. Although the monoclonal anti-ISP antibody was clearly not as sensitive as the polyclonal anti-cytochrome c1 antibody (no protein was detected in fractions 1, 7, or 8), the Western blot demonstrates that the ISP is distributed in a similar pattern in the gradient with a peak indicated in fraction 4 (density range 1.176-1.186 g/cm3). Most of the imported, 35S-labeled cytochrome c1 protein (75%) was found in four upper fractions of the gradient as follows: fraction 1 contained 10% of the total protected protein; fraction 2, 28%; fraction 3, 20%; and fraction 4, 17% (density range 1.15-1.186 g/cm3). Interestingly, although the imported cytochrome c1 protein overlapped with the mitochondrial membranes as detected by Western blot, the fraction that contained the most imported cytochrome c1 (fraction 2; density between 1.155 and 1.165 g/cm3) was shifted relative to the fraction that contained the most mitochondrial membranes (fraction 4; density between 1.176 and 1.186 g/cm3). This observation may suggest that the majority of the well sealed, actively respiring mitochondria are found in the lower density fraction.

An analysis of a gradient containing the mitochondria from a labeled ISP import reaction (Fig. 7C) gave similar results in terms of distribution; most of the labeled, imported protein (59%) and most of the mitochondrial membranes were found in fractions 2-4. The peak fraction for the import of labeled protein, fraction 3, contained 32% of total labeled protein. The imported, labeled ISP in these fractions was also processed to the intermediate and mature size as expected.

The cytochrome c1 distribution in the gradient containing the cytochrome c1 + CCCP import reaction shows some differences relative to the standard cytochrome c1 and ISP gradients just described. The Western blot in Fig. 7D shows that there are two peaks of mitochondrial membranes as detected by the cytochrome c1 antibody: one peak in the top fraction of the gradient; a second, larger, and more broad peak in fractions 3-5. (Note that fraction 1 in Fig. 7B contains very little native cytochrome c1 protein.) The imported, labeled cytochrome c1 was also found as two peaks in the gradient: fraction 1 contained 30% of the total imported protein, and fractions 3-5 contained a combined 28% of the total protein (11, 9, and 8%, respectively). We think it possible that the observed shift of some of the mitochondria to a lighter density in the presence of the CCCP inhibitor (resulting in a second peak of imported protein in fraction 1 that is not seen in the absence of inhibitor) may reflect the swelling of some of the well sealed mitochondria. CCCP-induced swelling of actively respiring mitochondria has been reported previously (51) in other systems.

Thus, in each gradient we were able to separate a peak of imported, labeled protein from the two fractions (7 and 8) that contained the dense glycosomes and microbodies (density range 1.207 to 1.227 g/cm3). Given that the imported, labeled proteins co-fractionate with the mitochondrial membrane marker proteins (as detected by Western blot) and that the densities of these fractions are consistent with the density range reported previously for trypanosome mitochondria, we believe that the cytochrome c1 protein is imported into vesicles of mitochondrial origin even in the absence of a membrane potential (CCCP reaction, Fig. 7D).

Cytochrome c1 Import Is Not Affected by Fluctuations in Matrix ATP Levels-- By using various inhibitors of ATP synthesis and ATP transport, others (12) have demonstrated that matrix ATP is not an absolute requirement for the import of native cytochrome c1 into N. crassa and yeast mitochondria. By using atractyloside (a specific inhibitor of the ADP/ATP carrier protein), oligomycin (an inhibitor of the mitochondrial F1F0-ATPase), and added external ATP and ADP, we attempted to determine the relevance of matrix ATP levels to the import of T. brucei cytochrome c1. In the first set of experiments, ATP and ADP were added to the import reaction in the absence of an ATP regeneration system and in the absence of any additional respiration substrates in an attempt to manipulate the mitochondrial matrix ATP pool via the ADP/ATP carrier protein. As shown in Fig. 8A, inclusion of 2 mM ATP caused a slight increase in the level of import of both the HSP70Delta RV and cytochrome c1 proteins. The addition of both 2 mM ATP and 400 µM atractyloside to the import reaction caused a dramatic increase in the level of HSP70Delta RV import (from 115 to 315% of the control level) but had no similar effect on cytochrome c1 import. This atractyloside-specific stimulation was not peculiar to the HSP70Delta RV protein; the conservatively sorted ISP behaved in a similar manner when ATP and atractyloside were added to the import reaction (about a 4-fold stimulation, data not shown). Atractyloside by itself slightly stimulated the import of HSP70Delta RV protein (45% increase over basal level) but had no such effect on cytochrome c1 import (Fig. 8B). We believe that the ADP present in the reticulocyte lysate translation mixture may, in the presence of a functional ADP/ATP carrier protein, rapidly deplete the ATP pool within the mitochondrial matrix. In fact, when an additional 2 mM ADP was added to the import reaction, we observed a 60% decrease in the level of HSP70Delta RV import, but only a 20% decrease in the level of cytochrome c1 import (Fig. 8A). The effect of ADP on HSP70Delta RV import could be completely reversed by the inclusion of atractyloside to block carrier protein-mediated exchange, but no stimulation in the import of cytochrome c1 was noted. As would be expected for an equilibrium-driven phenomenon, the effect of ADP on import of the HSP70Delta RV protein was largely negated by the inclusion of an equimolar amount of ATP in the reaction (Fig. 8A).


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Fig. 8.   Import of cytochrome c1 is not significantly inhibited by reduction of matrix ATP levels. A, 20-µl aliquots of mitochondria were preincubated at room temperature for 10 min in basal buffer either with or without atractyloside (Atractyl.) (400 µM final concentration). After the preincubation, 2 mM ADP and 2 mM ATP were added to the indicated reactions, and import was initiated by the addition of 5 µl of labeled cytochrome c1 (upper panel) or HSP70Delta RV (lower panel) in reticulocyte lysate. Import reactions were incubated for 7.5 min at room temperature and then centrifuged at 4 °C for 10 min. Mitochondrial pellets were digested with 30 µg/ml proteinase K in SME-20 buffer containing 30 µM oligomycin and 15 µM antimycin A (50 µl) for 15 min at room temperature and treated and analyzed as described previously. The amounts of the imported, labeled proteins were determined by storage phosphor autoradiography and expressed as a percentage of the control level of import (no additions). B, import assays were conducted as in A except that 30 µM oligomycin (Oligo.) and 2 mM ATP with an ATP regeneration (ATP + regen.) system were added to the indicated reactions after the preincubation.

In a second set of experiments, we examined the effects of an external ATP regeneration system on import, and we attempted to reduce further matrix ATP levels with oligomycin. We noted that ATP and an ATP regeneration system together were much more effective at stimulating import of the HSP70Delta RV protein than was ATP alone (compare the 15% increase over basal level in Fig. 8A and to the 665% increase in Fig. 8B). In contrast, import of cytochrome c1 was only slightly more efficient in the presence of the regeneration system (10% increase versus 50% increase, respectively). Inclusion of an ATP regeneration system in the HSP70Delta RV import reaction also significantly reduced the stimulatory effect observed with atractyloside. In the presence of ATP alone (shown in Fig. 8A), atractyloside caused a 1.7-fold increase in the level of HSP70Delta RV import (115-315% of the basal level of import), but, in the presence of ATP and the regeneration system, only a 0.4-fold increase was observed (765-1080% of the basal level). Taken together, these results are consistent with the proposed role of external ADP in the depletion of the matrix ATP pool and further suggest that cytochrome c1 is not sensitive to the matrix ATP concentration.

Others (52, 53) have demonstrated that the T. brucei ATPase can be inhibited by about 50% by oligomycin. As shown in Fig. 8B, oligomycin in the absence of added ATP resulted in a 60% inhibition of HSP70Delta RV import but only a 15% inhibition of cytochrome c1 import. In reactions containing either ATP and an ATP regeneration system to minimize the amount of efflux of ATP from the matrix, atractyloside to block ATP/ADP exchange, or both ATP and atractyloside, HSP70Delta RV import was consistently more sensitive to inhibition by oligomycin than cytochrome c1 import (32% inhibition versus 6% stimulation, 38% inhibition versus 21% inhibition, and 37% inhibition versus 12% inhibition, respectively).

Depletion of the External ATP Blocks Cytochrome c1 Import-- The 50% increase in import of cytochrome c1 observed when both ATP and an ATP-regenerating system were added to the import reaction (shown above) suggested that the import of this protein might be sensitive to the external ATP concentration. When potato apyrase was used to hydrolyze the ATP in the reticulocyte lysate prior to the import reaction and efflux of matrix ATP was prevented by inhibition of the ATP/ADP carrier protein with atractyloside, the import of HSP70Delta RV was essentially abolished (5% of control) and that of the cytochrome c1 was decreased to 15% of the control value (Fig. 9). Heat-treated apyrase enzyme had no effect on import (data not shown). Addition of ATP and the regenerating system essentially restored the import of the HSPDelta RV protein but had very little impact upon cytochrome c1 import. To rule out the possibility that contaminating activities in the apyrase preparation might be destroying essential cofactors required for cytochrome c1 import (9), we repeated the reactions in the presence of 5 mM NADH and 10 µM FMN. Although the cofactors did stimulate import in the control reaction (18-32% increase), they were not able to restore import in the reaction that was treated with apyrase and supplemented with ATP and the regeneration system (data not shown).


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Fig. 9.   External ATP is required for import of both cytochrome c1 and HSP70Delta RV. Import assays were conducted as described in Fig. 8 except that 2 mM ATP and an ATP-regenerating system (ATP + regen.) were added to the indicated reactions after the preincubation. In addition, the labeled rabbit reticulocyte lysates of the indicated reactions were treated with apyrase (10 units/ml incubated for 15 min at 30 °C and 15 min at room temperature) prior to addition. Atractyl., atractyloside.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the current work, we compared the import of the T. brucei cytochrome c1 protein to that of a truncated form of the C. fasciculata mitochondrial HSP70 protein. Based on our analysis of the trypanosome import system, we would suggest that the HSP70Delta RV protein is using a typical, presequence-directed matrix import pathway identical to the first segment of the conservative sorting pathway reported previously for the trypanosome ISP (37). In contrast, the cytochrome c1 protein is likely using an alternative pathway similar to that described for the yeast and N. crassa AAC and other inner membrane carrier proteins (31-36, 54). As shown in Fig. 10, HSP70Delta RV and cytochrome c1 both require external ATP in order to interact with protein receptors on the mitochondrial surface. Whereas the import of both the HSP70Delta RV and cytochrome c1 proteins was eliminated by apyrase pretreatment of reticulocyte lysate, only HSP70Delta RV import could be fully restored by the addition of ATP and a regenerating system. We know that both proteins require the co-translational association of chaperone-type factors found in the rabbit reticulocyte extract for import because neither protein was imported from a wheat germ extract translation even after the addition of reticulocyte lysate.2,4 We hypothesize that the cytochrome c1 protein may be released from these chaperone proteins upon ATP depletion and, perhaps because of the hydrophobic residues in the carboxyl-terminal region of the protein or because of the lack of an amino-terminal presequence, may become locked in an import-incompetent conformation that cannot be restored upon the re-introduction of ATP. This observation may be indicative of some unique characteristic of cytochrome c1 import into trypanosome mitochondria because apyrase inhibition of protein import into N. crassa mitochondria could be fully restored even for the AAC and cytochrome c1 proteins (36, 55).


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Fig. 10.   Proposed T. brucei mitochondrial import pathways for HSP70Delta RV and cytochrome c1. Both cytochrome c1 and HSP70Delta RV are translated in the cytosol and are associated with cytosolic factors or chaperones (not shown). Import of both proteins requires an association with a surface-exposed protein component (RECEPTOR) and external ATP. Import of HSP70Delta RV requires a membrane potential (Delta psi ) and matrix ATP and likely occurs through contact sites where the mitochondrial outer membrane (MOM) and mitochondrial inner membrane (MIM) complexes are in association. Once in the matrix, the amino-terminal presequence of the HSP70Delta RV protein (open box) is cleaved by a processing protease to yield the mature protein. In contrast, import of cytochrome c1 does not require a membrane potential or matrix ATP. The protein is likely imported through a MOM complex into the intermembrane space (IMS), and when a membrane potential is restored, the protein is transported to the MIM complex and inserted into the inner membrane. In the presence of heme and reducing equivalents, cytochrome c1 heme lyase covalently attaches heme (H) to form the holoprotein that is then assembled into the cytochrome c reductase complex.

We do not yet know if the HSP70Delta RV and cytochrome c1 proteins utilize the same mitochondrial surface receptor, but if the trypanosome system is like the other eukaryotic systems thus far examined, proteins that have cleaved presequences use a MOM-19-like receptor, whereas the AAC protein and others lacking presequences use a MOM-72-like receptor (reviewed in Refs. 2 and 3). Both proteins are then transferred to the mitochondrial outer membrane (MOM) import complex (32, 56). The major difference between the AAC pathway and the presequence-directed conservative pathway (or the conservative pathway segment leading to the matrix) is that the former can import proteins in the absence of a membrane potential. Proteins imported along the AAC pathway are moved into the intermembrane space, either in association with a membrane-bound, multisubunit complex (ATP/ADP carrier protein) or as a freely soluble protein (dicarboxylate carrier protein) (33-35, 54), whereas proteins imported into the matrix require the association of the mitochondrial inner membrane (MIM) complex, an intact membrane potential, and the ATP-dependent activity of mitochondrial matrix HSP70. As noted in Fig. 1, the T. brucei cytochrome c1 protein (and other kinetoplastid proteins as well) does have a two-part, carboxyl-terminal sequence that is consistent with the signal sequence identified by Arnold et al. (23) at the end of the yeast cytochrome c1 protein. The trypanosome sequence contains a region predicted to span the membrane (residues 222-238) and an 18-amino acid sequence that may form an amphipathic alpha -helix with the positively charged and hydrophobic residues distributed on opposite sides of the structure. As in the yeast system, this sequence may mediate an interaction with a still undefined MIM complex and, in the presence of a membrane potential, may direct the insertion of the protein from the IMS into the membrane in the correct orientation for complex assembly and holoenzyme formation.

As expected for a matrix-localized protein, HSP70Delta RV demonstrated a clear dependence upon matrix ATP for import; HSP70Delta RV import was quite sensitive to fluctuations in matrix ATP caused by the addition of external ADP (exchanged by the mitochondrial ATP/ADP carrier protein and blocked by atractyloside) and was inhibited by about 50% by the addition of oligomycin. Given that isotonically isolated mitochondria are essentially depleted of internal ATP (53), these results would suggest that the rabbit reticulocyte translation mixture, by itself, contains sufficient ATP and/or respiratory substrates to support a low level of import in basal buffer. The observed level of inhibition of the mitochondrial F1F0-ATPase by oligomycin (30-60%) is similar to that reported by others (52, 53). In contrast to the HSP70Delta RV, import of the cytochrome c1 was largely unaffected by the addition of ADP or oligomycin. Even though neither of these experiments can be considered definitive (100% inhibition was not achieved), the results certainly suggest that the trypanosome cytochrome c1 requires little or no matrix ATP for import. In this respect, the trypanosome cytochrome c1 appears to be similar to most other eukaryotic c1 cytochromes (11, 12, 14).

In light of the recent discovery of the carboxyl-terminal signal in the yeast cytochrome c1 and our description of an alternative cytochrome c1 import pathway in trypanosomes, it is interesting to speculate that the other eukaryotic c1 cytochromes are, in fact, using a stop-transfer pathway (or some related version) for import; the first part of the pathway may resemble the conservative pathway in that the amino-terminal presequence may direct the membrane potential-dependent import of a portion of the protein through the outer and inner membranes; the second part of the pathway may resemble the AAC pathway in that, once across the outer membrane, the carboxyl-terminal signal may direct membrane potential-dependent insertion of the mature portion of the protein into the inner membrane in the proper Nout-Cin orientation. How much of the protein enters the matrix and whether the cytochrome c1 heme lyase might play a role in import (either by "pulling" the protein across the outer membrane as with cytochrome c or by preventing further translocation into the matrix) remain to be determined. This hybrid pathway would explain the absence of a matrix ATP requirement and would be consistent with the cleavage of the bipartite, amino-terminal signal sequence on opposite sides of the inner mitochondrial membrane. The trypanosome system, therefore, is a "natural" experiment that demonstrates that the amino-terminal presequence-driven entry of the protein into the matrix is not essential for cytochrome c1 import and/or assembly.

Finally, it must be stated that, although we are certain that there is no cleaved amino-terminal presequence, we cannot be certain that there is no mitochondrial targeting signal at the amino terminus of the cytochrome c1. We and others (37, 47, 48) have noted that trypanosome amino-terminal, cleaved presequences are unusually short (as short as 8-9 residues) and that they often contain a MX1-2(K/R)(K/R) motif where X1-2 is one or two amino acid that are usually hydrophobic. All of the kinetoplastid c1 cytochromes thus far examined have 3-4 positively charged amino acids with the first 11 residues and a sequence that would seem to satisfy this motif, M(A/G)G(K/R)(K/R) (see Ref. 24 and this work). We do not believe these residues are necessary for cytochrome c1 import for two reasons. First, the E. gracilis cytochrome c1 protein also lacks a cleaved amino-terminal presequence yet does not have any positively charged residues in the first 20 residues of the sequence. Second, we have mutated the MAGKK sequence of the trypanosome protein to MAGQQ and have shown that it has no effect on import either with or without a membrane potential.2 We tend to believe that the import signals for cytochrome c1 are internal and most likely downstream of the heme-binding site. We are currently working to elucidate further the signals involved in recognition and import.

    ACKNOWLEDGEMENTS

We thank Kathy Hancock, Karen Bertrand, Lynn Sherrer, and the members of the Hajduk laboratory for their assistance, comments, and helpful discussions. We also thank the Adler and Hajduk families for being gracious hosts during the completion of this work.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants (to S. L. H.) and Postdoctoral Fellowship AI08259 (to J. W. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Division of Parasitic Diseases, Centers for Disease Control and Prevention, Mail Stop F-13, Bldg. 23, Rm. 1025, 4770 Buford Hwy. N.E., Atlanta, GA 30341-3724.

§ To whom correspondence should be addressed. Tel.: 205-934-6033; Fax: 205-934-6096; E-mail: shajduk@uab.edu.

Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M212956200

2 J. W. Priest and S. L. Hajduk, unpublished observations.

3 Z. A. Wood and S. L. Hajduk, unpublished observations.

4 K. Bertrand and S. L. Hajduk, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: IMS, intermembrane space; ISP, Rieske iron-sulfur protein subunit of the mitochondrial cytochrome c reductase complex; AAC, ATP/ADP carrier protein; HSP70Delta RV, truncated form of mitochondrial heat shock 70-kDa protein; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid detergent; CCCP, carbonylcyanide m-chlorophenylhydrazone; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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