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
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ABSTRACT |
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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.
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
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.
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
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 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 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).
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 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,
HSP70
No processing products were noted for the cytochrome
c1. The HSP70
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 HSP70 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 HSP70
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 HSP70
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.
Of the kinetoplastid proteins that have thus far been used in import
assays (ISP, HSP70 Import of Cytochrome c1 Is Time- and
Temperature-dependent--
Fig.
5A shows that the accumulation
of both the cytochrome c1 and the HSP70 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 HSP70 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 HSP70
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 HSP70
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 HSP70
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 HSP70 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 HSP70 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 HSP70
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(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
(HSP70
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).
-helical domains using the
PHDhtm program (44).
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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
-helix are indicated in boldface. B,
-helical plot of residues 239-256 of trypanosome cytochrome
c1. Positively charged residues are indicated by + and hydrophobic residues are circled.
-helix (Fig.
1A, amino acids 222-238 indicated by
underlines). An
-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
-helix with the positively charged residues and the hydrophobic
residues localized on opposite sides of the structure (Fig.
1B). The membrane spanning
-helix (Fig.
1A, underlines) and the amphipathic
-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.
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 HSP70
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 HSP70
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 HSP70
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
HSP70 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 HSP70
RV
(lanes 5-8) proteins were visualized by fluorography. The
positions of molecular weight markers are indicated.
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 HSP70
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.
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 HSP70
RV and
cytochrome c1 to a protease-protected location.
RV protein
(Fig. 3A,
HSP70
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 HSP70 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
HSP70
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.
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.
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Fig. 4.
Import of cytochrome
c1 and HSP70 RV in
the presence of different concentrations of CCCP. A,
cytochrome c1 and HSP70
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).
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.
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 HSP70
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 HSP70
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 HSP70 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.
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 HSP70
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 HSP70 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 HSP70
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).
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.
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 HSP70
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 HSP70
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 HSP70
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 HSP70
RV import, but only a 20% decrease in the level
of cytochrome c1 import (Fig. 8A).
The effect of ADP on HSP70
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
HSP70
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 HSP70 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.
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 HSP70
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 HSP70
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.
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, HSP70
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).
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 HSP
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
HSP70 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
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, HSP70
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 HSP70
RV and cytochrome
c1 proteins was eliminated by apyrase
pretreatment of reticulocyte lysate, only HSP70
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
HSP70 RV and cytochrome
c1. Both cytochrome
c1 and HSP70
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
HSP70
RV requires a membrane potential (
) 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 HSP70
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 HSP70RV 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
-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, HSP70RV demonstrated a
clear dependence upon matrix ATP for import; HSP70
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 HSP70
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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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;
HSP70RV, 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.
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