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INTRODUCTION |
Mitochondria in mammalian organisms are important subcellular
targets for a variety of xenobiotics including drugs, carcinogens, and
environmental contaminants (1-6). Structurally diverse chemicals and
pharmacologically important drugs, which alter mitochondrial membrane
properties and affect mitochondrial enzyme complexes or gene
expression, are thought to be contributing factors in a variety of
human disorders (3, 5-8). Efforts in our laboratory have been focused
on the characterization of mitochondrial class I and class II drug
metabolizing enzymes to determine their roles in modulating the toxic
effects of various xenobiotic chemicals on the mitochondrial genetic
system. Our studies showed the presence of five different
xenobiotic-inducible P4501
monooxygenases in rat liver mitochondria, which cross-react with antibodies to similarly induced microsomal P450 isoforms (9-14). Enzyme reconstitution and immunochemical studies also showed the presence of several P450 isoforms, resembling the liver mitochondrial forms, in induced rat brain and human brain mitochondria (15-17). Additionally, reports from various groups have shown the occurrence of
different isoforms of glutathione S-transferases in the
mitochondrial membrane compartment of rat liver, brain, and lung
(18-21), suggesting the existence of both class I and class II enzymes
in mitochondria.
Recent studies in our laboratory showed that the two
BNF-inducible hepatic mitochondrial P450s, designated as P450MT2A
and MT2B, are derived by differential proteolytic processing of
the similarly induced microsomal P4501A1 with cleavage sites past the
4th and 32nd amino acid residues, respectively (22). Transient transfection in COS cells and site-specific mutagenesis studies showed
that the N terminus of P4501A1 contains a chimeric signal for targeting
the protein to both the ER and mitochondrial compartments. About
20-25% of the nascent P4501A1 chains in both BNF-induced liver and
COS cells transfected with expression cDNA constructs escape ER
targeting by an unknown mechanism and are processed by an endoprotease
to activate a cryptic mitochondrial targeting sequence (22, 23). The
nature of the protease and also steps involved in redirecting this
otherwise predominantly ER-targeted protein to mitochondria remain
unclear. Enzyme reconstitution experiments with purified mitochondrial
P450 from BNF-treated liver and bacterially expressed P450 forms showed
that P450MT2 exhibits high ERND activity in an Adx + Adr-supported
system. In light of the established evidence that P4501A1 in a NADPH
P450 reductase-supported system has low ERND activity (23, 24), the
observed high ERND activity of the mitochondrial P450 form suggested
significant difference in the conformation of the two forms. This
possibility was further supported by a high affinity domain-specific
interaction of P450MT2 with Adx under in vitro conditions
(24).
In the present study, the difference in the catalytic properties of
P4501A1 targeted to the two subcellular compartments was used as a
marker to further investigate the dual targeting of P4501A1 to the ER
and mitochondria and also to elucidate the physiological significance
of mitochondrial targeting. Erythromycin is a potent antibiotic with
known inhibitory effects on the bacterial ribosome system and minimal
effects on the eukaryotic cytoplasmic ribosomes. Because of the
reported prokaryotic nature of the mitochondrial ribosomes with respect
to sensitivity to various antibiotics (25-30), we tested the effects
of P450MT2 overexpression in COS cells on the mitochondrial
erythromycin metabolism and erythromycin-mediated inhibition of
mitochondrial translation. Our results provide further supporting
evidence for the dual ER and mitochondrial targeting of P4501A1 in
addition to suggesting the role of the mitochondrial targeted P450MT2
in erythromycin detoxification and protection against the drug-induced
inhibition of mitochondrial protein synthesis. Additionally, we
demonstrate that erythromycin treatment not only induces CYP1A1
mRNA accumulation but also causes an enhanced accumulation of
P450MT2 in the mitochondrial compartment and an associated increase in
ERND activity.
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MATERIALS AND METHODS |
Subcellular Fractionation and Isolation of
Mitochondria--
Mitochondria and microsomes from BNF-induced rat
livers were isolated as described before (9, 13). Mitochondria and
microsomes from transfected COS cells and C6 glioma cells were isolated
by sucrose density banding and digitonin treatment as described
recently (22). Cells from about 10 plates (100 mm) were pooled and
homogenized in a sucrose-mannitol buffer (13) with 10-12 strokes
(about 5,000 revolutions/min) of a Teflon fitted glass homogenizer, and subcellular membrane fractions were isolated by differential
centrifugation. The crude mitochondrial fraction was suspended in the
sucrose-mannitol buffer by gentle homogenization and further purified
by sedimentation through a discontinuous sucrose gradient.
Mitochondrial particles banding at the interface of 1.35 and 1.6 M sucrose were recovered, washed twice with the
sucrose-mannitol buffer, and used for further analysis. As shown
recently, this method yields intact mitochondria free of detectable
microsomal marker proteins (23). In some experiments, mitochondria were
subjected to digitonin treatment (75 µg/mg protein for 5 min on ice)
as described (13).
cDNA Constructs and Expression in COS Cells--
The
cDNA constructs expressing full-length P4501A1 cDNA, N-terminal
deletions, and point mutations were cloned in pCMV4 as described before
(22). The pCD mammalian expression cDNA construct for bovine Adx
was generously provided by Dr. Michael Waterman (31, 32). Transfection
of CsCl-banded plasmid DNAs was carried out as described before (22).
Transfection efficiencies were monitored by co-expression with 2 µg/plate of cytomegalovirus-
-galactosidase and assaying the cell
extracts for
-galactosidase activities. About 62 h after
transfection, cells were homogenized, and the mitochondrial and
microsomal fractions were isolated by the differential centrifugation
method described above. The mitochondrial and microsomal fractions were
resuspended in 50 mM potassium phosphate buffer (pH 7.4)
containing 20% glycerol, 0.5 mM dithiothreitol, 1 mM EDTA, and 0.1 mM phenylmethylsulfonyl
fluoride and used for reconstituting the enzyme activity.
Reconstitution of Enzyme Activities--
Enzyme activities were
reconstituted in a reaction mixture (150-200-µl final volumes)
containing the appropriate buffer system, 100-200 µg of microsomal
or sonic disrupted mitochondrial protein, either using 400 pmol/ml
purified rat microsomal P450 reductase or 180 pmol/ml bovine Adx
reductase essentially as described before (9, 23, 24). ERND and EROD
activities were measured according to published methods (33, 34).
In Vitro Protein Import into Isolated
Mitochondria--
cDNAs encoding +5/1A1 and +33/1A1 were cloned in
pGEM7Zf plasmid and used as templates to program the synthesis of
35S-labeled in vitro translation products in a
Promega TNT coupled system essentially as described (22). In
vitro mitochondrial import was carried out in 200-µl reaction
volumes consisting of 2-4 µl of reticulocyte lysate translation
products (~80,000 cpm), 500 µg of freshly isolated rat liver
mitochondria, 60 µl of energy mix, and 70 µl of transport buffer as
described previously (35, 36). The final mitochondrial pellet was
dissolved in Laemmli sample buffer (37), analyzed by SDS-polyacrylamide
gel electrophoresis, and subjected to fluorography. Digitonin
fractionation of mitochondria before or after in vitro
incubation was carried out essentially as described before using 75 µg of digitonin/mg of protein (13, 38), which selectively eliminates
the outer membrane without any measurable loss of the inner membrane
matrix components (38). Fractionation of mitochondrial proteins into
alkaline-soluble and -insoluble components was carried out using 0.1 M Na2CO3 buffer (pH 11) as
described before (23, 39).
In Vitro Protein Synthesis with Isolated
Mitochondria--
Protein synthesis with isolated mitochondria was
carried out essentially as described before (40). Digitonin-treated
mitochondria from transfected COS cells and erythromycin-treated C6
glioma cells were suspended in a medium containing 0.25 M
sucrose, 10 mM HEPES (pH 7.4), 100 mM KCl, 10 mM MgCl2, 10 mM potassium phosphate (pH 7.4), and 5 mM
-mercaptoethanol at 2 mg of
protein/ml. The medium was supplemented with 2 mM ADP, 2 mM GTP, 5 mM creatine phosphate, 0.2 mg/ml
creatine phosphokinase, 2 mM succinate, 1 mM
isocitrate, 200 µg/ml cycloheximide, and 100 µM each of
19 L-amino acids except methionine. When required, ATP was
added at a final concentration of 2 mM. The mixture was
preincubated by gentle shaking for 5 min at 32 °C with added
erythromycin or chloramphenicol as indicated in the legend to Fig. 3.
At the end of preincubation, protein synthesis was initiated by adding
100 µCi/ml [35S]Met (1175 Ci/mmol; NEN Life Science
Products), and the incubation was continued for 45 min. At intervals,
aliquots (2 µl) were removed and used for determining the extent of
[35S]Met incorporation by the hot trichloroacetic acid
method (40). The labeled proteins (100 µg) were dissociated in
Laemmli (37) sample buffer at 37 °C for 60 min and electrophoresed
on 15-20% exponential gradient SDS-polyacrylamide gel
electrophoresis. The translation products were quantitated by using a
Bio-Rad GS-525 molecular imager.
In Vivo Mitochondrial Protein Synthesis in 1A1-transfected COS
Cells--
COS cells were transfected with Adx and P4501A1 cDNAs
as described before (22). After 62 h of transfection, cells were
washed with Met-free medium and incubated for 10 min with cycloheximide (200 µg/ml). This was followed by a 1-h incubation with varying concentrations of erythromycin, and labeling was carried out with [35S]Met (100 µCi/plate) for 4 h. Cells were
washed and homogenized gently in mitochondrial isolation buffer, and
mitochondria were isolated by differential centrifugation as described
above. 50 µg of mitochondrial protein was subjected to
electrophoresis on a 15-20% exponential gradient SDS-polyacrylamide
gel (41, 42). The translation products were quantitated by using a
Bio-Rad GS-525 molecular imager.
Northern and Western Blot Analysis--
Northern hybridization
was carried out using 30 µg of total RNA from treated or untreated C6
glioma cells using moderate stringency conditions as described before
(19). Western immunoblots were developed with polyclonal antibody
against P4501A1 (23) using the Pierce Super Signal ULTRA
chemiluminescent substrate kit and imaged, and the blots were
quantitated in a Bio-Rad Fluor-S imaging system.
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RESULTS |
Membrane Topology and Intramitochondrial Location of
P450MT2--
As reported recently, P450MT2 purified from the similarly
induced liver mitochondria resolves as two closely migrating proteins of about 51-54 kDa, both of which cross-react with antibody to P4501A1
(22, 23). Furthermore, N-terminal and also internal peptide sequencing
(22) showed that the two mitochondrial P450 species have primary
sequence similar to the microsomal P4501A1. The slower migrating
P450MT2A lacks the terminal 4 amino acid residues (+5/1A1), while the
faster migrating P450MT2B lacks the first 32 residues (+33/1A1).
The intramitochondrial location of the two P450MT2 components and their
topological organization in the mitochondrial membrane compartment was
studied by using a combination of digitonin treatment, trypsin
digestion, and extraction with alkaline buffers. It is seen from Fig.
1A that both
35S-labeled +5/1A1 and +33/1A1 are imported into isolated
rat liver mitochondria and rendered relatively insensitive to trypsin
digestion. It is also seen that both +5/1A1 and +33/1A1 proteins are
resistant to trypsin even after digitonin treatment (Fig.
1A), which removes over 80% of the mitochondrial outer
membrane, leaving relatively intact inner membrane, suggesting that
they are localized inside the inner membrane compartment. Nearly 80%
of the labeled proteins are extracted in the alkaline-soluble fraction,
suggesting that both proteins may be organized in a membrane-extrinsic
topology. Fig. 1B represents immunoblot analysis of
mitochondrial protein from +5/1A1- and +33/1A1-expressing COS cells
using CYP1A1-specific antibody. It is seen that both with +5/1A1 and
+33/1A1, over 80% of the antibody-reactive protein associated with the
mitochondrial membrane fraction was resistant to trypsin digestion and
was soluble in alkaline Na2CO3 buffer. In
support of our observation with BNF-induced rat liver mitochondria
(23), the results of the present study with the isolated mitochondrial
import system (Fig. 1A) and also transient transfection in
COS cells (Fig. 2B) show that
both of the in vivo expressed MT2 components (+5/1A1 and +33/1A1) are localized within the mitochondrial inner membrane, probably as membrane-extrinsic proteins. These results provide further
confirmation for the mitochondrial targeting of P4501A1 and also
possible differences in the topological properties of the P450 protein
targeted to the two membrane compartments.

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Fig. 1.
Membrane topology of N-terminal truncated
P4501A1 targeted to mitochondria under in vitro and
in vivo conditions. A,
35S-labeled +5/1A1 and +33/1A1 proteins were generated in
the transcription-linked TNT system and used for in vitro
transport in isolated rat liver mitochondria as described under
"Materials and Methods." Reisolated, washed mitochondria ( Trypsin Control) were subjected to trypsin
digestion before (+ Trypsin) or after (+ Digitonin + Trypsin) digitonin fractionation. The
digitonin-treated and trypsin-treated mitochondria were further
subjected to alkaline extraction. 100 µg each of mitochondrial
samples or a 100-µg equivalent of alkaline-soluble and -insoluble
proteins was subjected to electrophoresis and imaging in a Bio-Rad
GS525 molecular imager. B, COS cells were transfected with
+5/1A1 or +33/1A1 cDNA constructs. Mitochondrial isolates from the
resulting cells were subjected to treatments similar to that in
A. 50 µg of mitochondrial protein or a 50-µg protein
equivalent of alkaline-soluble and -insoluble fractions was subjected
to Western immunoblot analysis using polyclonal antibody against
P4501A1 as described under "Materials and Methods."
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Fig. 2.
EROD activity of P4501A1 targeted to
mitochondria and microsomes. COS cells were transfected with
vector alone (Mock transfected) or WT/1A1
(1A1 cDNA) and Adx cDNA constructs and used for
isolating mitochondrial and microsomal fractions by the sucrose density
banding method described under "Materials and Methods." The protein
fractions were assayed for EROD activity either in the presence of
added Adr or P450 reductase as indicated. The mean ± S.E. was
calculated from three separate transfection experiments, assayed in
duplicate.
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Catalytic Profiles of P450 Targeted to the Mitochondrial and ER
Compartments in 1A1-expressing COS Cells--
Previous results from
our laboratory showed that whole mitochondrial isolates and also
partially purified P450MT2 from BNF-induced liver exhibit high ERND
activity in an Adx + Adr-supported system. The bacterially expressed
+33/1A1, resembling P450MT2, also showed high ERND activity, while the
nearly intact +5/1A1 showed low activity (24). In the present study,
direct evidence for the differential catalytic properties of P450
targeted to the two subcellular membrane compartments was sought by
using the COS cell expression system. COS cells contain relatively low
endogenous levels of Adx, Adr, and P450 reductase and no detectable
P450 (43), thus providing a valuable cell system to study the catalytic properties of the transiently expressed P450s and their electron transport protein requirements. COS cells were transfected with the
WT/1A1 cDNA, or various mutant constructs with or without co-transfection with the bovine Adx cDNA construct. Mitochondria and microsomes were isolated by a combination of differential centrifugation and density banding methods (13, 22), which was shown to
yield a nearly qualitative separation of the two membrane fractions
with no detectable cross-contaminating marker proteins. The effects of
overexpression of the mitochondrial targeted P450MT2 alone or in
combination with Adx were examined by reconstituting the activity
either with Adr or P450 reductase. We have assayed ERND activity and
compared it with the EROD activity of the two subcellular fractions,
since the latter is believed to be a marker for the microsomal P4501A1
(44).
Results in Table I show that expression
of intact 1A1 or +33/1A1 proteins, and reconstitution with Adr yielded
marginal ERND activity (0.1-0.2 nmol) in both mitochondria and
microsomes. Coexpression with Adx cDNA in both cases yielded about
5-7-fold higher Adr-dependent activity with the
mitochondrial but not the microsomal fractions. These results are
consistent with the ERND activity profiles of purified microsomal
P4501A1 in a P450 reductase-supported system and purified P450MT2 or
bacterially expressed +33/1A1 in Adx + Adr-supported systems,
respectively (23, 24). These results are also in accordance with the
observations that cells transfected with wild type 1A1 yield the two
forms of mitochondrial P450s, including the putative +33/1A1 (MT2b)
with high ERND activity in an Adx + Adr-supported system. Following the
entry into mitochondria, the latter species interacts efficiently with
mitochondrially targeted Adx to metabolize erythromycin, while the
+5/1A1 has very low ERND activity (24). Expression of mutant forms,
Mut/1A1 and +33/Mut, yielded activities similar to those of cells
transfected with the Adx cDNA alone. The mitochondrially targeted
P450MT2 also showed a significant but reduced ERND activity when
reconstituted with the P450 reductase electron donor system. As
expected, the P450 reductase-supported activity was not affected
by co-expression with Adx cDNA. The microsomal fraction in both Adx + Adr-supported and NADPH P450 reductase-supported systems showed a
marginal activity. These latter results are consistent with the
reported low ERND activity of the microsomal P4501A1 (23, 24).
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Table I
Altered substrate specificity of P450 targeted to the mitochondrial
compartment by transfection in COS cells
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The EROD activities of the mitochondrial and microsomal fractions from
COS cells transfected with P4501A1 cDNA in the Adx + Adr-supported
and P450 reductase-supported systems are presented in Fig. 2. In
1A1-expressing cells, the mitochondrial EROD activity when
reconstituted with Adr was induced about 3-fold by co-expression with
the Adx cDNA. As expected, Adx co-expression had no significant effect on the microsomal EROD activity. Reconstitution with P450 reductase protein, however, yielded a low, but significant activity (2.5 pmol/min/mg protein), while the microsomal fraction showed a high
activity in the range of 70-80 pmol/min/mg protein. It should be noted
that P450 reductase-supported EROD activity with both the mitochondrial
and microsomal enzymes was not affected by Adx cDNA co-expression.
These results show that the mitochondrial localized P450 can receive
electrons from both P450 reductase and Adx + Adr electron transfer
systems. Our results also show that in an Adx + Adr-supported system,
the truncated mitochondrial form, +33/1A1 exhibits substrate
specificity and catalytic activity somewhat different from its more
intact microsomal counterpart, reconstituted with the P450 reductase system.
Reversal of Erythromycin-induced Mitochondrial Translation by
P450MT2--
The inhibitory effects of chloramphenicol, streptomycin,
and related macrolide antibiotics, including erythromycin, on the mitochondrial translation systems from various cell sources have been
well established (25-30). Because of the observed ERND activity of
P450MT2, we investigated its ability to provide protection against
erythromycin-induced inhibition of mitochondrial translation. Fig.
3A shows the effects of 120 µg/ml erythromycin on protein synthesis by mitochondria from COS
cells expressing Adx alone or Adx plus wild type or Mut/1A1 proteins.
The pattern of total [35S]Met incorporation in isolated
mitochondria shows nearly complete reversal of mitochondrial
translation inhibition by co-expression with WT/1A1 and Adx cDNAs,
but co-expression with Mut/1A1 cDNA had no protective effect. As
shown in the autoradiogram in Fig. 3A, mitochondria from
Adx-expressing cells incorporated [35S]Met into
characteristic mitochondrial translation products. The identification
and designation of 13 individual polypeptide species is based in part
on immunoprecipitation with specific antibodies (45, 46) and follows
the nomenclature and electrophoretic patterns reported by Chomyn
et al. (41). It is seen that in mitochondria from
Adx-expressing cells, erythromycin (120 µg/ml) inhibited the
mitochondrial translation by 70-80% as seen by the reduced band
intensities and also the total trichloroacetic acid-insoluble radioactivity presented in Fig. 3B. The gel pattern in Fig.
3A also shows that ND3 and ATPase 8 proteins appear to be
major translation products, suggesting a relatively higher translation
efficiency of these subunits under the in vitro conditions.
Chloramphenicol (200 µg/ml), which is a well known inhibitor of
mitochondrial specific translation also inhibited the activity of
mitochondria from control (Adx-expresssing) cells (see Fig.
3A, last lane). Co-expression with
Mut/1A1 cDNA with altered mitochondrial targeting ability had no
effect on the erythromycin-induced inhibition of mitochondrial
translation (see lanes 3 and 4).
Co-expression with WT/1A1 cDNA, however, resulted in a nearly
complete reversal of translation inhibition. As shown in Fig.
4, A-F, autoradiograms from
four independent cell transfection and mitochondrial translation experiments, as in Fig. 3A, were used to quantitate the
levels of [35S]Met incorporation into individual
mitochondrial translation products, namely CO II + CO III (Fig.
4A), ND1 (B), ND2 + cytochrome b5 (C), ND4 + CO I (D),
ND3 (E), and ATPase 8 (F). Results show that all
of these subunits were inhibited by 120 µg/ml of erythromycin by
about 70-80%, and their inhibitory effects were fully reversed by
co-expression with WT/1A1 but not Mut/1A1 cDNA.

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Fig. 3.
Effects of mitochondrial targeted P450MT2 on
erythromycin-mediated translation inhibition. COS cells were
transfected with Adx alone or in combination with Mut/1A1 (R34D and
K39I) or WT/1A1 cDNAs, and mitochondria were isolated by sucrose
density banding. A, isolated mitochondria were used for
in vitro translation in the presence of added
[35S]Met, with or without added 120 µg/ml erythromycin
as described under "Materials and Methods." In one reaction, 120 µg/ml chloramphenicol was added as a positive control. Labeled
proteins (100 µg each) were resolved by electrophoresis on a 15-20%
exponential SDS-polyacrylamide gel, and the gel was imaged in a Bio-Rad
GS-525 molecular imager. B shows the total
[35S]Met incorporation by isolated mitochondria from
various cDNA-transfected COS cells in the presence of varying
levels of erythromycin.
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Fig. 4.
Extent of erythromycin inhibition and
reversal on individual mitochondrial translation products.
In vitro translation products with isolated COS cell
mitochondria as in Fig. 6, with or without the indicated added levels
of erythromycin were resolved on 15-20% exponential gels.
Radioactivity in individual bands was quantitated by scanning the gel
in a Bio-Rad GS-525 phosphor imager. A, CO II + CO III;
B, ND1; C, ND2 + cytochrome
b5; D, ND4 + CO I; E, ND3;
F, ATPase 8.
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The ability of P450MT2 to reverse the erythromycin inhibition of
mitochondrial translation in whole cells was investigated using the COS
cell expression system. As shown in Fig.
5, cells transfected with various
cDNA constructs for 62 h were labeled with
[35S]Met in the presence of 200 µg/ml cycloheximide to
suppress cytosolic translation or in combination with cycloheximide and
the indicated amounts of erythromycin. The results show that in
cycloheximide-treated cells, the mitochondrial translation continues as
seen by the characteristic electrophoretic pattern (lane
1) and size distribution of proteins, similar to the gel
profile of in vitro translation products in Fig.
3A. A notable difference from the in vitro
translation pattern (Fig. 3A) was that ND4L and ATPase 8 are
translated as minor products under the in vivo translation
conditions (see Fig. 5A). Currently, reasons for this
difference remain unclear. The gel pattern in Fig. 5A and
quantitation in Fig. 5B also show that mitochondrial
translation was inhibited by 85-90% by the addition of 120 µg/ml
chloramphenicol or thiamphenicol (lanes 8 and
9). Erythromycin inhibited mitochondrial protein synthesis
in a dose-dependent manner (lanes 2 and 3). Co-expression with the WT/1A1 construct (lanes 6 and 7) but not the Mut/1A1
cDNA (lanes 4 and 5) reversed the
erythromycin effect in these cells. These results collectively show
that in both intact cells and isolated mitochondria, P450MT2 can
provide protection against erythromycin-induced mitochondrial translation possibly by inducing the metabolic inactivation of erythromycin into its demethylated form.

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Fig. 5.
Reversal of erythromycin-mediated inhibition
of mitochondrial translation in vivo in COS cells
overexpressing P4501A1. COS cells were transfected with various
cDNA constructs as in Fig. 6 and at the top of
individual lanes of the gel in A. 62 h after
transfection, cells were labeled for 4 h with
[35S]Met in the presence of 200 µg/ml cycloheximide to
suppress cytoplasmic translation. Erythromycin, thiamphenicol, and
chloramphenicol were added about 15 min before the addition of
[35S]Met. +, indicates 60 µg of the inhibitor; ++, 120 µg of the inhibitor. Mitochondrial isolates from labeled cells (50 µg of protein each dissociated in Laemmli sample buffer) were
subjected electrophoresis and imaging as described in Fig. 5.
B shows the total [35S]Met incorporated in
mitochondrial proteins. The values represent mean ± S.E. from
three separate transfection experiments.
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Induction of mRNA and Mitochondrial P450MT2 Levels by
Erythromycin Treatment--
A hallmark of class I family
drug-metabolizing monooxygenases is that the steady state levels of a
given P450 enzyme are induced severalfold by its cognate xenobiotic
substrate (47). Induction of 1A family enzymes by diverse polycyclic
aromatic hydrocarbons, of the 2B family by phenobarbital, and of the 3A
family by dexamethazone and related glucocorticoids (48) are some
examples. We therefore decided to see if erythromycin had any effect on
CYP1A1 mRNA and its steady state levels in the mitochondrial
fraction. We have used rat glioma C6 cells for this study because of
the known response of these cells to CYP1A1-specific inducers (49, 50).
Results in Fig. 6A
(top) show that erythromycin doses that are shown to inhibit
mitochondrial translation in COS cells (see Figs. 3 and 5) also induced
the steady state levels of P4501A1 mRNA (lane 3) compared with untreated control cells, which contain a
low level of the mRNA (lane 1). The extent of
induction with erythromycin was similar to that obtained with the well
known P4501A1 inducer, BNF. The 18 S rRNA hybridization pattern in Fig.
6A (bottom) indicate a similar RNA loading in all
three cases. Neither erythromycin nor BNF had any inducible effect on
the P4503A1/2 mRNA level (Fig. 6A, middle).
This was surprising in view of the fact that P4503A1 is a marker enzyme
for the microsomal ERND activity (47, 48).

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Fig. 6.
Induction of P4501A1 mRNA and
mitochondrial P450MT2 by erythromycin in C6 glioma cells. C6
glioma cells were cultured with added erythromycin (30 µM) or BNF (12 µM) for 92-96 h, and the
cells were used for total RNA isolation or isolation of mitochondria
using the sucrose density gradient method. A, Northern blot
analysis using 30 µg of total RNA from each of the untreated or
treated cells. The same blot was sequentially hybridized with
32P-labeled rat CYP1A1 cDNA probe, followed by
stripping and hybridization with 32P-labeled rat 3A1
cDNA and 18 S rDNA probes. B, mitochondrial and
microsomal proteins (50 µg each) from Me2SO
(DMSO) control cells or cells treated with BNF or
erythromycin (ERM) were subjected to Western blot analysis
using polyclonal antibody to P4501A1. The blot was developed with the
Super Signal Ultra chemiluminiscence kit as described under
"Materials and Methods" and imaged and quantitated in a Bio-Rad
Fluor-S imager. The relative levels of antibody-reactive P450MT2 in the
mitochondrial fractions of treated and untreated cells are presented at
the bottom of the immunoblot. The values are averages of two
separate immunoblots. C, the ERND activities of
mitochondrial isolates from control Me2SO-treated cells,
and cells treated with BNF and erythromycin. The results represent the
average of two independent experiments. D shows the effects
of 120 and 240 µg of erythromycin on the [35S]Met
incorporation by mitochondrial isolates from control
Me2SO-treated cells and cells treated with 50 µM erythromycin for 92 h as described for
A above. Total [35S]Met incorporation was
measured by extraction with hot trichloroacetic acid as described under
"Materials and Methods."
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The Western blot results in Fig. 6B using antibody to
P4501A1 show that erythromycin-treated cells contain 6-fold higher
levels of P4501A1 antibody-reactive protein in the mitochondrial
compartment as against a marginal (~2-fold) increase in the
microsomal fraction. BNF treatment, on the other hand, resulted in a
higher level of induction of the microsomal 1A1 protein pool, as
compared with the mitochondrial fraction. These results suggest a
preferential induction of P450MT2 by erythromycin treatment. In keeping
with the increase in antibody-reactive protein level, the mitochondrial ERND activity in erythromycin-treated cells was also increased by over
4-fold (Fig. 6C). As expected, the increased ERND activity in treated cells rendered protection against erythromycin-mediated inhibition of mitochondrial translation (Fig. 6D). These
results together demonstrate that in susceptible cells, erythromycin
induced both the CYP1A1 mRNA level and also preferentially elevated
the level of mitochondrial P450MT2.
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DISCUSSION |
Erythromycin, a 14-membered ring macrolide, is well known for its
antibacterial and anti-inflammatory effects in different target
tissues. Its pharmacological potency is known to reside in its ability
to selectively inhibit bacterial protein synthesis (27, 28). In keeping
with the hypothesis on the endosymbiotic origin of mitochondria (51,
52), erythromycin also inhibits mitochondrial translation (26-29). It
is also suggested that the anti-inflammatory action of this antibiotic
may reside in its ability to induce NO production in endothelial cells
and also its ability to modulate interleukin-8 gene expression (53). In
this paper, we demonstrate that overexpression of P4501A1 either by its
cognate inducer or by transfection with expression cDNA constructs
effectively rendered protection against erythromycin-mediated inhibition of mitochondrial protein synthesis. The protective effects
of 1A1 specifically stem from the N-terminal truncated P450 targeted to
mitochondria, which exhibits a remarkably high ability to metabolize
erythromycin into inactive demethylated product.
Studies reported over the past 10 years showed that hepatic
mitochondria from BNF- and PB-treated rats contain P450 proteins cross-reacting with the antibody to similarly induced major microsomal P450 forms (9, 14, 23). Although the putative mitochondrial forms
exhibited electrophoretic migration similar to the microsomal counterparts, the former species showed preference for mitochondrial specific electron transfer proteins, Adx and Adr, for enzyme
reconstitution, suggesting that they may be different molecular forms.
A recent study (22) on P4501A1 in our laboratory showed that N-terminal truncation of the protein by a cytosolic endoprotease activates the
cryptic mitochondrial targeting signal at the N-terminal 32-44 sequence region of the protein. Our results also showed that the bacterially expressed +33/1A1 not only had a higher affinity for Adx
binding but also high ERND activity in an Adx- and Adr-supported system, suggesting a possible conformational change in the P450 protein
devoid of the N-terminal transmembrane domain. Results presented in
this study confirm and extend our previous observations on the
mitochondrial targeting of P4501A1 and also its altered activity in the
mitochondrial compartment. Our results show that expression of wild
type 1A1 or +33/1A1 by transient transfection in COS cells results in
the accumulation of antibody-reactive protein in mitochondria and
increased ERND activity in the mitochondrial compartment.
Interestingly, coexpression with mitochondrially targeted Adx protein
resulted in a severalfold higher activity, further confirming the
intramitochondrial location of the P450 and hence the ERND activity.
Furthermore, mutations targeted to the putative mitochondrial targeting
sequence abolished both mitochondrial accumulation of antibody-reactive
protein and the ERND activity, further confirming the mitochondrial
targeting of the N-terminal modified version of P4501A1.
Consistent with the ERND activity of the mitochondrial targeted P4501A1
in an Adx + Adr-supported system, P450 targeted to the COS cell
mitochondrial compartment under the transient transfection conditions
is detected as membrane-extrinsic protein (see Fig. 2B).
Similar is the case with both +5/1A1 and +33/1A1 proteins imported into
isolated mitochondria. It is not clear why +5/1A1 with nearly complete
transmembrane domain assumes a membrane extrinsic topological
arrangement within the mitochondrial membrane compartment, although the
mitochondrial matrix environment may favor this organization. In any
case, this mode of organization is highly conducive for interaction
with soluble electron transfer proteins Adx and Adr. These results
support and extend previous observations of our own and others that
mitochondrial monooxygenase activities with either total mitochondrial
membrane fragments or purified proteins (9-17, 23, 24) are
preferentially supported by soluble electron transfer proteins Adx + Adr but very poorly by P450 reductase.
Until recently, it was widely believed that the microsomal P450s
require microsomal cytochrome P450 reductase for transfer of electrons
from NADPH, while the mitochondrial P450s require mitochondrial
specific Adx + Adr for substrate oxidation. A number of exceptions to
this generality have recently been reported. Bacterially expressed
microsomal P450c17 was fully active with bacterial flavodoxin and
flavodoxin reductase, and also flavodoxin induced a change in the spin
state of P450 indicating productive physical association (54).
Similarly, Dong et al. (55) showed that N-terminal truncated
bacterially expressed human P4501A2 is active with bacterial flavodoxin
as well as mitochondrial Adx and Adr electron transfer proteins. In
extension of these observations, we showed that wild type Adx but not
mutant forms physically interact with the bacterially expressed,
N-terminal truncated P4501A1 as well as that purified from BNF-induced
mitochondria as shown by chemical cross-linking, a change in the spin
state of P450, and reconstitution of enzyme activity (24). Parallel
results from various laboratories also show that N-terminal modified
mitochondrial P450c27 targeted to the ER can be efficiently
reconstituted with microsomal P450 reductase
(56).2 Similarly, removal of
N-terminal transmembrane domain of microsomal P4501A2 and -2E1 resulted
in a 40-50% reduced activity in a P450 reductase-supported system,
suggesting inefficient interaction (55, 57). Furthermore (57, 58),
N-terminal truncation of P450 reductase significantly reduced its
ability to support the activity of microsomal P450s (57-59),
suggesting that membrane anchoring of both the P450 and the reductase
proteins is necessary for optimal activity. Results emerging from
various studies, including ours, therefore suggest that some of the
microsomal P450 forms can effectively interact with Adx + Adr electron
transfer proteins when they are organized in a membrane-extrinsic mode.
Some of the mitochondrial P450s attached to a transmembrane anchor
domain and organized in a transmembrane topology, on the other hand, can interact with membrane-anchored P450 reductase.
Although some of the xenobiotic-inducible hepatic microsomal P450 forms
have been shown to exhibit varied levels of steroid hydroxylase
activities (60), the precise physiological roles in most cases remain
unclear. A number of studies suggest that some of the
xenobiotic-inducible P450s may have roles in the manifestation of
oxidative stress, while other studies imply a protective role against
oxidative stress (61, 62). We therefore decided to determine if the
altered activity of the mitochondrial targeted P4501A1 has any
physiological significance. An exciting new observation of this study
is that erythromycin treatment not only induced the mRNA level but
also caused a preferential accumulation of antibody-reactive protein in
the mitochondrial compartment (Fig. 6). Erythromycin is known to
modulate or potentiate the pharmacological effectiveness of various
drugs, including astemizole, carbamazepine, corticosteroids, warfarin,
etc., by yet unknown mechanisms (63-65). The finding of this study
that erythromycin acts as a classical P450 inducer provides a rational
bases for its reported modulatory effects on other drugs.
Many hepatic P450 forms such as P450IID family members play a central
role in modulating the pharmacological potencies and efficacies of many
drugs and antibiotics. Thus, evaluating interpatient differences in
P450 levels and genotyping specific P450 forms are becoming major
developments in human medicine. In fact, Watkins et al. (66)
have recently developed a breath test to measure the rate of metabolism
of [14C]erythromycin as a measure of
glucocorticoid-inducible hepatic P4503A1/2 level. In view of our
present results showing high ERND activity of P4501A1 targeted to
mitochondria, mitochondrial targeting of various P450 forms, and their
altered substrate specificity (24), the catalytic properties of the
mitochondrial targeted forms should also be taken into account both in
the development of tests and in the interpretation of test results. In
summary, this study provides the first evidence for the physiological
role of mitochondrial targeted P450MT2 in drug detoxification and
protection against erythromycin-induced inhibition of mitochondrial
protein synthesis.