Laboratories of Biochemistry, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced
by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b,
are also found localized in mitochondria from -naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal
that facilitates the targeting of the protein to both the
ER and mitochondria. The NH2-terminal 30-amino
acid stretch of P4501A1 is thought to provide signals
for ER membrane insertion and also stop transfer. The
present study provides evidence that a sequence motif
immediately COOH-terminal (residues 33-44) to the
transmembrane domain functions as a mitochondrial
targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting.
Results suggest that 25% of P4501A1 nascent chains,
which escape ER membrane insertion, are processed by
a liver cytosolic endoprotease. We postulate that the
NH2-terminal proteolytic cleavage activates a cryptic
mitochondrial targeting signal. Immunofluorescence
microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as
tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as
to the mitochondria.
PROTEIN targeting to the mitochondrial and ER compartments follow distinct pathways and involve different primary translation sites, targeting signals,
and transport machinery (Schatz and Dobberstein, 1996 The hepatic cytochrome P450s are mostly localized on
the ER (hereafter referred to as microsomes), though
some of the constitutive as well as inducible forms are also
found in the mitochondrial compartment. The occurrence
of xenobiotic inducible cytochrome P450 (P450) forms in
the hepatic (Niranjan and Avadhani, 1980 Tissue Fractionation and P450 Purification
Mitochondria and microsomes were isolated from the treated or untreated Sprague Dawley rat livers described before (Niranjan et al., 1984 Bacterial expression of +5/1A1 and +33/1A1 was carried out using the
PCW vector as described before (Imai et al., 1993 Peptide Fingerprint Analysis and Amino
Acid Sequencing
Purified P450MT2 and the microsomal P4501A1 were resolved by electrophoresis on 14-16% gradient polyacrylamide gels by overrunning. Protein
bands (P450MT2a/MT2b and P4501A1) were transferred to PVDF membrane and subjected to NH2-terminal sequencing (model 475A sequencer;
Applied Biosystems, Inc., Foster City, CA). Proteins immobilized on
polyvinyldifluoride (PVDF) membranes were subjected to trypsin digestion, and the peptides were recovered and resolved on a reverse-phase microbore HPLC column using 80% acetonitrile and 10% TFA solvent system. Different peak fractions were collected, lyophilized, and subjected to
NH2-terminal sequencing.
Preparation of Antibody and Subcellular
Protein Fractions
A high titer polyclonal antibody in rabbits was raised against >95% homogeneous P450MT2 using standard protocols (Parkinson and Gemzik,
1991 The rat liver cytosolic fraction, without added protease inhibitors, was
fractionated with (NH4)2SO4, and the proteins precipitating between 18 and 40% saturation were collected by centrifugation at 100,000 g for 30 min. The protein preparation was dialyzed against 20 mM KH2PO4, pH
7.4, 20 mM KCl, 1 mM EDTA, and 0.5 mM DTT, suspended in the same
buffer, and stored in Construction of Expression Plasmids
P4501A1 cDNA (Yabusaki et al., 1984 In Vitro Protein Transport into Isolated Mitochondria
cDNAs encoding the wild-type P4501A1, various NH2-terminal truncated
forms, point mutations, 1A1Mut, +33/Mut, and 1A1M32/33 constructs
generated as described above were cloned in pGEM7Zf plasmid
(Promega Biotech, Madison, WI), and were used as templates for generating the 35S-labeled translation products. 1-2 µg of circular plasmid DNAs were used as templates in a Sp6 or T7 polymerase-coupled reticulocyte lysate transcription translation system in the presence of [35S]methionine
(40 µci/50µl/reaction, 1,000 Ci/mmol; Amersham Corp., Arlington Heights,
IL) using the protocol recommended by Promega Biotech. Import of the
in vitro-synthesized proteins into the mitochondria was carried out by a
procedure modified from that of Gasser et al. (1982) Expression of cDNA in COS Cells
cDNAs encoding the wild-type protein, NH2-terminal deletions, and point
mutations were cloned in the proper orientation in the HindIII and Xbal
sites of a mammalian expression vector pCMV4 (Andersson et al., 1989 Indirect Immunofluorescence Microscopy
COS cells were grown on coverslips, transfected with various cDNA constructs, and processed for antibody staining essentially as described by
Rizzolo et al. (1985) Sequence Characteristics of the
Mitochondrial P450MT2
Using a high resolution gradient polyacrylamide gel system, initially we found that the mitochondrial and microsomal P450s purified from BNF-induced rat livers differed in electrophoretic mobility. The Coomassie-stained
gel pattern in Fig. 1 A shows that the mitochondrial
P450MT2 resolves into two distinct components marked
as MT2a and MT2b. The larger component, MT2a, comigrated with bacterially expressed purified +5/1A1 and
with intact 1A1 purified from BNF-induced rat liver microsomes. MT2a (+5/1A1) and P4501A1 were not separable on the gel electrophoresis system used in this study.
MT2b comigrated with bacterially expressed purified +33/
1A1. The Western blot in Fig. 1 B shows that both MT2a
and MT2b cross-react with antibody to microsomal P4501A1. As expected, bacterially expressed +33/1A1, +5/1A1, and
purified, intact P4501A1 cross-reacted with the antibody
(Fig. 1 B). Although +33/1A1 and intact +1A1 vary by ~4
kD in mass, we experienced difficulty in clearly resolving
these two proteins because of the unusual migration of the
truncated form on SDS gels. As shown in Fig. 1 C, mitochondria from BNF-treated rat livers contained two differently migrating 1A1 antibody-reactive proteins that comigrated with the major components of purified P450MT2.
Additionally, a companion Western blot using antibody to
P450c27 detected an intact 52-kD protein that comigrated
with purified P450c27. The two antibody-reactive bands in
the purified P450MT2 closely resemble the proteins detected in intact mitochondria from BNF-induced liver and
the mitochondrial species generated by in vivo expression of intact 1A1 protein in COS cells, as described later in
this section (see Fig. 3). These results therefore suggest
that the two closely migrating proteins in the purified
P450MT2 are not likely caused by proteolytic degradation
during protein purification.
As seen from Fig. 2 A, the protein recovered from the
faster migrating MT2b band yielded peptide fragments
nearly identical to that of P4501A1. Although not shown,
the slower migrating MT2a also yielded a nearly similar
peptide pattern. NH2-terminal sequencing, as shown in
Fig. 2 B, and sequencing of internal peptides (results not
shown) revealed that both MT2a and MT2b have primary
sequences to that of P4501A1, except that MT2b lacks the
NH2-terminal 32-amino acid residues and MT2a lacks the
NH2-terminal four-amino acid residues. Different P450MT2
preparations from induced livers showed the +5/1A1 to
+33/1A1 ratios from 20:80 to 40:60. Although the precise
reason for this variation remains unknown, the age of the
animals and the length of BNF treatment are some of the
factors that affect this ratio. However, both of these species are true mitochondrial forms since they are resistant
to limited protease digestion of both mitochondrial and
mitoplast preparations under conditions when >95% of
the microsomal P4501A1 is degraded (Anandatheerthavarada et al., 1997
As shown in Fig. 2 C, the NH2-terminal transmembrane
domain of P4501A1 is followed by a Pro-rich region that
acts as a hinge between the membrane anchor domain and
the rest of the molecule, which is folded in the form of a
globular structure. A number of xenobiotic-inducible P450s,
including P4501A1, also contain two to three positively
charged residues between the NH2-terminal hydrophobic
domain and the Pro-rich hinge region. As seen in Fig. 2, B
and C, P4501A1 contains three positively charged residues at positions 34, 39, and 42, thus, mimicking some of the
known mitochondrial targeting sequences (Roise et al.,
1988 Mitochondrial Targeting of P450MT2 under
In Vivo Conditions
The possibility that the sequence 1-44 of P4501A1 acts as
a chimeric signal sequence with residues 33-44 of the protein functioning as a mitochondrial-specific targeting sequence was tested by in vivo transient expression of
cDNA constructs in COS cells. Cells were transfected with
cDNAs encoding intact P4501A1, and its truncated forms,
+5/1A1 and +33/1A1, in pCMV4 expression vector (Andersson et al., 1989 To determine the extent of cross-contaminating membrane fragments in the mitochondrial and microsomal
fractions isolated from transfected COS cells, we carried
out a control experiment in which cells were transfected
with intact 1A1 reporter construct and were coexpressed
with human P450 reductase and bovine Adx expression
constructs. P450 reductase is targeted exclusively to the
ER, and Adx targeted exclusively to mitochondria (Mitani, 1979
Mitochondrial targeting of P4501A1 was further ascertained using immunofluorescence microscopy. COS cells
transfected with 1A1 cDNA or +33/1A1 cDNA constructs
were subjected to double immunostaining with rabbit
polyclonal antibody to 1A1 and mouse mAb to COX subunit I. As seen from Fig. 5 A, COS cells transfected with
+33/1A1 cDNA show P4501A1 antibody cross-reactivity
mainly in punctuate filamentous to wormiform extranuclear structures that have characteristic mitochondrial
morphology. The 1A1 antibody-reactive protein also colocalized with the mitochondrial-specific COX subunit I
(Fig. 5, B and C), further supporting the mitochondrial targeting of +33/1A1 protein. In intact 1A1-expressing cells,
however, the 1A1 antibody stains were localized in granular to wormy membrane structures and more spread-out
microsomal membranes (see Fig. 5 D). As seen from Fig.
5, E and F, the COX subunit I-specific antibody colocalized with the granular/wormy membrane structures, further ascertaining that they represent mitochondrial membranes. The mock-transfected cells, on the other hand,
show minimal staining with the 1A1 antibody but characteristic granular staining with the COX subunit I antibody
(Fig. 5, G and H). Additionally, Texas red-conjugated secondary antibody yielded no significant staining in the absence of added COX subunit I antibody, indicating the specificity of the antibody staining. These results are consistent with the cell fractionation data in Figs. 3 and 4, and
they support the dual targeting of P4501A1 to both the mitochondrial and microsomal membranes under in vivo
conditions.
In Vitro Transport of Intact and Truncated P4501A1
into Mitochondria
The sequence requirements for the mitochondrial transport of P4501A1 were further tested using an in vitro mitochondrial transport system in which resistance to limited
proteolysis and energy requirements are used as criteria
for the transport of proteins into the mitochondrial membrane compartment. Results in Fig. 6 A show that although
intact 1A1 and +5/1A1 proteins bind to mitochondrial
particles, only ~2-4% of the former and 12-15% of the
latter species were resistant to protease digestion, suggesting negligible and low levels of transport, respectively. The
+33/1A1 protein, on the other hand, exhibited a high level
of binding and a high level (40-55%) of transport, as
judged by the band intensities and radiometric quantitation. More progressive NH2-terminal deletion products,
+53/1A1 and +73/1A1 (Fig. 6 B), showed varying levels of
binding to mitochondria, although neither was transported
at significant levels inside the membrane compartment.
Consistent with the in vivo transfection data in Fig. 3, the
in vitro transport efficiency of 1A1Mut and +33/Mut proteins carrying R34D and K39I mutations (Fig. 2 B) into isolated mitochondria was extremely low, suggesting the
importance of these positively charged residues for mitochondrial targeting. These results provide further evidence
that the 12-amino acid sequence between 33 and 44 functions as a putative mitochondrial-targeting signal, and that
removal of the first 4- or 32-amino acid residues from the
intact protein positions the mitochondrial targeting signal
for efficient binding to the mitochondrial import receptors under the in vitro conditions.
As shown in Fig. 6, C and D, ~40% of the labeled +33/
1A1 protein incubated with intact mitochondria is refractory to protease digestion. Disruption of mitochondrial
membrane by treatment with Triton X-100, however, renders the in vitro-imported protein sensitive to protease action (Fig. 6 D). It is seen that labeled protein incubated
with the microsomal fraction under these conditions is
nearly completely degraded in 30 min of protease digestion (see Fig. 6 C). As shown in Fig. 6 D, both CCCP (carbonyl cyanide-m-chlorophenylhydrazone), which disrupts
the mitochondrial membrane potential, and also oligomycin, which inhibits the mitochondrial ATP synthase leading to the depletion of the ATP pool, inhibit the transport.
Consistent with the established views on energy requirements for mitochondrial protein transport (Glick and
Schatz, 1991 Processing of P4501A1 by a Cytosolic Protease
The site of conversion of P4501A1 to +5/1A1 and +33/
1A1 was investigated by incubating 35S-labeled translation
products with (NH4)2SO4-fractionated rat liver cytosolic
protein. It is seen in Fig. 7 A that the addition of protein
fraction from the rat liver cytosol converted P4501A1 protein into components that resemble P450MT2a and MT2b
(+5/1A1 and +33/1A1, respectively). However, the same
protein fraction had no effect on the migration pattern of
+5/1A1 protein, suggesting that intact 1A1 NH2 terminus
is important for the proteolytic processing. These results
are in accordance with our in vivo transfection data, showing that only intact 1A1 protein, but not +5/1A1 protein, was converted to faster migrating components when tested
in the whole-cell extract and mitochondrial fraction (Fig.
3). Additionally, as seen in Fig. 7 A, the liver cytosolic protein was unable to process 1A1 protein carrying V32A and
T33I mutations (referred to as 1A1M32/33) into faster migrating components. Results in Fig. 7 B show that the processing activity of the control cytosolic protein fraction
(marked C) was abolished by incubation at 95°C for 5 min
(marked B), or by addition of 5 mM EDTA. Also, a protein inhibitor mix containing leupeptin, pepstatin, chymostatin, and PMSF inhibited the processing activity. In Fig.
7 C, we tested the effects of cytosolic extract on 1A1 protein, which is in association with the ER membrane. In this
experiment, 1A1 protein was translated in the presence of
an added 2.5 Eq ER/50 µl of reaction volume, and the soluble (supernate) and membrane (pellet) fractions were separated by ultracentrifugation. The results in Fig. 7 C
show that 1A1 protein from the soluble phase was efficiently converted by the cytosolic protein fraction into
components that comigrated with +5/1A1 and +33/1A1,
while 1A1 protein associated with the ER was refractory
to processing. Although not shown, >90% of radioactivity partitioning with the membrane fraction was resistant to
alkaline Na2CO3 extraction, suggesting its transmembrane
organization. In support of the results in Fig. 7 B, only the
control (C), but not the heated extract (B), was able to
process the membrane-free 1A1 protein (Fig. 7 C). These
results demonstrate that only membrane-free (but not
membrane-bound) P4501A1 is processed by the cytosolic protease. Finally, both wheat germ and reticulocyte translation extracts did not contain this protease activity (data
not shown), suggesting that it is either inactivated during
the lysate preparation or it occurs in a tissue-specific manner.
The cytosolic location of the processing protease suggested a leaky nature of the ER targeting or membrane insertion of 1A1 nascent chains. We investigated this possibility by quantitating the distribution of 1A1 nascent
chains in the soluble and membrane compartments under
in vitro conditions in the presence of saturating levels of
the ER membrane. P4501A1 and P450 reductase, a protein exclusively targeted to the ER, and DHFR, a soluble
cytosolic protein, were cotranslated in the presence of different added concentrations of canine microsomal ER.
The membrane-integrated and nonintegral labeled proteins were quantitated by extraction with 0.1 M Na2CO3,
pH 11.5. The autoradiogram in Fig. 8 A and quantitation
of radioactivity in Fig. 8 B show that in the absence of
added ER, all three proteins (i.e., 1A1, P450 reductase,
and DHFR) were quantitatively extracted in the aqueous
phase. The patterns in both the alkaline-soluble (top) and
alkaline-insoluble (bottom) fractions show an intermediary band marked ns, which we believe represents premature termination product of P450 reductase. At increasing
concentrations of added ER, increasing amounts of P450
reductase and 1A1 proteins remained in association with
the membrane fraction, while DHFR was quantitatively
extracted in the soluble fraction, even at the highest level
of ER added. At saturating levels of ER between 2.5 and 5 Eq/50 µl of reaction, P450 reductase was quantitatively
partitioned in the membrane fraction, as expected of a
protein exclusively targeted to the ER. However, under
saturating ER levels, a maximum of only 75% of 1A1 protein is detected in this fraction. Similar fractional ER association of both rabbit P4501A1 and rat P4502B1 proteins under the in vitro conditions were reported before (Sakaguchi et al., 1987
Mammalian mitochondria are evolved to carry out a variety of generalized and tissue-specific metabolic functions.
A number of biosynthetic and metabolic enzyme activities
that are found in the nuclear-cytosolic-ER compartments
are also duplicated in the mitochondrial compartment,
possibly as part of the eukaryotic evolutionary process.
Nevertheless, in almost all cases, the mitochondrial proteins are encoded by distinctly different sets of genes, and they are designed to carry either an NH2-terminal or internal signal that is recognized by the mitochondrial import
machinery. In a limited number of cases, where the mitochondrial and cytosolic proteins are encoded by the same
genes (reviewed in Jaussi, 1995 Matrix Targeting Property of the NH2-terminal Signal
Sequence of P4501A1
A majority of the mitochondrial targeting signals consist
of NH2-terminal cleavable helical amphipathic sequences
(Roise et al., 1988 Mechanism of NH2-terminal Processing and
P450MT2 Biogenesis
Based on our results suggesting extramitochondrial processing of P4501A1 and the current knowledge of protein
targeting to the ER membrane, we considered two alternate working models for the biogenesis of P450MT2 (Fig.
9). The mechanism in model 1 proposes the initial targeting of the P450 apoprotein to the ER membrane through
the SRP pathway and insertion into the membrane through a single transmembrane domain (Fujii-Kuriyama et al.,
1979
Experiments in Figs. 7 and 8 aimed at testing the validity
of these two models show that ~25-33% of 1A1 nascent
chains resist membrane insertion and remain in the soluble pool. Our results also show that this pool of 1A1,
which is amenable for processing by the cytosolic endoprotease, is also comparable with the 30% or so antibody-reactive proteins associated with the mitochondrial fraction under the transient transfection conditions. The
NH2-terminal 32-amino acid region of 1A1 appears to be the
target of the cytosolic endoprotease since removal of
the NH2-terminal four residues or mutations targeted to
the 32/33 positions of the protein (1A1M32/33) inhibit the
processing activity. Additionally, only 1A1 protein with
free NH2 terminus, but not that associated with the ER
membrane, is a substrate for this endoprotease. The inability of the endoprotease to process +5/1A1 suggests
that the same protease may be involved in processing past
the 4th and 32nd residues of the protein, and processing at
the former site may be conditional for the second processing at the latter site. We postulate that the extent of processing in different tissues and the efficiency with which the internal site is processed are rate-limiting steps that
determine the level of mitochondrial P450MT2. Purification and further characterization of the endoprotease is required to understand the details of the two-site processing
and differential activity of the enzyme. Thus, our experimental results clearly support model 2, although it is not
clear if the nascent chains are in the SRP-bound or unbound form. The model also postulates that the NH2-terminal cleaved protein is chaperoned to the mitochondria
by an ATP-dependent pathway involving HSP 70-type
proteins (Glick and Schatz, 1991 Physiological and Evolutionary Significance
The occurrence of chimeric signal sequences and transportation to two different cytoplasmic membrane compartments is not restricted to P4501A1; our preliminary results
show that other inducible P450 forms, including P4502B1,
3A2, 2E1, and 2C6, as well as the sex-specific 2C12, exhibit
similar dual transport modalities, although in some cases,
intact unprocessed polypeptide chains are transported into
the mitochondria. Thus, we believe that the mechanism
described for P4501A1 in this study represents a novel and
more general pathway designed to target the same primary translation products to two distinct membrane compartments. Our recent results also show that these mitochondrially targeted, microsomal P450s can functionally
interact with Adx (Anandatheerthavarada, H.K., S. Addya, J. Mullick, and N.G. Avadhani, manuscript submitted for publication) and catalyze the metabolism of various
xenobiotic chemicals, suggesting a physiological function
for the imported P450s. Our results also suggest an interesting coevolutionary pattern of mitochondrial metabolic
functions in parallel with the microsomal mixed function
oxidase systems. Therefore, these results have implications in some of the fundamental issues of eukaryotic evolution. It should be noted that the coevolution includes the
development of a chimeric signal for mitochondrial targeting and also the inclusion of structural domain(s) for interaction with the mitochondrial electron transport chain
proteins Adx and Adx reductase.
).
A majority of proteins targeted to the ER reach their destination through a cotranslational mechanism that requires the association of the NH2-terminal signal sequence
with signal recognition particle (SRP)1 and association of
the translation complex with the ER membrane (Walter et
al., 1981
; Gilmore et al., 1982
; and see Isenman et al., 1995
for a recent review). Exceptions to the cotranslational mechanisms that do not follow the SRP pathway have also
been reported for a limited number of proteins targeted to
the ER (Andersson et al., 1983
; Wickner and Lodish,
1985
). The membrane topology and mechanisms of targeting of the hepatic P450 isoenzymes that remain bound to
the ER have been studied extensively (Bar-Nun et al.,
1980
; Black and Coon, 1982
; Sakaguchi et al., 1987
; Monier et al., 1988
; Szcesna-Skorupa et al., 1988); it is generally
believed that the enzyme is anchored through a single
transmembrane domain with most of the catalytic domains
facing the cytosolic side of the membrane (Monier et al.,
1988
; Szczesna-Skorupa and Kemper, 1993
). Various studies (Fujii-Kuriyama et al., 1979
; Bar-Nun et al., 1980
; Sakaguchi et al., 1987
; Monier et al., 1988
; Szczesna-Skorupa et al., 1988
) have shown that the NH2-terminal hydrophobic sequence functions as an unclipped signal for targeting
to the ER through the cotranslational SRP pathway. In
particular, the NH2-terminal 25-30 hydrophobic residues
have been shown to provide the signal for membrane insertion and stop transfer, in addition to serving as the
transmembrane anchor (Sakaguchi et al., 1987
; Monier et al., 1988
; Szczesna-Skorupa et al., 1988
). Mitochondrial
protein transport, on the other hand, occurs through a
posttranslational mechanism and involves a complex series of interactions of the protein with various cytosolic
factors (Ohta and Schatz, 1984
; Murakami et al., 1988
;
Kang et al., 1990
; Hachiya et al., 1995
), as well as interaction of the NH2-terminal or internal mitochondrial specific
signal sequence with the multisubunit outer and inner membrane receptors (Sollner et al., 1989
; Lithgow et al.,
1994
; Hachiya et al., 1995
; Lill et al., 1996
; Schatz and Dobberstein, 1996
). In this paper, we demonstrate that the rat
cytochrome P4501A1 protein contains an unusual chimeric signal that facilitates its targeting to both the ER
and mitochondria through a novel pathway.
; Niranjan et al.,
1984
; Honkakowski et al., 1988
; Raza and Avadhani, 1988
;
Anandatheerthavarada et al., 1997
) and brain (Bhagwat et al., 1995
; Iscan et al., 1990
) mitochondria have been reported by many groups, although their primary sequence
and gene structure remain unclear. It is well documented
that the rat genome contains a single or limited number of
gene copies for some of the xenobiotic inducible P450
forms, including P4501A1/2, P4503A1/2, and P4502E1 (Gonzalez, 1990
). This apparent limitation raises questions on the precise nature and sequence properties of the
similarly inducible mitochondrial P450 forms that exhibit
immunological cross-reactivity to the major microsomal
forms. In the present study, we have extensively characterized the
-napthoflavone (BNF)-inducible mitochondrial P450MT2 and surprisingly found that it exists in two electrophoretically separable molecular forms that are different NH2-terminal truncated versions of the microsomal
P4501A1. Our results also suggest that the NH2-terminal
processing past the 4th or 32nd amino acid residues by a
cytosolic endoprotease exposes a cryptic mitochondrial
targeting sequence that directs the protein into the mitochondrial compartment. We postulate that this mode of
protein targeting represents a novel mechanism for the
biogenesis of not only P450MT2, but for other mitochondrial drugs metabolizing P450 enzymes as well.
Materials and Methods
).
Freshly isolated mitochondria from untreated livers were washed three
times with the sucrose-mannitol buffer and used for in vitro protein import assays. For purifying P450MT2, mitochondria from BNF-treated livers (Raza and Avadhani, 1988
) were fractionated with 75 µg/mg digitonin
(Niranjan et al., 1984
) under conditions that yield mitoplasts with <1%
microsomal-specific NADPH cytochrome c reductase (rotenone insensitive), 0.7% outer membrane-specific monoamine oxidase, and >95% mitochondrial-specific cytochrome c oxidase (COX) and isocitrate dehydrogenase activities (Anandatheerthavarada et al., 1997
). The mitochondrial
P450MT2 and microsomal P4501A1 were purified as described previously
(Raza and Avadhani, 1988
). The postmitochondrial supernatant was used
to prepare the microsomal fraction as described (Niranjan et al., 1984
).
). Details of induced expression and purification of proteins have been described elsewhere
(Anandatheerthavarada, H.K., S. Addya, J. Mullick, and N.G. Avadhani,
manuscript submitted for publication).
). The antibody was affinity purified against bacterially expressed, purified +33/1A1 protein conjugated to a solid matrix.
80°C. Canine pancreatic ER was generously provided by Dr. Reid Gilmore (University of Massachusetts Medical Center,
Worcester, MA).
) containing a 5
HindIII site and a
3
XbaI site was generated by the reverse transcriptase-based PCR. Various NH2-terminal deletions were generated by PCR amplification of the
parent cDNA using the appropriate sense primer containing an ATG
codon and upstream Kozak consensus (Kozak, 1986
) for translation initiation. The 1A1Mut and +33/Mut cDNAs with internal mutations (R34D
and K39I) and 1A1M32/33 cDNA with V32A/T33I mutations were generated by overlap PCR. All constructs were engineered to contain a Kozak
consensus ATG codon, as well as a 5
HindIII site and a 3
Xbal site. cDNAs
were cloned in pCMV4 or pGEM vectors, and the sequence properties of
all the plasmid constructs were ascertained by manual sequencing using
the dideoxy termination method. Plasmid pCDADX encoding the bovine
adrenodoxin (Zuber et al., 1988
), pMT2 vector containing full-length human NADPH cytochrome P450 reductase (P450 reductase), and pSP-DHFR plasmid containing full-length cDNA for the mouse dihydrofolate
reductase (Skerjanc et al., 1990
) were generously provided by Drs. Michael
Waterman (Vanderbilt University School of Medicine, Nashville, TN),
Frank Gonzalez (National Institutes of Health, Bethesda, MD), and Gordon Shore (McGill University, Montreal, Quebec, Canada), respectively.
, using freshly isolated mitochondria. The import assays were carried out in a 200-µl final
vol and contained 4 µl of 35S-labeled translation product (105 cpm), 500 µg
mitochondria, or microsomes (from a 10 mg/ml suspension in sucrose-mannitol buffer), 60 µl energy mixture (10 mM ATP, 10 mM GTP, 2.5 mM CDP, 2.5 mM UDP, 50 mM malate, 20 mM isocitrate), 70 µl transport
buffer (0.6 M mannitol, 20 mM Hepes, pH 7.4, 1 mM MgCl2, 2.5 mg/ml
BSA with or without added inhibitors), as indicated in the figure legends.
After incubation at 28°C for 60 min, the reaction mixtures were cooled on
ice for 5 min, and each mixture was divided into two or three equal portions. One portion was mixed with 20 µl of protease inhibitor mix to yield a final concentration of 1 mM PMSF, 25 µg each of antipain, chymostatin, leupeptin, and pepstatin, and was stored on ice. The other portions were
incubated with pronase (125-250 µg/ml of reaction) or trypsin (150-375
µg/ml of reaction) for 30-90 min on ice as specified in the figure legends.
The protease-treated samples were mixed with the protease inhibitor mix
as described above. Mitochondria were reisolated from both protease-treated and untreated samples by sedimentation through 1.35 M sucrose,
and were washed twice with sucrose-mannitol buffer. Mitochondrial proteins were dissociated in Laemmli's sample buffer at 95°C for 5 min, and
were analyzed by SDS-gel electrophoresis and fluorography.
).
CsCl banded plasmid DNAs were used for transfecting COS-M6 cells by
the DEAE dextran method (Zuber et al., 1988
). About 48-60 h after
transfection, cells from 10 plates (100 mm) were pooled, homogenized in a
Teflon fitted glass homogenizer (10 strokes at 5,000 revolutions), and used
for the isolation of mitochondrial and microsomal fractions by the differential centrifugation method (Niranjan et al., 1984
). The mitochondrial fraction was resuspended in the sucrose-mannitol buffer by gentle homogenization and further purified by banding 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 the Western blot analysis. In all cDNA expression experiments, the transfection efficiencies were monitored by coexpression with
2 µg/plate of cytomegalovirus
-galactosidase and assaying the cell extracts for
-galactosidase activities. Only plates showing activities within
the range of 85-100% were chosen for the isolation of subcellular fractions. In some experiments, mitochondria from transfected cells and those used for in vitro import were subjected to digitonin fractionation, as described above for liver mitochondria.
, except that after permeabilization with 0.1% Triton
X-100, cells were blocked with 5% goat serum for 1 h at 25°C. Cells were
double immunostained with 1:100 dilution of rabbit antibody to P4501A1
and 1:50 dilution of mouse mAb to the mitochondrial genome encoded
human COX subunit I (Molecular Probes, Inc., Eugene, OR) as a positive
control. Cells were washed repeatedly with PBS, and were incubated with
FITC-conjugated anti-rabbit donkey IgG for the detection of P4501A1
and Texas red-conjugated anti-mouse donkey IgG for the detection of
COX subunit I protein. Incubation with both the secondary antibodies
(purchased from Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA) was carried out for 1 h at 37°C at 1:100 dilution. Unbound secondary antibodies were removed by repeated washing with PBS. Fluorescence microscopy was carried out under a TCS laser scanning microscope (Leica Inc., Deerfield, IL). 0.5-µm optical sections were scanned at the z-axis
with both FITC and Texas red channels fully open to prevent any shifting
or distortion of the images.
Results
Fig. 1.
Electrophoretic resolution of the
two forms of mitochondrial P450MT2. P450s
from BNF-induced rat liver mitochondria and
microsomes and bacterially expressed +5/
1A1 and +33/1A1 were purified, and ~1-2
µg of protein sample in each case was resolved by electrophoresis on a 14-16% gradient SDS-polyacrylamide gel. (A) Coomassie
Blue stained pattern. (B) A companion gel
was subjected to Western blot analysis using
20 µg/ml of affinity-purified P4501A1 antibody. (C) The purified P450MT2 was compared with antibody-reactive proteins from BNF-induced liver mitochondria. Indicated amounts of mitochondria from BNF-induced
liver and 2 µg of P450MT2 were subjected to electrophoretic resolution and Western blot analysis using either affinity-purified P4501A1 antibody or mAb against P450c27 (Addya et al., 1991), as indicated.
[View Larger Versions of these Images (30 + 68K GIF file)]
Fig. 3.
Targeting of
P4501A1 to the microsomal
and mitochondrial compartments in vivo in COS cells.
COS cells were transfected
with 10 µg/plate of the various wild type, mutant cDNA
constructs, or pCMV4 vector
without any insert (Mock
transfected), together with 2 µg/plate of CMV -galactosidase plasmid. Cells from 8-10 companion plates were used
to isolate the mitochondrial
and microsomal membrane
fractions or whole-cell extracts
as described in Materials and
Methods. 2 µg of purified
P450MT2 and 25 µg protein
from each of the cell fractions were resolved by electrophoresis on 14-16% gradient polyacrylamide gels and
were subjected to Western
blot analysis using the affinity-
purified P4501A1 antibody.
[View Larger Version of this Image (26K GIF file)]
).
Fig. 2.
The sequence properties of P450MT2. (A) 3-6 µg of
P450MT2 and 1A1 proteins were resolved as described in Fig. 1
and transferred to PVDF membranes. Bands corresponding to
P450MT2b and 1A1 were excised and subjected to proteolytic digestion and HPLC analysis. The absorbency profiles at 260 nm
are presented. (B) The microsomal P4501A1 and mitochondrial
P450MT2a and MT2b protein bands were transblotted to PVDF
membrane and sequenced by Edman degradation as described in
Materials and Methods. The nature of 1A1Mut and +33/Mut
cDNA constructs carrying R34D and K39I substitutions, as well
as NH2-terminal deletion clones are indicated. (C) The chimeric
signal properties of P4501A1 are indicated. The NH2-terminal-most luminal region is indicated in dark, and the transmembrane
region is indicated in gray. The sequence 33-44 of the protein
containing three basic amino acid residues at positions 34, 39, and
42 is predicted to function as a putative mitochondrial-targeting
sequence.
[View Larger Version of this Image (39K GIF file)]
; von Heijne et al., 1989
).
), and the subcellular fractions were purified by differential centrifugation and sucrose density
banding. The Western blot in Fig. 3, developed with 1A1
antibody, shows that the whole-cell extract (Fig. 3 A, lane
3) and mitochondrial proteins (Fig. 3 B, lane 3) from cells
transfected with wild-type 1A1 cDNA contain two antibody-reactive bands that migrated similarly to purified P450MT2. Although the precise composition of the slower
migrating MT2a band in Fig. 3 A, lane 3, remains unknown, it is likely to contain both intact 1A1 and +5/1A1,
which migrate very similarly on the SDS gel, as shown in
Fig. 1. The microsomal fraction from these cells showed a
single protein band (Fig. 3 C, lane 3) consistent with intact
P4501A1. Surprisingly, all three fractions from +5/1A1-expressing cells showed a single band that migrated similarly to the MT2a band in Fig. 3 C, lane 1. The whole-cell
extract from cells transfected with 1A1Mut cDNA construct, with mutations targeted to the two basic residues at
34 and 39, showed an unusual pattern in that it contained a
major band comigrating with MT2a and a very minor MT2b-like band, in addition to a number of faster migrating bands that may represent degradation products. Furthermore, mitochondria from cells transfected with 1A1Mut
cDNA (Fig. 3 B, lane 4) contained no detectable antibody-reactive protein, while the microsomal fraction (Fig. 3 C,
lane 4) contained normal levels of 1A1-like protein. Although reasons for the rapid degradation of the mutant protein in the whole-cell extract remain unknown, it is
possibly more open to attack by proteases, or more likely,
the degraded component may represent proteins blocked
at the level of mitochondrial transport because of a mutated matrix targeting sequence. Transfection with the
+33/1A1 cDNA construct (Fig. 3 C, lane 6), however,
yielded antibody-reactive protein comigrating with the
MT2b band in the whole-cell extract, as well as in the mitochondrial isolate, but not in the microsomal preparations from these cells. These results are consistent with
reports suggesting the importance of the NH2-terminal 30-
amino acid residues for the microsomal targeting of P4501A1 (Monier et al., 1988
; Sakaguchi et al., 1988; Szczesna-
Skorupa and Kemper, 1993). Expression of +33/Mut protein, which carries mutations at the same two positively
charged residues (Nos. 34 and 39), however, yielded no detectable antibody-reactive protein in the mitochondrial
and microsomal preparations (Fig. 3, B and C, lane 7). The
whole-cell extract of cells transfected with the mutant construct, however, contained antibody-reactive protein similar in size and intensity to the +33/1A1-expressing cells, indicating that the mutant protein is indeed translated. In
repeated attempts, we observed that cells transfected with
+33/1A1 and +33/Mut constructs show lower levels of antibody-reactive proteins, as compared to 1A1- or +5/1A1-expressing cells, possibly because of lower translation or
faster turnover rates.
; Black and Coon, 1982
; Zuber et al., 1988
).
Western blot analysis showed that the microsomal fraction
of transfected COS cells contains proteins cross-reacting
with P4501A1 antibody (Fig. 4 B) and rat P450 reductase
antibody (Fig. 4 A), but no detectable Adx antibody-reactive protein (see Fig. 4 C). The mitochondrial fraction, on
the other hand, showed proteins reactive to the 1A1 antibody (Fig. 4 B) and Adx antibody (Fig. 4 C), but lacked
significant P450 reductase antibody-reactive protein (see
Fig. 4 A). Additionally, in agreement with the results presented in Fig. 3, the P4501A1 antibody-reactive protein
associated with the mitochondrial particles (Fig. 4 B) migrated as two components characteristic of P450MT2, while the antibody-reactive component in the microsomal
fraction migrated as a single slow migrating band, consistent with intact P4501A1. The results in Fig. 4 B also show
that both of the antibody-reactive proteins associated with
the intact mitochondrial particles are resistant to limited
protease digestion, while the addition of 0.3% Triton X-100
renders them protease sensitive, suggesting their intramitochondrial location. The 1A1 protein associated with the
microsomal membrane fraction, on the other hand, is highly sensitive to proteolytic attack, even in the absence of added Triton X-100. In addition to providing further support for
the mitochondrial targeting of P4501A1 under the in vivo
conditions, these results provide evidence for the qualitative separation of the two membrane components under
the fractionation scheme used in this study. Although not
shown, the mitochondrial content of P450MT2 was unaffected by outer membrane stripping by limited digitonin
fractionation (see Materials and Methods). Additionally, both MT2a and MT2b were nearly quantitatively extracted
with alkaline 0.1 M Na2CO3 under conditions when transmembrane Fo components of the mitochondrial ATPase
remained associated with the inner membrane fraction,
suggesting that the mitochondrial-targeted P450s exist as
extrinsic membrane proteins.
Fig. 4.
Specificity of the
mitochondrial targeting of
P4501A1 under in vivo conditions. In lanes marked T,
COS cells were transfected with full-length 1A1 construct (10 µg/plate) and
cotransfected with 10 µg/
plate of the human P450 reductase cDNA and 5 µg/
plate of bovine Adx cDNA
constructs. In lanes marked
M, cells were mock transfected with equivalent amount
of pCMV-4 plasmid DNA.
Mitochondrial and microsomal fractions were isolated as
described in Materials and
Methods, and 20 µg of microsomal and 30 µg of mitochondrial proteins were resolved on a 14-16% gradient polyacrylamide gel and were
subjected to Western blot analysis using the indicated antibodies.
(A and C) Mitochondrial and microsomal fractions in duplicate
lanes were derived from two separate transfections. (B) Microsomal and mitochondrial proteins were digested with 100 µg/ml
(+) or 200 µg/ml of reaction (++) with pronase for 30 min on ice
before electrophoresis.
[View Larger Version of this Image (30K GIF file)]
Fig. 5.
Subcellular localization of P4501A1 and +33/1A1 by
immuno-fluorescence microscopy. COS cells were grown on coverslips and transfected with +33/1A1 cDNA (top), intact
1A1cDNA (middle), or mock transfected with pCMV-4 vector
without insert (bottom). Cells in A-H were double immunostained with rabbit polyclonal antibody to P4501A1 and mouse
mAb to COX subunit I protein, and were developed with FITC-conjugated anti-rabbit IgG and Texas red-conjugated anti-
mouse IgG, respectively. Cells in I represent control without
added primary antibody, developed with Texas red-conjugated IgG as in H. A, D, and G represent FITC staining, showing patterns of 1A1 protein distribution. B, E, and H represent Texas
red staining showing COX subunit I patterns. C and F represent
superimposed patterns of A and B and D and E, respectively.
The micrograph in H was scanned at a threefold higher magnification compared to those in the other panels.
[View Larger Version of this Image (87K GIF file)]
Fig. 6.
In vitro transport of P4501A1
into mitochondria. 35S-labeled in vitro
translation products were programmed with
the various cDNA constructs in a transcription-linked reticulocyte lysate system, and
were used for in vitro transport in isolated
rat liver mitochondria, as described in Materials and Methods. In each case, 200 µg of
mitochondrial protein was used for electrophoresis, and the gels were subjected to
fluorography. (A) 35S-labeled, full-length
1A1, +5/1A1, +33/1A1, +33/Mut, and
1A1Mut proteins were used for the in vitro
transport. Faster migrating bands in some of
the lanes probably represent proteolytic
fragments generated as a result of endogenous mitochondrial or externally added
protease action. A single + at the top of
lanes represents treatment with 125 µg pronase, and ++ represents treatment with 250 µg pronase/ml of reaction. (B) In vitro
transport of +33/1A1, +53/1A1, and +73/
1A1 proteins into isolated rat liver mitochondria. Protease treatment was carried
out using 125 µg of pronase/ml of reaction.
(C) The specificity of the in vitro transport
system was tested using 35S labeled +33/1A1 protein, as well as mitochondrial and microsomal fractions isolated from rat liver. Incubations with mitochondrial or microsomal membranes were carried out as described in A. Protease digestion was carried out for various
time periods as indicated, using 125 µg pronase/ml of reaction. (D) Effects of mitochondrial inhibitors on the in vitro protein transport were tested using 35S-labeled +33/1A1 protein. The reaction mixtures were preincubated with or without added inhibitors (50 µM
CCCP or 50 µM oligomycin) at 25°C for 10 min before initiating the in vitro transport by adding the 35S-labeled +33/1A1 translation
product. Triton X-100 was added to the sample marked +Triton at a final concentration of 0.3% at the start of protease digestion. Protease digestion was carried out for 60 min with 125 µg pronase/ml of reaction. Radioactivity in Pronase samples in the case of 1A1,
+5/1A1, and +33/1A1 proteins represent ~50-60% of the input counts, indicating efficient binding to mitochondria. The mutant proteins showed varied levels of binding as follows: +33/Mut = 10%, 1A1Mut and +73/1A1 = 30%, and +53/1A1 = 4% of input.
[View Larger Versions of these Images (44 + 28K GIF file)]
; Schwartz and Neupert, 1994
; Neupert, 1997
),
these results demonstrate that the transport of +33/1A1
requires membrane potential and that it is dependent on
the mitochondrial ATP pool.
Fig. 7.
Processing of
P4501A1 by a cytosolic endoprotease. (A) P4501A1,
+33/1A1, and 1A1M32/33
(V32A and T33I) translation
products in reticulocyte lysate (25 µl) were incubated
with 15 µg of (NH4)2SO4-fractionated rat liver cytosolic protein. Incubation was
carried out for 30 min at
30°C, and 3-µl aliquots each
were electrophoresed on a 14-16% gradient SDS-
polyacrylamide gel. A mixture of 35S-labeled +5/1A1
and +33/1A1 were run as
markers. (B) P4501A1 translation product was incubated
with control cytosolic extract (C) or extract incubated at
95°C for 5 min (B) in the
presence or absence of added
EDTA (5 mM) or protein inhibitor mix (to a final concentration of 1 mM PMSF and 20 µg/ml
each of leupeptin, pepstatin, and chymostatin), as described in
Materials and Methods. The incubation and electrophoresis conditions were as described in A. (C) The effects of membrane association of P4501A1 on the processing activity of the cytosolic
protein extract was tested. 1A1 protein was translated in the
presence of added canine pancreatic ER (2.5 Eq/50 µl reaction),
and the membrane-bound fraction (pellet) was isolated by centrifugation over a layer of 0.5 M sucrose containing 100 mM KCl,
50 mM Hepes, pH 7.4, and 5 mM MgOAc2 at 100,000 g for 30 min. The soluble fraction remaining over the sucrose cushion was
aspirated and used as the supernate fraction. Details of incubation with the control and heated cytosolic protein fractions were
as described in A.
[View Larger Version of this Image (24K GIF file)]
; Monier et al., 1988
). Furthermore, in
transfected COS cells, ~28-33% of the 1A1Mut protein
with mutated matrix targeting sequence, but unaltered ER
targeting property remains in the soluble cytosolic fraction
(results not presented). These results suggest that 25-33%
of the 1A1 proteins escape ER targeting under both in
vivo and in vitro conditions because of an unknown mechanism.
Fig. 8.
Extent of ER membrane targeting of P4501A1 under in
vitro conditions. (A) P4501A1, P450 reductase, and DHFR were
cotranslated in rabbit reticulocyte lysate in the presence or absence of unwashed canine pancreatic ER. The reaction mixtures
were extracted with 0.1 M Na2CO3, pH 11.5, and the alkaline-soluble and -insoluble fractions were recovered and concentrated as
described recently (Anandatheerthavarada et al., 1997). Protein
fractions equivalent of 5 µl of initial reaction mixture in each case
were subjected to electrophoresis and autoradiography. (B) The
gel patterns for the alkaline-soluble and -insoluble fractions in A
were quantitated in a PhosphorImager to determine the percentage of recovery in the membrane integral (Membrane Fraction)
and extrinsic (Aqueous Fraction) fractions, respectively. Values
represent the average of two experiments.
[View Larger Versions of these Images (46 + 15K GIF file)]
Discussion
; Neupert, 1997
), mRNAs
coding for the mitochondrially destined proteins are transcribed differently using alternate transcription start sites,
alternate translation initiation sites, or differential splicing
of precursor RNAs such that the translation products contain an NH2-terminal cleavable signal sequence for mitochondrial targeting (Boguta et al., 1994
; Jaussi et al., 1995;
Neupert, 1997
). The results presented in this study on the
targeting of P4501A1 to mitochondria by using multiple
approaches, therefore, describe yet another pathway by
which the same primary translation product, encoded by
the same mRNA, can be targeted to both the ER and mitochondria.
; Lemire et al., 1989
; Isenman et al., 1995
),
although uncleaved NH2-terminal and internal signals
have also been reported in some cases (for a review see
Isenman et al., 1995
). Recently, the signal sequence characteristics of proteins targeted to the mitochondrial outer
and inner membrane compartments have been described.
The yeast Mas70p and the mammalian protooncogene
product Bcl-2 are two outer membrane proteins organized
in opposite orientations (Li and Shore, 1992
; Nguyen et
al., 1993
) which contain signal anchor sequences with
flanking positively charged putative mitochondrial-targeting domains. In the case of yeast mitochondrial NADH cytochrome b5 reductase, the same primary translation
product is targeted to both the outer membrane and intermembrane space (Hahne et al., 1994
) as 34- and 32-kD
species, respectively. The NH2 terminus of this protein
contains a variation of the signal anchor domains of the
Mas70p and Bcl-2 proteins, with an extended positively charged region resembling the matrix-targeting sequence
on the NH2-terminal side of the transmembrane domain,
as well as a sequence rich in charged residues on the
COOH-terminal side. Recently, Hahne et al. (1994)
have
proposed a novel mechanism in which incomplete translocation arrest in the outer membrane results in the targeting of the same translation product to two different submitochondrial compartments. Yet another example of mitochondrial-targeted protein with membrane anchor domain
is BCS1 protein, the Reiske FeS protein of the cytochrome
bc1 complex, which is organized in a transmembrane Nout-Cin
topology on the inner membrane (Folsch et al., 1996
). The
putative transmembrane domain at residues 45-68, immediately followed by an amphipathic helical structure with
positively charged residues (positions 69-83), together
function as an internal signal for mitochondrial targeting
and correct topological positioning in the inner membrane
(Folsch et al., 1996
). Interestingly, the NH2-terminal signal
components of +5/1A1 (and also intact 1A1) closely resemble that of the internal signal of BCS1 protein. In contrast to the integral membrane orientation of the BCS1
protein, however, both +5/1A1 and +33/1A1 are extrinsic
proteins localized inside the inner membrane compartment. Additionally, although results are not shown, the mitochondrial-targeting sequence of P4501A1 differs from
that of the BCS1 protein in that it is functional only when
placed at the NH2 terminus, and it is not processed by the
mitochondrial endoprotease.
; Bar-Nun et al., 1980
; Sakaguchi et al., 1987
). As shown
for the hepatitis B protein (Garcia et al., 1988
), cleavage at
the NH2-terminal signal region or past the transmembrane
domain by endoprotease may result in the aborted translocation or release of the protein to the cytosol. In model 2, a fraction of the nascent 1A1 chains escape insertion on
the ER either because of slippage of the SRP recognition
(SRP-unbound 1A1), or post-SRP (SRP-bound 1A1) steps (Belin et al., 1996
), that are translated as membrane-free
polypeptides. The NH2-terminal segment of the polypeptide is then cleaved by a cytosolic protease, thus activating
the mitochondrial-targeting sequence.
Fig. 9.
Models for the biogenesis of P450MT2.
[View Larger Version of this Image (31K GIF file)]
; Schwartz and Neupert,
1994
; Ryan and Jensen, 1995
). Members of the HSP 70 family of cytosolic proteins are known to play critical roles
in the ATP-dependent unfolding of the protein, as well as presentation to the mitochondrial import receptor(s) in a
conformational state amenable for importation (Glick and
Schatz, 1991
).
Received for publication 6 May 1997 and in revised form 12 August 1997.
We are thankful to Drs. Michael Waterman, Byron Kemper, Michael Atchison, and Steven Liebhaber for critically reading the manuscript. We also thank Drs. Reid Gilmore, Michael Waterman, Frank Gonzalez, and Gordon Shore for generously providing some of the reagents and expression plasmids used in this study, and Dr. Lee Peechy and Ms. Gladys Gray-Board for the use of their confocal microscopy facility. Finally, the suggestions and help of members of the Avadhani laboratory, particularly those of Drs. C. Vijayasarathy and Nibedita Lenka on many aspects of this work, are gratefully acknowledged.
Adx, adrenodoxin;
BNF, -napthoflavone;
COX, cytochrome c oxidase;
DHFR, dihydrofolate reductase;
PVDF, polyvinyldifluoride;
P450, cytochrome P450;
P450 reductase, NADPH cytochrome P450 reductase;
SRP, signal recognition particle.
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