From the Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden
Received for publication, September 21, 2000, and in revised form, December 19, 2000
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ABSTRACT |
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Cytochrome P450 2E1 (CYP2E1) lacking the
hydrophobic NH2-terminal hydrophobic transmembrane
domain is specifically targeted to mitochondria, where it is processed
to a soluble and catalytically active form ( The majority of the mitochondrial proteins are encoded from
nuclear DNA, synthesized in the cytosol as precursors and
posttranslationally targeted to the mitochondria (1-3). Many of the
proteins that are destined for import into the mitochondrial matrix
contain a signal sequence in their NH2 terminus that
directs these proteins to the mitochondria (4), although these signal
sequences are also found in the COOH terminus of the protein (5, 6).
Despite the fact that there is no general consensus for mitochondrial matrix targeting sequences, many of these sequences were demonstrated to be rich in positively charged and hydrophobic amino acid residues and usually are able to form an amphiphilic secondary structure (7, 8).
The mitochondrial matrix targeting sequence initially interacts with
the import receptor (Tom20) that is part of the translocase of the
outer membrane (TOM)1 complex
present in the mitochondrial outer membrane (9, 10). The surface of
Tom20, which is rich in negatively charged amino acid residues, is
thought to recognize and bind the positively charged residues present
in these signal sequences; however, additional binding forces such as
hydrophobic interactions also play an important role (10). After
binding, the protein is then allowed to cross the lipid bilayer through
the general translocation pore formed by the TOM complex (11). Proteins
destined for the matrix are then transferred to the translocase of the
inner membrane (TIM) complex and translocated into the matrix space
(12, 13), where the NH2-terminal signal sequence of the
protein is proteolytically removed by proteases present in the matrix
(14, 15).
The NH2-terminal hydrophobic transmembrane domain of
microsomal cytochrome P450s (P450s) was shown not only to be
responsible for the cotranslational targeting in a signal recognition
particle (SRP)-dependent manner to the endoplasmic
reticulum (ER) membrane (16, 17), but also to be responsible for
anchoring and retention in the ER membrane (18, 19). Not all P450
enzymes are retained in the ER membrane by the same mechanisms; as was
recently demonstrated, CYP2C1 is retained by static retention
preventing exit from the ER, while CYP2E1 is retained by a retrieval
mechanism allowing it to be recycled back from post-ER compartments
(20, 21). In contrast to microsomal P450s, mitochondrial P450s are,
like most of the nuclear encoded mitochondrial proteins,
posttranslationally targeted to mitochondria by means of their
amphiphilic NH2-terminal signal sequence (22, 23).
Cytochrome P450 2E1 (CYP2E1) is the alcohol-inducible member of the
P450 family and has been suggested to be involved in gluconeogenesis because of its ability to metabolize ketone bodies such as acetone (24,
25). In addition, a wide variety of small hydrophobic xenobiotics
including many well known carcinogenic and toxic compounds are
metabolized by CYP2E1 (24). CYP2E1 is also known for its ability to
cause oxidative stress that could ultimately lead to apoptosis (26) and
to alcoholic liver disease (27, 28).
Although CYP2E1 is predominantly located in the membrane of the ER, it
has also been demonstrated to be present in lysosomes (29), peroxisomes
(30), Golgi apparatus (31), and on the outer surface of the plasma
membrane (32, 33). Previously we reported that an
NH2-terminally truncated form of CYP2E1 was specifically
targeted to the mitochondria (34). Deletion or mutation of the
NH2-terminally hydrophobic transmembrane domain of CYP2E1
resulted in the mitochondrial localization of the protein when these
constructs were transiently expressed in a mouse hepatoma cell line. It
was demonstrated that this mitochondrially localized form of CYP2E1 was
a NH2-terminally truncated form of CYP2E1 of ~40 kDa
named In the present investigation, the mitochondrial targeting sequence of
CYP2E1 was identified by constructing several NH2-terminal deletion mutants of CYP2E1, which lacked one or both putative mitochondrial targeting sequences that are present in the
NH2 terminus of CYP2E1. Moreover, the role of the
positively charged and hydrophobic amino acid residues present in this
sequence in the mitochondrial targeting was investigated by mutational analysis.
Expression Vectors--
The CYP2E1 cDNAs encoding the
truncated CYP2E1 variants Cell Culture, Transient Transfection, and Western
Blotting--
H2.35 cells were grown and transfected as described (33,
34). Western blot analysis was performed as described (31) and
immunoreactive bands were visualized using the Fuji Las-1000 luminescent image analyzer (Fujifilm, Sweden).
Immunofluorescent Microscopy--
After transfection, the H2.35
cells were fixed and processed for immunostaining as described (33,
34). Transfected proteins were detected by using CYP2E1-specific
antibodies and visualized by anti-rabbit fluorescein
isothiocyanate-conjugated antibodies. Cells were double-immunostained
by sequential incubation with CYP2E1-specific rabbit polyclonal
antibodies, which were visualized by anti-rabbit fluorescein
isothiocyanate-conjugated antibodies, followed by incubation with
mitochondrial heat shock protein 70 (mHsp70)-specific monoclonal
antibodies (Affinity Bioreagents Inc., Goldon, CO), which were
visualized by anti-mouse tetramethyl rhodamine
isothiocyanate-conjugated antibodies. Cells were viewed under a Nikon
Eclipse E600 fluorescent microscope equipped with a camera using the
60× oil immersion objective.
Catalytic Activity--
The catalytic activity of several of the
CYP2E1 variants was determined by monitoring the hydroxylation of
chlorzoxazone (31, 34). The post-nuclear supernatant of cells
transfected with The mitochondrial targeting of a NH2-terminally
truncated form of CYP2E1, 2E1) with a mass of
about 40 kDa. Small amounts of
2E1 were also observed in
mitochondria isolated from rat liver, indicating that this form of
CYP2E1 is also present in vivo. In the present study the
mitochondrial targeting signal was identified and characterized by the
use of several NH2-terminally truncated and mutated forms
of CYP2E1 that were expressed in the mouse H2.35 hepatoma cell line.
Two potential mitochondrial targeting sequences were identified in the
NH2 terminus of CYP2E1. Deletion of the first potential
mitochondrial targeting sequence located between amino acids 50 and 65, as in
(2-64)2E1, still resulted in mitochondrial targeting and
processing, but when, in addition to the first, the second potential
mitochondrial targeting sequence located between amino acids 74 and 95 was also deleted, as in
(2-95)2E1, the mitochondrial targeting was
abolished. Mutation of the four positively charged Arg and Lys residues
present in this sequence to neutral Ala residues resulted in the
abrogation of mitochondrial targeting. Deletion of a hydrophobic
stretch of amino acids between residues 76 and 83 also abolished
mitochondrial targeting and import. Once imported in the mitochondria,
these constructs were further processed to the mature protein
2E1.
It is concluded that mitochondrial targeting of CYP2E1 is mediated
through a sequence located between residues 74 and 95 and that
positively charged residues as well as a hydrophobic stretch present in
the beginning of this sequence are essential for this process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2E1, which was shown to be soluble, catalytically active, and
localized inside the mitochondria. In addition,
2E1 was present in
low levels in vivo in mitochondria isolated from rat liver.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(2-29)2E1,
(2-64)2E1,
(2-76)2E1,
(2-82)2E1, and
(2-95)2E12 were generated
by polymerase chain reaction amplification using Pfu DNA
polymerase (Stratagene, La Jolla, CA), the appropriate sense primer
containing an ATG codon, primers 1a, 1b, 1c, 1d, and 1e, respectively
(Table I); the wild-type antisense primer 2a; and CYP2E1 cDNA as a template. The RK
A cDNA containing
the internal mutations R75A, R76A, K84A, and K87A and the K
A
cDNA containing K84A and K87A were generated by polymerase chain
reaction amplification using the sense primers starting from position
193 (corresponding to amino acid position 65) that incorporated the mutations, primer 1f for RK
A and primer 1g for K
A, the wild-type antisense primer 2a, and CYP2E1 cDNA as a template. The resulting cDNAs were cloned between the EcoRI and XbaI
sites of the mammalian pCMV5 expression vector. The constructs R75A and
RR
AA were prepared as follows. The pCMV5 vector contains a unique
BsgI restriction site located outside the cloning box, 235 bases 3' of the XbaI site, and CYP2E1 cDNA also has a
unique BsgI restriction site at position 213. The vector
containing the insert
(2-29)2E1 was digested with BsgI,
thereby cutting out the
(2-29)2E1 cDNA from position 127 until
the BsgI site after the cloning box, thereby leaving the
first 126 5' bases of
(2-29)2E1 attached to the vector. The CYP2E1
cDNAs containing the mutations were generated by polymerase chain
reaction amplification using the sense primer containing a
BsgI site and the appropriate mutations, primer 1h for R75A and primer 1i for RR
AA, the antisense primer that anneals just after
the BsgI site of the vector primer 2b, and pCMV-CYP2E1 as a
template. The resulting cDNA fragments were cloned in between the
two BsgI sites of pCMV5 vector still containing the cDNA
coding for the first amino acids of the construct. The correct
sequences of all inserts were confirmed by DNA sequencing using the ABI PRISM® dye terminator cycle sequencing kit from
PerkinElmer Life Sciences.
Sequences of the oligonucleotides used as polymerase chain reaction
primers
(2-29)2E1,
(2-64)2E1,
(2-95)2E1, or empty
plasmid was prepared, and organelles were disrupted by sonication
(three bursts of 10 s with 30-s intervals) on ice. Mitochondria
were isolated from cells transfected with empty plasmid or
(2-29)2E1 and disrupted by sonication as described previously (34).
The incubation mixture consisted of 250 µg of sonic disrupted
post-nuclear supernatant or 100 µg of sonic disrupted mitochondria,
50 µM chlorzoxazone, a NADPH generating system (0.2 mM NADPH, 2.0 mM glucose 6-phosphate, and 3 units/ml glucose-6-phosphate dehydrogenase) and 50 mM
phosphate buffer, pH 7.4, in a final volume of 250 µl in the presence
or absence of 1.0 nmol of adrenodoxin (Adx), 0.1 nmol of adrenodoxin reductase (AdR), kindly supplied by Prof. Rita Bernhardt (University of
Saarbrücken, Germany). After 30 min of incubation at 37 °C, the reaction was terminated by the addition of orthophosphoric acid,
and samples were extracted and analyzed on an Varian ProStar HPLC
system (Varian, Walnut Creek, CA) equipped with an amperometric detector (Bioanalytical Systems Inc, West Lafayette, IN).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(2-29)2E1, was investigated by
constructing several NH2-terminal deletion mutants of
CYP2E1 (see Fig. 1). Examination of the
first 100 amino acids of CYP2E1 revealed that there are two amino acid
stretches that could serve as potential mitochondrial targeting signals
(the underlined sequences shown in Fig. 1). Both
these sequences are rich in positively charged and hydrophobic amino
acids and are potentially able to form an amphiphilic secondary structure, features that are characteristic for a mitochondrial targeting signal.
View larger version (18K):
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Fig. 1.
NH2-terminal amino acid sequence
of the CYP2E1 constructs used in this study. The potential
mitochondrial targeting sequences are underlined, and the
hydrophobic transmembrane domain is represented in a box.
The positively charged amino acid residues that were mutated are
depicted in a gray box and mutated residues in a
black box.
H2.35 cells transfected with (2-29)2E1 were analyzed by Western
blotting and a NH2-terminally truncated form of CYP2E1 of around 40 kDa named
2E1 (Fig. 2) was
detected (34). Subcellular localization by immunofluorescent microscopy
confirmed the mitochondrial localization of
2E1 (Fig.
3). Indeed, the staining pattern observed using CYP2E1-specific antibodies (Fig. 4,
upper left panel) was identical to
that observed using antibodies recognizing the mitochondrial protein,
mHsp70 (Fig. 4, upper right panel). To
identify the targeting sequence that was responsible for the
mitochondrial targeting and import, two constructs were designed that
lacked one or both potential targeting sequences.
(2-64)2E1, in
which the first potential targeting signal (amino acids 51-64) was
deleted, was transfected into H2.35 cells, which were analyzed by
Western blotting. In addition to
2E1, a protein with the expected
full length of the construct was observed (Fig. 2), and both these
proteins were recovered in the mitochondrial fraction (data not shown).
Immunofluorescent microscopy demonstrated that cells transfected with
(2-64)2E1 displayed a similar staining pattern to that observed for
(2-29)2E1-transfected cells (Fig. 3) and was shown to colocalize
with mHsp70 (Fig. 4), confirming its mitochondrial localization. These
results clearly demonstrated that deletion of the first 64 amino acids
of CYP2E1 did not affect the mitochondrial targeting and that the
region between amino acids 51 and 64 was not responsible for directing
(2-29)2E1 to the mitochondria.
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(2-95)2E1, in which in addition to the first potential
mitochondrial targeting signal, also the second potential targeting sequence between amino acids 75 and 95 was deleted, was expressed in
H2.35 cells. A protein with a slightly lower mobility than
2E1 was
observed by Western blotting (Fig. 2), which corresponds to the
expected full-length size of the construct. Immunofluorescent microscopy demonstrated that
(2-95)2E1 was not associated with the
mitochondria, but instead displayed a diffuse staining pattern throughout the whole cell, most likely representing a cytosolic localization of this protein (Fig. 3). These data demonstrated that the
region between amino acids 74 and 95 was responsible for the
mitochondrial targeting and that this region served as a mitochondrial
targeting signal.
Previously we showed that 2E1 was catalytically active in the
presence of the mitochondrial electron carrier proteins Adx and AdR
(34). The post-nuclear supernatant of cells transfected with
(2-29)2E1,
(2-64)2E1, and
(2-95)2E1 was assayed for
catalytic activity (Fig. 5A).
As expected, cells transfected with
(2-29)2E1 displayed catalytic
activity in the presence of both Adx and AdR. Additionally, cells
transfected with
(2-64)2E1 had significant chlorzoxazone
hydroxylation activity with levels well over those of cells transfected
with empty plasmid (mock). When incubated in the absence of Adx and
AdR, no significant increase in catalytic activity was observed,
indicating that these truncated CYP2E1 variants were not able to couple
with the endogenous NADPH cytochrome P450 reductase. Despite the
extensive deletion in
(2-95)2E1, some residual catalytic activity
could still be detected, although the observed levels were very low.
The expression levels of these three truncated proteins were found to
be very similar (see Fig. 2), which indicated that only a part of the
(2-95)2E1 expressed was catalytically active. Mitochondria isolated
from cells transfected with
(2-29)2E1 displayed a 3-fold increase
in catalytic activity over mitochondria isolated from mock transfected
cells (Fig. 5B), indicating that the catalytic activity is
associated with the mitochondria as was also previously reported
(34).
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The involvement of the positively charged residues that are present in
the mitochondrial targeting sequence identified above, Arg at positions
75 and 76 and Lys at positions 84 and 87, was studied by mutating all
four these residues to uncharged Ala residues, mutant RKA. Western
blotting revealed that only the full length of the construct was
expressed in cells transfected with RK
A; no
2E1 protein could be
observed (Fig. 2). The subcellular localization was shown to be similar
to cells transfected with
(2-95)2E1, a diffuse staining pattern
indicative of a cytoplasmic localization (Fig. 3). This showed that
together these four positively charged residues present in the
mitochondrial targeting signal are essential for the mitochondrial
targeting and subsequent import of this protein. In addition, the data
hitherto presented demonstrate that, once the protein is imported into
the mitochondria, the mitochondrial import signal is removed thus
forming the mature protein
2E1.
Mutation of only the two Lys residues to Ala (KA) caused decreased,
although significant mitochondrial import and processing (Fig. 2).
Thus, most of the protein expressed was the full-length construct
(unprocessed form), whereas a significantly smaller amount was found to
be truncated to
2E1. When K
A-transfected cells were examined by
immunofluorescent microscopy, two populations of staining patterns were
observed (Fig. 3), some cells displayed typical mitochondrial staining,
which was confirmed by colocalization with mHsp70 (Fig. 4), while
others had a more diffuse staining pattern resembling the staining
observed for RK
A.
Mutation of only the two positive Arg residues to the uncharged Ala
residues (RRAA) had no effect on the mitochondrial localization of
(2-29)2E1 (Figs. 3 and 4). Unfortunately, because of the low levels
of expression of RR
AA, analysis by Western blot proved to be
difficult. These data indicated no absolute requirement for these two
residues alone in the mitochondrial targeting. When Arg-75 was mutated
to the hydrophobic residue Ile and Arg-76 mutated to Ala (RR
IA), a
similar subcellular distribution as for RR
AA was observed. In
addition, Western blot analysis revealed that these transfected cells
expressed
2E1, indicating that mitochondrial targeting and import
was not affected by these mutations (data not shown). Additionally,
substitution of Arg-75 with Ala (R75A) had no effect on the
mitochondrial targeting and processing, the transfected cells expressed
2E1 (Fig. 2) and displayed the typical mitochondrial staining
pattern (Figs. 3 and 4). These results indicated that the two Arg
residues at positions 75 and 76 alone were not essential for the
mitochondrial targeting and import of
(2-29)2E1.
Partial disruption of the mitochondrial targeting signal as in
(2-76)2E1 and
(2-82)2E1 resulted in low expression levels that
were difficult to detect by Western blot analysis. The few transfected
cells were, however, easily detected by immunofluorescent microscopy.
Some of the cells transfected with
(2-76)2E1, in which the
NH2 terminus including Arg-75 and Arg-76 was deleted, displayed the typical mitochondrial staining pattern, while others had
a more diffuse staining pattern, a pattern similar to that observed for
K
A-transfected cells (Figs. 3 and 4). This was in good agreement
with the results obtained above, which showed that Arg-75 and Arg-76
had only a minor effect on mitochondrial targeting. Further disruption
of the mitochondrial targeting signal as in
(2-82)2E1 resulted in
the diffuse cytoplasmic staining pattern in cells transfected with this
construct, indicating that the hydrophobic region between amino acids
76 and 83 was essential for the mitochondrial targeting of
(2-29)2E1 (Fig. 3).
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DISCUSSION |
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The hydrophobic NH2 terminus of P450s is responsible
for the cotranslational targeting of the protein to the membrane of the ER (16, 17). Deletion of the hydrophobic ER targeting signal as in
(2-29)2E1 results in targeting of this truncated protein to the
mitochondria (34). In the present study, it was demonstrated, by
sequential deletion of the NH2 terminus and by mutational
analysis, that the region between amino acids 74 and 95 is responsible
for the mitochondrial targeting of CYP2E1. The presence of four
positively charged amino acid residues as well as a stretch of
hydrophobic residues at the beginning of this targeting sequence were
shown to be essential for the mitochondrial targeting and import of
(2-29)2E1. Western blot analysis showed that
2E1 displayed a slightly higher mobility than
(2-95)2E1, suggesting that
2E1 is
a NH2-terminally truncated form of CYP2E1 lacking
approximately the first 100 amino acids.
It is well known that many of the proteins that are targeted to mitochondria contain a mitochondrial targeting signal, also called a presequence, which is usually located at the NH2 terminus of the protein (1-4). Attempts to define a general consensus sequence for these mitochondrial targeting signals have not resulted in a well defined motif as is known for proteins targeted to other intracellular organelles such as peroxisomes (35) or the endocytic compartment (36). General features valid for most of the mitochondrial targeting sequences are that they are usually rich in positively charged and hydrophobic amino acid residues, they do not contain any or very few negatively charged residues, and they have the potential to form an amphiphilic secondary structure (7, 8, 37). It is believed that the positively charged residues together with the hydrophobic residues are essential for binding to the negatively charged surface of the Tom20 receptor (10, 11). Recently, however, NMR studies have shown that the initial interaction between the signal sequence of rat aldehyde dehydrogenase and the rat Tom20 receptor was entirely mediated by hydrophobic interactions (38). The initial binding was shown not to be dependent on ionic interactions, thereby reducing the role for the positively charged residues of the presequence in the initial binding. This agreed well with the observation made in the present study; the hydrophobic region at the beginning of the mitochondrial targeting signal (amino acids 77-82) of CYP2E1 is essential for targeting to and import into the mitochondria. The positive charges of the presequence were shown to be important for transport across the mitochondrial outer and inner membrane by sequential binding to several components of the TOM and TIM complex (the "acid chain hypothesis") (11-13) and also are thought to be necessary for recognition by the mitochondrial processing peptidase (39).
Mutation of all four positively charged residues present in the
mitochondrial targeting signal of CYP2E1 to uncharged Ala residues
effectively prevented mitochondrial targeting and subsequent import as
shown by both immunofluorescent microscopy and Western blot analysis.
However, mutation of only one or both Arg residues at the beginning of
this signal did not have any major effect on the mitochondrial
targeting and import. Mutation of only the two Lys residues resulted in
an intermediate result; although mitochondrial targeting and import was
still observed, it was less efficient as compared with the construct
(2-64)2E1 where the two Lys residues were still present. Deletion
of the NH2 terminus until just after the two Arg residues,
as in
(2-76)2E1, still resulted in mitochondrial targeting,
although less efficient, indicating that these Arg residues are not
essential for mitochondrial targeting and import. These results implied
that all four positively charged residues together are essential for
mitochondrial targeting, and subsequent import and processing of
(2-29)2E1. The construct
(2-82)2E1, where, in addition to the
two Arg residues, a hydrophobic stretch of amino acids was deleted,
displayed a similar staining pattern as observed for
(2-95)2E1,
i.e. a cytosolic localization. This strongly suggests that
the hydrophobic region between Arg-76 and Lys-84 is critical for the
mitochondrial targeting of
(2-29)2E1.
Despite the fact that (2-95)2E1 displayed catalytic activity, it
was lower than that observed in cells transfected with
(2-29)2E1 and
(2-64)2E1, which expressed
2E1. In addition, it was further demonstrated that the catalytic activity is localized in the
mitochondria. These data suggest that to efficiently assemble a
catalytically active
2E1 the folding and processing machinery
present in the mitochondrial matrix is required. It should, however, be
emphasized that the observed activities as well as the protein
expression levels are very low when compared with those observed for
wild-type CYP2E1. The demonstration that the catalytic activity is
associated with isolated mitochondria, together with the observation
that all the mitochondrial localized CYP2E1 variants are processed to
the mature
2E1, strongly indicates that
2E1 is present in the
mitochondrial matrix.
The results obtained in the present study allow the construction of a
model that can explain the mitochondrial targeting of CYP2E1, where
binding of the SRP and subsequently the cotranslational targeting
pathway has been compromised by either deletion or mutation of the
hydrophobic NH2-terminal transmembrane domain. The
translation of CYP2E1 is normally initiated on ribosomes present in the
cytosol and binding of the SRP to the hydrophobic NH2
terminus arrests this translation as soon as the nascent chain emerges
from the ribosome (16, 17, 40). Translation only commences when the arrested complex is properly docked to the translocation complex present in the membrane of the ER (40). Deletion or modification of the
hydrophobic NH2 terminus of CYP2E1 compromises the binding of the SRP, resulting in the translation of the entire protein on the
cytosolic ribosomes. After synthesis, these proteins are most likely
bound by cytosolic chaperones, targeted to the mitochondria by the
exposed mitochondrial targeting sequence located between residues 74 and 95. After interaction with the TOM and TIM complex, CYP2E1 is
translocated into the matrix of the mitochondria were the presequence
is removed by the mitochondrial processing peptidase resulting in the
mature protein, 2E1.
Analysis of the secondary structure of the region of CYP2E1 between
amino acids 74 and 95 revealed that the region from 83 to 95 was able
to form an -helical structure, while the region from 76 to 80 preferentially forms a
-sheet. The presence of the
-helical part
alone is not sufficient for targeting of
(2-29)2E1 to the
mitochondria. Deletion of the hydrophobic part between residues 76 and
83 abolished the mitochondrial targeting as observed for
(2-82)2E1,
emphasizing the importance of this hydrophobic stretch in the initial
targeting and binding of the protein to the mitochondria. It can be
suggested that, for the
-helical structure to bind to the
hydrophobic groove present in the Tom20 receptor, an initial
interaction of the hydrophobic residues located between residues 76 and
83 is necessary for the binding of the presequence to the mitochondrial
import receptor. After proper binding of the presequence to Tom20, the
presence of the positively charged amino acid residues becomes
important for the actual translocation over the outer and inner
membrane of the mitochondria and also for the interaction with the
proteolytic and folding machinery in the mitochondrial matrix.
A model has been proposed for the mitochondrial targeting of an
NH2-terminally truncated form of CYP1A1 expressed in both -naphthoflavone-induced rat liver and transiently in COS cells (41).
By using an in vitro translation assay in the presence or
absence of canine pancreatic microsomes and isolated rat liver cytosolic proteins, it was suggested that up to 25% of the nascent chains were able to escape cotranslational insertion into the ER
membrane. It was further suggested that these escaped chains were
processed by a cytosolic endopeptidase, resulting in the exposure of a
mitochondrial targeting signal that directed the truncated CYP1A1 to
the mitochondria. Our data, however, do not support this hypothesis;
previous results showed that, when wild-type CYP2E1 was transfected in
H2.35 cells, no mitochondrially localized
2E1 could be detected,
although the expression levels were significantly higher than for the
constructs used in the present study (34). Only removal, as in
(2-29)2E1, or modification, as in N++2E1, of the
hydrophobic NH2-terminal transmembrane domain of CYP2E1 resulted in mitochondrial targeting. Small amounts of
2E1 were detected in isolated rat liver mitochondria suggesting that in vivo the fidelity of the SRP binding and cotranslational targeting pathway can be compromised.
Cyclic AMP-dependent phosphorylation of CYP2B1 at serine 128 was demonstrated to cause mitochondrial targeting of this phosphorylated protein (42). It was shown by using an in vitro translation system that phosphorylation of CYP2B1 interfered with the binding of the SRP and subsequent insertion into the ER membrane. Additionally, CYP2E1 has a serine at position 129 that can be phosphorylated in a cAMP-dependent manner, a modification that has been proposed to be involved in the degradation process (43). No mitochondrial targeting could be observed when cells expressing wild-type CYP2E1 were treated with a stable cAMP analogue, indicating that for CYP2E1 phosphorylation does not seem to play a role in the mitochondrial targeting.3
In conclusion, when SRP binding to the hydrophobic
NH2-terminal transmembrane domain of CYP2E1 is compromised,
protein translation is not arrested and the entire CYP2E1 protein is
translated in the cytosol. A mitochondrial targeting signal located
between amino acid residues 74 and 95 is being exposed, probably after binding of the translated CYP2E1 protein with cytosolic chaperones, and
the protein is targeted to the mitochondria. The mitochondrial targeting and import of (2-29)2E1 is strongly dependent on the presence of positively charged amino acid residues Arg-75 and Arg-76
and Lys-84 and Lys-87 and of a hydrophobic region between residues 76 and 82. After mitochondrial import the signal sequence of
(2-29)2E1
is removed in the mitochondrial matrix, thereby forming the mature
soluble and catalytically active protein
2E1.
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ACKNOWLEDGEMENT |
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We thank Prof. Rita Bernhardt for the generous gift of adrenodoxin and adrenodoxin reductase.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Medical Research Council and AstraZeneca.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Molecular
Toxicology, Inst. of Environmental Medicine, Karolinska Institutet, Box
210, S-171 77 Stockholm, Sweden. Tel.: 46-8-7287735; Fax: 46-8-337327;
E-mail: magnus.ingelman-sundberg@imm.ki.se.
Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M008640200
2 The numbering of the amino acids corresponds to that of the full-length CYP2E1.
3 E. P. A. Neve and M. Ingelman-Sundberg, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: TOM, translocase of the outer membrane; ER, endoplasmic reticulum; P450, cytochrome P450; SRP, signal recognition particle; TIM, translocase of the inner membrane; mHsp70, mitochondrial heat shock protein 70; Adx, adrenodoxin; AdR, adrenodoxin reductase.
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REFERENCES |
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