Originally published In Press as doi:10.1074/jbc.M203292200 on August 20, 2002
J. Biol. Chem., Vol. 277, Issue 43, 40583-40593, October 25, 2002
Bimodal Targeting of Microsomal CYP2E1 to Mitochondria through
Activation of an N-terminal Chimeric Signal by cAMP-mediated
Phosphorylation*
Marie-Anne
Robin
,
Hindupur K.
Anandatheerthavarada,
Gopa
Biswas,
Naresh Babu V.
Sepuri§,
Donna M.
Gordon¶,
Debkumar
Pain¶, and
Narayan G.
Avadhani
From the Department of Animal Biology and the Mari
Lowe Center for Comparative Oncology, School of Veterinary Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104, the
§ Department of Biochemistry and Pharmacology, Thomas
Jefferson University Medical School, Philadelphia, Pennsylvania 19107, and the ¶ Department of Pharmacology and Physiology, University of
Medicine and Dentistry of New Jersey, Newark, New Jersey 07103
Received for publication, April 5, 2002, and in revised form, August 8, 2002
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ABSTRACT |
Cytochrome P450 2E1 (CYP2E1) plays an important
role in alcohol-induced toxicity and oxidative stress. Recently, we
showed that this predominantly microsomal protein is also localized in rat hepatic mitochondria. In this report, we show that the N-terminal 30 amino acids of CYP2E1 contain a chimeric signal for bimodal targeting of the apoprotein to endoplasmic reticulum (ER) and mitochondria. We demonstrate that the cryptic mitochondrial targeting signal at sequence 21-31 of the protein is activated by
cAMP-dependent phosphorylation at Ser-129. S129A mutation
resulted in lower affinity for binding to cytoplasmic Hsp70,
mitochondrial translocases (TOM40 and TIM44) and reduced mitochondrial
import. S129A mutation, however, did not affect the extent of binding
to the signal recognition particle and association with ER membrane
translocator protein Sec61. Addition of saturating levels of signal
recognition particle caused only a partial inhibition of CYP2E1
translation under in vitro conditions, and saturating
levels of ER resulted only in partial membrane integration. cAMP
enhanced the mitochondrial CYP2E1 (referred to as P450MT5) level but
did not affect its level in the ER. Our results provide new
insights on the mechanism of cAMP-mediated activation of a cryptic
mitochondrial targeting signal and regulation of P450MT5 targeting to mitochondria.
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INTRODUCTION |
Accurate targeting of proteins to their designated
subcellular compartments is critical for maintaining the distinctive
structural and functional characteristics of individual cellular
components. At least three major types of import/transport systems have
been described for targeting proteins translated in the cytoplasm to different organelles in the eukaryotic cells. 1) Proteins destined for
the ER,1 Golgi, plasma
membrane, and also those secreted out of cells, are targeted to the ER
through a signal recognition particle (SRP)-dependent mechanism. This pathway is mostly co-translational and involves the
delivery of nascent chains by SRP to the translocon complex on the ER
membrane (1, 2). 2) Protein targeting to mitochondria occurs mostly by
a post-translational mechanism, although exceptions to this generality
have been reported (3, 4). As part of the mitochondrial targeting
pathway, an unfolded polypeptide is brought in contact with the outer
and inner membrane translocase complexes (TOM and TIM, respectively)
(5). The protein is unidirectionally translocated through transmembrane
protein channels formed of TOM40 and TIM23/TIM17 subunits, and its
entry into the matrix space is finally facilitated by an
ATP-dependent pull exerted by the mitochondrial Hsp70
chaperone protein (6). 3) The peroxisomal protein targeting, although
it occurs post-translationally, involves a distinct set of cytosolic
receptors. These proteins not only guide the precursor proteins to the
peroxisomal membrane receptor Pex, they also lead the polypeptide into
the matrix compartment and eventually recycle back to the cytosol for
reutilization (7, 8). As predicted by the signal hypothesis, the
targeting specificity of proteins is dictated by the signal sequences
that function as specific "address codes" (2). The signal sequences
required for protein targeting to the ER, mitochondria and peroxisomes are quite different, and it is generally believed that a protein may
carry only one type of signal, thus ensuring its targeting to the
correct membrane compartment.
In contrast to this general view, recent studies from our laboratory
showed that xenobiotic inducible CYP1A1 and CYP2B1 contain "atypical" signals at the N termini, which we named "chimeric signals," that were capable of targeting the apoprotein chains to
both the ER and mitochondria under in vitro and in
vivo conditions (9, 10). In the case of CYP1A1, the protein chains
that escaped ER targeting (~25% of the total pool) were cleaved past
N-terminal residues 4 and 32, thereby activating a cryptic
mitochondrial targeting signal (9). In the case of CYP2B1, an intact
but Ser-128-phosphorylated protein was transported to mitochondria with
high efficiency (10). Ser-128-phosphorylated CYP2B1 nascent chains
showed vastly reduced efficiency for binding to SRP, but increased
mitochondrial transport. These latter results suggested that protein
kinase A (PKA)-dependent phosphorylation at Ser-128 activates the cryptic mitochondrial targeting signal. Dual localization of yeast fumarase with identical N terminus in the cytosol and mitochondria has been reported to involve a novel mitochondrial processing of the primary translation product for subsequent export to
the cytosol (11).
Recently we studied the nature of hepatic mitochondrial CYP2E1
(alternatively referred to as P450MT5), which exhibits immunological and molecular properties similar to microsomal CYP2E1 (12). The
ethanol- and pyrazole-inducible CYP2E1 is thought to play important
roles in the metabolism of ethanol, acetone, and induction of oxidative
stress (13, 14). Our results with combinatorial approaches showed that
mitochondrial P450MT5 has identical primary structure to that of
microsomal CYP2E1, although the former exhibited a much higher level of
Ser-129 phosphorylation, and significantly different helical and
-sheet contents (12). In the present study we demonstrate that
CYP2E1 contains a cryptic mitochondrial targeting signal at amino acid
residues 21-31, in addition to the previously demonstrated ER
targeting signal within the first 30 residues of the protein (15, 16).
A comparison of chimeric signals of CYP1A1, CYP2B1, and CYP2E1 shows
subtle differences in terms of affinity for binding to SRP and
mitochondrial translocases, but all of them show a distinct resistance
to translational arrest in response to saturating levels of SRP, and
propensity for translation in a membrane-free form. Our results also
show for the first time that Ser-129 phosphorylation increases the
affinity of the protein for binding to cytoplasmic Hsp70 (CyHsp70)
family chaperones as a possible basis for increased mitochondrial
targeting of P450MT5 but does not affect the binding of nascent chains
to SRP or the major ER translocator protein Sec61.
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EXPERIMENTAL PROCEDURES |
Construction of Expression Plasmids--
Full-length CYP2E1
cDNA (17) was generated by reverse transcriptase-based PCR.
N-terminal and point mutations containing a start codon preceded by a
Kozac consensus sequence were constructed essentially as described
before (9, 10), and cloned in the pCMV4 and pGEM7zf (Promega Biotech,
Madison, WI) vectors. Various fusion cDNA constructs were generated
as described before (10) and cloned in pGEM7zf vector.
In Vitro Import of Labeled Proteins into Mitochondria--
Rat
liver or yeast mitochondria were isolated and used for in
vitro import using a system described before (9, 10). In vitro translation products were generated in the TNT rabbit
reticulocyte lysate system (RRL, Promega, Madison, WI) in presence of
added [35S]Met (40 µCi/50-µl reaction, PerkinElmer
Life Sciences), according to the recommended protocol of the
manufacturer. In some experiments the translation products were
phosphorylated for 45 min at 30 °C by supplementing the translation
mix with 10 units of PKA (Sigma) and 100 µM ATP as
described (18). The import assays were carried out in a 200-µl final
volume as described before (9) and treated with 0-300 µg/ml trypsin
for 20 min on ice. Trypsin-treated samples were mixed with 10 M excess of trypsin inhibitor, and mitochondria were
recovered by sedimentation through 1 M sucrose as described (9). Mitochondrial proteins were solubilized in 2× Laemmli sample
buffer for 10 min at 75 °C and analyzed by SDS-PAGE (19) and fluorography.
Expression of CYP2E1 cDNAs in COS Cells--
Various
cDNAs cloned in pCMV4 vector were transfected in COS-7 cells using
FuGENE 6, a non-liposomal transfection reagent (Roche Molecular
Biochemicals), according to the recommended protocol of the
manufacturer. Cells from 6-8 plates (100 mm), after 24 h of
transfection, were homogenized in sucrose-mannitol buffer containing
protease inhibitor mixture with Teflon fitted glass homogenizer (~25
strokes). Isolation of mitochondria by differential centrifugation and
sucrose density banding, and isolation of microsomes were described
before (9).
SDS-PAGE and Immunoblot Analysis--
Proteins were resolved by
SDS-PAGE (19) and transferred to nitrocellulose membranes for
immunoblot analysis as described (20). Polyclonal antibodies against
CYP2E1 (Oxford Biomedical Research, Oxford, MI), mitochondrial
transcription factor A, calreticulin (ABR, Golden, CO), and monoclonal
antibody against CyHsp70 (Sigma) were used. Immunoblots were developed
with chemiluminescence Super Signal Ultra kit (Pierce), and the blots
were imaged and quantitated using a Fluor-S imaging system
(Bio-Rad).
Chemical Cross-linking of CYP2E1 with Mitochondrial Translocases,
SRP, and ER Translocator Protein Sec61--
For cross-linking with
mitochondrial translocator proteins, fusion proteins 1-160
CYP2E1/dihydrofolate reductase (DHFR) and 1-160 S129A/DHFR, subjected
to phosphorylation in presence of added PKA and used for in
vitro transport in isolated yeast mitochondria, and translocation
arrest was initiated by adding 1 µM methotrexate as
described before (6, 10). Translocation intermediates were cross-linked
with 500 µM
m-maleimidobenzoyl-N-hydroxysuccinimidate ester
(MBS), a membrane-permeable cross-linker (Pierce) for 20 min at room
temperature (6). Termination of reaction with 80 mM glycine
and 5 mM -mercaptoethanol, and immunoprecipitation with
anti-CYP2E1 antibody (1:50) or antibodies to mitochondrial translocase
proteins were essentially as described before (10, 21).
Immunoprecipitated proteins were subjected to SDS-PAGE, and the gels
were imaged in a Bio-Rad GS525 Molecular Imager.
Cross-linking of 35S-labeled CYP2E1 or CYP2B1 proteins with
SRP was carried out co-translationally (21) as modified recently (10).
In vitro translation was carried out in the wheat germ lysate (WGL) in the presence of increasing amounts of SRP (0.125-2 units). When phosphorylation was required, PKA (10 units/reaction) was
added to the reaction mixture. After 30 min at 30 °C, reactions were
stopped by adding 1 mM cycloheximide and the incubation was continued for 10 min to allow chain completion. Cross-linking was
performed as described above using the water-soluble
m-maleimidobenzoyl-N-hydroxysulfosuccinimidate ester (S-MBS) cross-linker. Immunoprecipitation was
performed using antibodies against CYP2E1, CYP2B1, or 54-kDa subunit of SRP. Immunoprecipitates were subjected to electrophoresis, and gels
were imaged and quantified using a Bio-Rad GS525 Molecular Imager.
Cross-linking of CYP2E1 and CYP2B1 with ER membrane translocator
protein Sec61 was carried out following the procedure of Greenfield and
High (22) with some modifications. COS cells were co-transfected with
both CYP2E1 and CYP2B1 cDNA constructs. Cells were harvested after
8 h, suspended in Hepes-sucrose (2 mM HEPES, pH 7.4, 70 mM sucrose, 210 mM Mannitol, 2 mM EDTA) at ~5 × 106 cells/ml,
subjected to cross-linking with 500 µM MBS for 1 h at room temperature, and used for isolating microsomes and cytosol fractions. Cross-linked CYP2E1, CYP2B1, and calreticulin proteins were
first immunoprecipitated with respective antibodies followed by
immunoblot analysis with antibody to Sec61
or CyHsp70.
Membrane Integration Assay--
Proteins were translated in WGL
with or without added PKA (10 units), in the presence of 2 units/25
µl of unwashed or KCl-washed canine reticular endoplasmic membranes
(10). Reactions were stopped by adding 1 mM cycloheximide,
membranes were extracted with 0.1 M
Na2CO3 (pH 11) for 1 h on ice and
fractionated in to soluble (200,000 × g supernatant)
and insoluble (pellet fractions) as described before (23). Both the
membrane pellet and the protein extract were resuspended in 40 µl of
10 mM Tris-HCl (pH 7) containing 2% SDS and subjected to
SDS-PAGE.
Interaction of CYP2E1 with CyHsp70--
Interaction of CYP2E1
with CyHsp70 under in vitro and in vivo
conditions was tested by co-immunoprecipitation. For studying in
vitro interaction, CYP2E1 protein was synthesized and
phosphorylated in RRL in the presence or the absence of PKA inhibitors
(H89 or a myristoylated peptide, PKI, Calbiochem-Novabiochem Corp., San Diego, CA). Interaction of the CYP proteins with CyHsp70 present in the
RRL under these conditions was estimated by co-immunoprecipitation. Reaction mixtures were immunoprecipitated with a polyclonal antibody against CYP2E1 raised in goat, and resolved by SDS-PAGE on a 10% gel.
The bottom half of the gel containing the 52-kDa CYP proteins was
subjected to autoradiography, and the top portion of the gel was
transferred to a nitrocellulose membrane and probed with a mouse
monoclonal antibody for Hsp70.
In vivo interaction was studied using post-mitochondrial
supernatant fraction from cells transfected with CYP2E1 WT, CYP2E1 S129A and CYP2E1 KKmut cDNAs, in the presence or absence of
dibutyryl cAMP (db-cAMP). CyHsp70 was co-immunoprecipitated with a
polyclonal antibody against CYP2E1 raised in goat. The
immunoprecipitated proteins were resolved by SDS-PAGE on a 12% gel and
transferred to nitrocellulose, and the membrane was probed
concomitantly with mouse monoclonal antibody directed against CyHsp70
and monoclonal antibody to CYP2E1.
Binding of CYP2E1 to Reconstituted TOM40--
Yeast TOM40
protein expressed in Escherichia coli BL21 cells (24) was
purified from the inclusion bodies to near homogeneity and used for
reconstitution in liposomes essentially as described (24). Liposomes
were prepared from azolectin (Sigma, type IVS) in MOPS-Tris (pH 6.9)
buffer using a Branson sonifier as described before (25). Liposomes
were freeze-thawed three times and solubilized by adding
n-octyl glucopyranoside (6% v/v) on ice for 30 min. Solubilized liposomes (10 mg) were mixed with 0.1 mg of TOM40 protein and diluted 2-fold with 10 mM MOPS-Tris, pH 6.9, containing 0.5 mM PMSF and 0.5 mM DTT.
Reconstitution was performed by dialysis of the mixture at 4 °C for
24 h against the same buffer. 35S-Labeled and
phosphorylated RRL translation products (80,000-160,000 cpm) were
incubated for 1 h at 30 °C with TOM40-containing or TOM40-free
liposomes (250 µg) in a buffer containing 20 mM KCl and
2% bovine serum albumin, followed by addition of 150 mM
KCl. Liposomes were isolated by centrifugation at 200,000 × g, washed once with 150 mM KCl containing buffer
(26), resuspended in Laemmli buffer, and subjected to SDS-PAGE on a
12% gel. The amounts of radioactive protein binding were measured by
imaging through the GS-525 imager (Bio-Rad).
Depletion of CyHsp70 from RRL--
CYP2E1 WT and 1-160
CYP2E1/DHFR were translated in RRL (100-µl reactions) and incubated
for 30 min on ice with 8 M urea (v/v). After dialysis for
6 h against three changes of 50 mM Tris-HCl buffer, pH
7.4, containing 1 mM MgCl2, 50 mM
KCl, and 0.1 mM DTT, the mixture was incubated overnight
with 10 µg of monoclonal anti-CyHsp70 antibody. CyHsp70 was depleted
by adding protein A-agarose, followed by pelleting the immune complex.
In vitro import with isolated rat liver mitochondria was
carried out using translation products formed in control RRL or in RRL
depleted of CyHsp70.
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RESULTS |
Characteristics of the N-terminal Signal Sequence of
CYP2E1--
Fig. 1 shows the known and
predicted microdomains at the N terminus of CYP2E1. The N-terminal
29-amino acid region of CYP2E1 contains an ER targeting and a
transmembrane helical domain, but it lacks an efficient ER retention
signal compared with other ER-targeted CYPs (27). The +33 to +39 region
of the protein contains the conserved proline-rich domain, which is
suggested to help fold the protein facing the cytoplasmic side of the
membrane. No canonical mitochondrial targeting signal preceding the
proline-rich domain, similar to those in CYP1A1 and CYP2B1 (9, 10) is seen in the N-terminal 39-amino acid region of CYP2E1. However, sequence 21-31, which is part of the transmembrane anchor domain, contains two positively charged amino acids (at positions 24 and 25),
which may potentially function as part of a cryptic mitochondrial targeting signal. As observed with other members of CYP family 2, CYP2E1 contains a unique PKA target phosphorylation site at Ser-129
(28, 29). Based on this general background, we generated a series of
N-terminal deletions and point mutations targeted to the positive
residues at positions 24 and 25 and to the phosphorylation site at
position 129 (see Fig. 1). We also generated fusion constructs of
sequence 1-160 of CYP2E1 (wild-type and phosphorylation site mutant
S129A) fused to DHFR reporter protein as shown. A construct of sequence
1-100 of CYP1A1 fused to DHFR was also generated as shown (Fig.
1).
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Fig. 1.
Chimeric signal properties of the N-terminal
end of CYP2E1. The predicted signal properties of the N-terminal
35 amino acid sequences of CYP2E1 and various mutations targeted to
this region (in bold) and fusion constructs used in this
study are shown.
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Localization of Mitochondrial Targeting Signal and Activation by
cAMP-dependent Phosphorylation--
Mitochondrial
targeting of CYP2E1 was studied using in vitro import of
35S-labeled proteins into isolated rat liver mitochondria
using resistance to limited trypsin digestion as a criterion for
protein import. ~9% of input wild-type CYP2E1 protein was found to
be associated with mitochondria banded through 1 M sucrose,
and only 1.5% of input protein was rendered resistant to trypsin
digestion, suggesting inefficient binding and import (Fig.
2A). Phosphorylation of
translation products by incubation with PKA and ATP strongly increased
both protein binding and import to 70 and 30% of input counts,
respectively (Fig. 2A). Addition of H89 (an inhibitor of
PKA) during phosphorylation (Fig. 2A) or mutations targeted to the phosphorylation site Ser-129 (Fig. 2B, construct
CYP2E1 S129A), reduced mitochondrial transport compared to the control unphosphorylated CYP2E1 level. Deletion of N-terminal 21 amino acid
residues (+21/2E1) increased the transport by ~10-fold compared with
intact and unphosphorylated CYP2E1 protein, and phosphorylation caused
an additional increase in import to ~45% of input protein (Fig.
2C). Truncation to residue 36, on the other hand, nearly completely abolished the import, suggesting that the putative mitochondrial targeting signal resides between amino acid residues 21 and 36 (Fig. 2B). N-terminal deletion to residue 31 caused a
similar impairment of transport, narrowing the potential mitochondrial targeting sequence from position 21 to 31 of the protein (data not
shown). Mutations targeted to the two positively charged residues at 24 and 25 abolished both the basal transport and the
PKA-dependent increase in import of wild-type and +21/2E1
(Fig. 2, A and C). Fig. 2D shows that
import of phosphorylated CYP2E1 protein was dependent on transmembrane
potential because 2,4-dinitrophenol and carbonyl
cyanide-m-chlorophenyl hydrazone, which disrupt
mitochondrial membrane potential, reduced the transport to 10 and 2%
of control reactions, respectively. The transport was also dependent on
ATP, because omission of energy mix reduced the transport. Activators (Mn2+ and Mg2+) and inhibitors (EDTA and
O-phenanthroline) of matrix processing peptidase (MPP) had
no significant effect on the efficiency of mitochondrial import of
phosphorylated CYP2E1 protein or the size of the imported protein,
further confirming that imported CYP2E1 is not proteolytically
processed (results not shown).
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Fig. 2.
Sequence requirements for the in
vitro transport of CYP2E1 protein.
35S-Labeled translation products generated in the presence
or absence of added PKA were used for the import into rat liver
mitochondria. Mitochondria from a companion set of reactions were
treated with trypsin (300 µg/ml), and the proteins were resolved by
SDS-PAGE on a 12% gel and subjected to fluorography. A-C,
wild-type and various mutant CYP2E1 proteins were used as indicated. In
D, mitochondria were pre-incubated for 20 min on ice with or
without added 2,4-dinitrophenol (2,4DNP, 25 µM), carbonyl cyanide-m-chlorophenyl hydrazone
(CCCP, 25 µM), or in the absence of added
energy mix (E mix) before initiating import reaction with
phosphorylated wild-type CYP2E1 translation product. The percentage
binding and import are presented.
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The role of PKA-dependent phosphorylation on the in
vivo targeting of CYP2E1 to mitochondria was investigated using
COS cells transfected with various wild-type and mutant cDNA
constructs shown in Fig. 1. The effects of cAMP analog db-cAMP, cAMP
inducer forskolin, and PKA-specific inhibitor myristoylated peptide
(PKI) on subcellular distribution of CYP2E1 were examined. After
24 h of transfection, mitochondria and microsomes were isolated
and analyzed by immunoblot analysis. As previously described, the cell
fractionation procedure yielded mitochondrial preparations containing
less than 1% microsomal contamination as tested by marker enzyme
assays (9, 10).
Immunoblot analysis in Fig. 3 was carried
out with equal amount (20 µg) of mitochondrial and microsomal
proteins, and the levels of antibody reactive proteins in different
subcellular compartments (Fig. 3, bottom) were calculated
based on a recovery of 2:1 ratio of microsomal and mitochondrial
proteins from COS cell homogenates. Results together show that, under
normal culture conditions, the microsomal CYP2E1 content was
~2-3-fold higher than the mitochondrial content. The immunoblots in
topmost panels show that addition of db-cAMP or
forskolin resulted in a 50-70% increase in the mitochondrial CYP2E1
level but did not affect the level of microsomal CYP2E1. PKI
caused a 70% inhibition of mitochondrial CYP2E1 but
did not affect the level of microsome-associated CYP2E1 (see
Fig. 3, top panels). Mitochondrial targeting of
CYP2E1 was drastically reduced by K24A and K25A substitutions
(2E1KKmut), but these mutations only marginally affected the microsomal
targeting of the protein. Forskolin and db-cAMP did not affect the
targeting of mutant protein to either of subcellular compartments (Fig. 3, top panels). Mutation at the Ser-129
phosphorylation site (2E1S129A) drastically reduced mitochondrial
targeting while leaving ER targeting unaffected (Fig. 3,
middle panels). As expected, db-cAMP, forskolin, or PKI did not affect the level of targeting of mutant protein either
to mitochondria or to the ER. Furthermore, +36/2E1, lacking the
transmembrane domain and also the putative mitochondrial targeting signal, was detected neither in the mitochondrial nor the microsomal fractions, although it was detected in the total homogenate
(middle panel). Deletion to residue 21 (+21/2E1)
reduced the microsomal targeting by ~70% but increased the
mitochondrial targeting by nearly 2-fold (bottom
panel). Mutations targeted to the positive residues at
positions 24 and 25 (+21/KKmut) and the phosphorylation site (S129A)
reduced the level of mitochondrial targeting of +21/2E1 by 70-80%
without any significant effect on the microsomal targeting. These
results are consistent with the in vitro results presented in Fig. 2, and show that Lys-24 and Lys-25 are critical for
mitochondrial targeting of CYP2E1.
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Fig. 3.
Effects of phosphorylation on in
vivo targeting of CYP2E1 protein to mitochondria and
microsomes. COS cells were transfected with indicated cDNA
constructs or empty vector (Mock), and subcellular fractions
were isolated as described under "Experimental Procedures."
Microsomal (A) and mitochondrial (B) proteins (20 µg each) or total homogenate fraction (100 µg) were resolved by
SDS-PAGE on a 12% gel and subjected to immunoblot analysis. Blots were
sequentially probed with a goat polyclonal antibody for CYP2E1 (1:2000
dilution) and with an anti-calreticulin (1:1000 dilution) antibody for
microsomal proteins (A) or with an anti-mitochondrial
transcription factor A antibody (1:3000 dilution) for mitochondrial
proteins (B) as loading controls. db-cAMP (10 µM) and forskolin (10 µM) were added 3 h after transfection. PKI was added 1 h before transfection at a
final concentration of 400 nM. The blots were quantitated
by imaging through a Bio-Rad Fluor S imager, and the relative
distribution of CYP2E1 in the microsomal and mitochondrial fractions
was calculated based on a 2:1 recovery of the microsomal and
mitochondrial protein from the total cell homogenate.
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The quantitation of immunoblot data based on a 2:1 recovery of
microsomal and mitochondrial proteins from transfected COS cells shows
that the total cellular CYP2E1 level was increased by ~25% in cells
treated with db-cAMP and forskolin and reduced by ~25% in cells
treated with PKI. Results also show that only the mitochondrial CYP2E1
content changed significantly under these treatment conditions, whereas
the microsomal content remained relatively unaltered. Mutations
targeted to Ser-129 did not affect the total cellular level of CYP2E1
protein, although the mitochondrial level was drastically reduced with
a marginal increase in the microsomal protein level.
Effects of Phosphorylation on the Binding of CYP2E1 Apoprotein to
SRP--
In Fig. 4A
(lanes 1-6), we compared the extent of ER
membrane integration of wild-type and S129A mutant proteins that were translated in the WGL in the presence of [35S]Met, with
or without added PKA (10 units), and 5 units of unwashed canine rough
ER or KCl-washed ER membranes. Preprolactin (PPL) as a marker protein
containing a classical ER targeting signal, and porin as a marker for a
non-ER-targeted protein were also co-translated. The extent of membrane
association was determined by extraction with alkaline
Na2CO3. In the absence of added PKA, both
wild-type and CYP2E1/S129A mutant proteins partitioned almost equally
in the insoluble (P) and soluble (S) fractions, indicating that ~50%
of the CYP2E1 was not efficiently inserted in the ER membranes and
instead remained in the soluble fraction (Fig. 4A, lanes 1 and 2). Although not shown,
in vitro translated CYP2E1 in RRL showed a similar
distribution. Addition of PKA did not alter the distribution of CYP2E1
and CYP2E1/S129A mutant between insoluble and soluble compartments
(data not shown). Notably, the S129A mutant protein also partitioned in
the membrane-bound and -free phases in a manner similar to the
wild-type phosphorylated protein, suggesting that phosphorylation has
no effect on the efficiency of CYP2E1 targeting to the ER. As expected,
more than 90% of ER-targeted PPL was recovered in the insoluble
fraction and more than 90% of the mitochondrial outer membrane protein porin was recovered in the soluble fraction (Fig. 4A,
lanes 1-4). When translation was performed in
the presence of KCl-washed ER membranes that are deficient in SRP,
almost 100% of the CYP2E1 protein partitioned in the soluble fraction,
indicating that insertion of CYP2E1 in the ER membranes was
SRP-dependent (Fig. 4A, lanes 5 and 6). Results also show that CYP2B1, another
similarly oriented transmembrane CYP, was almost completely recovered
in the membrane fraction under similar in vitro translation
conditions (Fig. 4A, lanes 7 and
8), suggesting that CYP2B1 is targeted to the ER more efficiently than CYP2E1.
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Fig. 4.
Effect of phosphorylation of CYP2E1 on ER
membrane association and SRP binding. A, extent of
integration of wild-type and S129A mutant CYP2E1 proteins into ER
membrane. Proteins were translated in WGL in the presence of
[35S]Met and 5 units of unwashed endoplasmic reticular
membranes (ERM, lanes 1-4) or
KCl-washed membranes (KERM, lanes 5 and 6). PPL and porin were co-translated. CYP2B1, another
N-terminally anchored membrane protein, was used also as a control
(lanes 7 and 8). B,
wild-type CYP2E1, CYP2E1 S129A mutant and DHFR proteins were translated
in WGL in presence of [35S]Met with or without added PKA
(10 units) and indicated amounts of SRP. Aliquots of translation
reaction (20 µl) were resolved by SDS-PAGE on 12% gels.
C, rates of SRP-mediated translation inhibition of CYP2E1,
CYP2B1, and PPL from reactions run as in B. The gels were
imaged and quantitated in a Bio-Rad Fluor S molecular imager. The
activity in the absence of added SRP for each protein was regarded as
100%. D and E, levels of SRP binding to
phosphorylated and unphosphorylated CYP2E1 by chemical cross-linking.
Translation of wild-type CYP2E1 (top panel of
D and also first two lanes
in E) or CYP2B1 (bottom panel of
D and last two lanes of
E) was carried out in WGL in presence of
[35S]Met, increasing amounts of SRP, with or without
added PKA (10 units). Cross-linking with S-MBS was performed
as described under "Experimental Procedures." Reaction products
with or without added cross-linker were immunoprecipitated with
polyclonal antibody to CYP2E1 or CYP2B1 as indicated in D.
Cross-linked products (CLP) are indicated. The extent of
cross-linking as a function of SRP concentration is presented
underneath panel D. In E,
the immunoprecipitation was carried out with antibody to the 54-kDa
subunit of SRP.
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Under in vitro conditions, SRP binds to nascent chains
containing an ER targeting signal, causing an arrest of chain
elongation and thus, translation inhibition (30). Elongation arrest is thought to be a physiologically important function of SRP (31). We
therefore used this property as an indicator of the affinity of SRP for
phosphorylated or unphosphorylated CYP2E1 nascent chains (Fig.
4B). Cytosolic protein DHFR was used as an internal control. In the first approach, CYP2E1, CYP2E1/S129A, or DHFR were translated in
the WGL system in presence of increasing concentrations of SRP. The
rate of translation of both wild-type and S129A mutant CYP2E1 was
inhibited with increasing amounts of SRP with a maximum inhibition of
70-80% at the highest SRP concentration (8 units) used. Addition of
PKA had no significant effect on the rate of inhibition by SRP, further
confirming that phosphorylation does not alter the extent of CYP2E1
binding to SRP. The in vitro translation of DHFR was not
inhibited under these conditions. A comparison of the rate of
SRP-mediated inhibition of CYP2E1, CYP2B1, and PPL is presented in Fig.
4C. It is noteworthy that, at the highest SRP concentration
of 4 units, the translation of both CYP2B1 and PPL was inhibited to
near 100%. Under these conditions, CYP2E1 was inhibited only by
~40-45%. These results show that the chimeric signals of the two
CYP proteins respond differently to added SRP suggesting possible
difference in binding affinities.
In a more direct approach, we studied the extent of association of
phosphorylated and unphosphorylated CYP2E1 with SRP and compared with
that of CYP2B1 by chemical cross-linking (Fig. 4, D and
E). Proteins were translated in WGL in the presence of
[35S]Met and increasing amounts of SRP and subjected to
cross-linking with S-MBS. The cross-linked products were
immunoprecipitated with antibody to CYP2E1 (Fig. 4D,
top panel) and CYP2B1 (Fig. 4D,
bottom panel), respectively. Immunoprecipitates
with both antibodies showed slow migrating cross-linked products of
~95 kDa, in addition to 52-kDa CYP proteins. As expected, the
immunoprecipitates showed increasing levels of cross-linked product
with increasing SRP in both cases reaching saturation levels at ~4
units of SRP (see the quantitation at the bottom of Fig.
4D). In the case of CYP2E1, phosphorylated (+ PKA) as well
as unphosphorylated (
PKA) translation products yielded similar levels
of cross-linking. However, in support of our recent results on extent
of ER targeting (10), the unphosphorylated CYP2B1 yielded 3-4-fold
higher level of cross-linking as compared with unphosphorylated CYP2B1.
The data on cross-linking (Fig. 4D and the quantitation
underneath) show that the association constant for CYP2B1 (0.41 nM) nearly doubled after phosphorylation (0.76 nM). However, the association constant for CYP2E1 (1.23 nM) was not significantly affected by phosphorylation (1.34 nM). The results of SRP mediated translation inhibition
(Fig. 4C) and cross-linking (Fig. 4D) together
show the following. 1) In marked variation from that observed with CYP2B1, phosphorylation has no effect on the extent of CYP2E1 binding
to SRP. 2) The chimeric N-terminal signals of CYP2E1 and CYP2B1 have
different affinities for SRP, with unphosphorylated CYP2E1 showing a
significantly higher association constant as compared with
unphosphorylated CYP2B1.
In Fig. 4E, the reaction mixtures with 2 units of SRP, and
with or without added PKA, were also immunoprecipitated with antibody to the 54-kDa subunit of SRP, confirming that the cross-linked product
indeed contains a component of SRP in addition to the CYP proteins.
Association of Phosphorylated CYP2E1-DHFR Fusion Protein with
Mitochondrial Translocases--
Chemical cross-linking was carried out
to determine the interaction of CYP2E1 signal sequence with components
of mitochondrial TOM and TIM complexes. We used fusion proteins
containing the first 160 amino acids of wild-type or S129A mutant
CYP2E1 proteins fused to DHFR (6). Fusion protein composed of the first
100 amino acids of CYP1A1 fused to DHFR served as a negative control, because the mitochondrial import of CYP1A1 is not regulated by phosphorylation. The translation products with or without added PKA
were used for import into yeast mitochondria, translocation was
arrested by adding methotrexate and translocation intermediates were
generated (6, 10). As expected, only phosphorylated 1-160 CYP2E1/DHFR
was imported into mitochondria and rendered resistant to trypsin (Fig.
5A). The unphosphorylated
fusion protein was not imported at a significant level, although it
bound to mitochondria at a significant level. The S129A mutant protein, on the other hand, was only marginally imported even when it was translated in the presence of added PKA. Finally, the
1-100:CYP1A1/DHFR fusion protein was imported inefficiently, whether
it was translated in the presence or absence of added PKA. Fig.
5B shows the formation of a translocation-arrested
intermediate in the presence of added methotrexate. No detectable
unphosphorylated 1-160 CYP2E1/DHFR fusion protein was resistant to
trypsin digestion, possibly because of inefficient binding of the
protein to mitochondrial translocases. The phosphorylated 1-160
CYP2E1/DHFR fusion protein, however, yielded an 18-kDa
trypsin-resistant product representing the translocation-arrested component. Fusion protein with mutated phosphorylation site, 1-160 S129A/DHFR, failed to yield any trypsin-resistant product, further confirming inefficient import of the protein.
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Fig. 5.
Interaction of phosphorylated CYP2E1 with
mitochondrial protein translocases. A, 1-160
CYP2E1/DHFR, 1-160 S129A/DHFR, and 1-100 CYP1A1/DHFR fusion proteins
translated in the RRL were used for in vitro import.
B, effect of methotrexate-mediated translocation arrest of
wild-type (1-160 CYP2E1/DHFR) and mutant (1-160 S129A/DHFR) fusion
proteins. Reaction mixtures were preincubated on ice for 20 min with 1 µM methotrexate before initiating the import.
C, chemical cross-linking of translocation-arrested 1-160
CYP2E1/DHFR and 1-160 S129A/DHFR fusion proteins with mitochondrial
translocases and MtHsp70. Both fusion proteins were translated in
presence of PKA (10 units), and translocation arrest was caused by
adding 1 µM methotrexate as in B. IPP, immunoprecipitation; ab, antibody.
Cross-linking with MBS cross-linker and immunoprecipitation were
carried out as described under "Experimental Procedures." In
A and B, proteins were resolved by SDS-PAGE on
14% gels and in C on 10% gel. Cross-linked products
(CLP) are indicated.
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In the next series of experiments, methotrexate-arrested intermediates
of wild-type and S129A mutant fusion proteins (with added PKA) were
subjected to chemical cross-linking with membrane-permeable cross-linker MBS, and the cross-linked products were immunoprecipitated with antibodies against subunits of translocase complexes or CYP2E1 antibody. Results in Fig. 5C show that pre-immune IgG did
not interact with 35S-labeled 1-160 CYP2E1/DHFR fusion
protein. In reaction mixture without added cross-linker, only the input
36-kDa fusion protein was immunoprecipitated by antibody to CYP2E1. A
companion reaction with added cross-linker yielded two major
cross-linked products of ~105 and ~75 kDa, and a number of minor
products in addition to the 36-kDa input fusion protein.
Immunoprecipitation with specific antibodies showed that the 75-kDa
band in lane 3 represents two similarly migrating
cross-linked products with TOM40 and TIM44, and the 105-kDa band
represents cross-linked product with mitochondrial Hsp70 (MtHsp70) from
the matrix side. Results also showed that the fusion protein did not
cross-link significantly with TOM20. This observation is consistent
with unpublished results in our laboratory that chimeric signals of
both CYP2B1 and CYP2E1 bypass interaction with TOM20 and directly
associate with TOM22.2 As
expected, the mutant protein 1-160 S129A/DHFR failed to cross-link significantly with any of the translocase subunits tested. Both the
wild-type and S129A mutant fusion proteins showed double bands, indicating alternate translation at an immediate downstream AUG codon.
These results suggest that Ser-129 phosphorylation is important for
CYP2E1 interaction with the mitochondrial translocase complexes and for
the mitochondrial import.
Phosphorylated CYP2E1 Binds to CyHsp70 and Mitochondrial TOM40
Proteins with Increased Affinity--
With a view to understand the
mechanism of cAMP-mediated increase in mitochondrial targeting of
CYP2E1, we studied two different extramitochondrial steps of protein
targeting, namely binding of nascent protein to CyHsp70 and its
subsequent binding to liposome reconstituted TOM40. Association with
CyHsp70 was tested by co-immunoprecipitation of in vitro
translated CYP2E1 with endogenous CyHsp70 present in the RRL (Fig.
6A). Interaction of CYP2E1
with CyHsp70 in vivo in transfected COS cells was tested by
immunoprecipitation of post-mitochondrial protein fraction (Fig.
6B). The in vitro translation products were
immunoprecipitated with CYP2E1 antibody, and subjected to immunoblot
analysis with CyHsp70 antibody. As seen from Fig. 6A
(lane 2), CYP2E1 antibody co-immunoprecipitated
the highest amount of CyHsp70 from reaction with phosphorylated
wild-type CYP2E1 (without added inhibitors). The pre-immune antibody,
on the other hand, did not precipitate either of the two proteins to a
significant level (lane 1). The inhibitors of
PKA, H89 and PKI, added during translation markedly reduced the
co-precipitation of CyHsp70 protein (lanes 3 and
4). Additionally, the S129A mutant protein also drastically
reduced the level of CyHsp70 co-precipitation, suggesting that
phosphorylation increases the CYP2E1 binding to CyHsp70
(lane 5). As expected, treatment with 4 M urea followed by dialysis (see lane
8) drastically reduced the binding of nascent protein to
CyHsp70 as compared with control samples not treated with urea but
dialyzed (lane 7). The levels of CYP2E1 proteins in these reactions were not limiting as indicated by the intensity of
bands in the lower panel of Fig.
6A.
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Fig. 6.
Ser-129 phosphorylation increases the
affinity of CYP2E1 for binding to CyHsp70 and reconstituted TOM40
proteins. In A, wild-type and S129A mutant CYP2E1
proteins were translated and phosphorylated in RRL in the presence or
absence of H89 (100 nM) or PKI (1 µM).
Treatment with 4 M urea was carried out at 30 °C for 25 min, followed by dialysis over two changes of 20 mM
Tris-Cl, pH 7.5, 1 mM MgCl2, 1 mM
DTT, 50 mM KCl. Control samples without added urea were
also incubated and dialyzed similarly, but with added 100 µM ATP. Translation products (25 µl) were
immunoprecipitated (IP) either with pre-immune serum or with
polyclonal antibody (ab) against CYP2E1, and the
immunoprecipitates were resolved by SDS-PAGE on a 10% gel. Purified
CyHsp70 (1 µg) was run alongside. The upper
part of the gel (containing Hsp70) was transferred to
nitrocellulose and probed with monoclonal antibody to CyHsp70. The
bottom part of the gel (containing CYP2E1) was
subjected to autoradiography. In B, post-mitochondrial
supernatants were isolated from COS cells transfected with WT/CYP2E1, CYP2E1/129A, or CYP2E1/KKmut in the absence or
presence of db-cAMP (10 µM). The supernatants (1 ml) were
immunoprecipitated either with pre-immune serum or with polyclonal
antibody against CYP2E1 and separated by SDS-PAGE on a 12% gel. After
transfer to nitrocellulose, the membrane was incubated with monoclonal
antibodies against Hsp70 and CYP2E1. In C,
35S-labeled +33/CYP1A1 and DHFR proteins (80,000 cpm each)
or phosphorylated CYP2E1 and CYP2E1/S129A proteins (80,000 and 160,000 cpm as indicated) were incubated with liposomes containing
reconstituted TOM40 or with an equal amount of liposomes lacking TOM40.
The amount of bound proteins was quantified by autoradiography. In
D, binding experiments were carried out as in C
using 160,000 cpm each of translation product in RRL: control untreated
translation product (lane 1), translation product
incubated without added urea followed by dialysis (lane
2), translation product incubated with urea followed by
dialysis (lane 3), and translation mix depleted
of CyHsp70 by immunoadsorption (lane 4) as
described under "Experimental Procedures." Treatment with urea and
dialysis were carried out as in A.
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Fig. 6B shows the pattern of co-immunoprecipitation using
post-mitochondrial protein fractions of COS cells transfected with WT
CYP2E1, CYP2E1/S129A, or CYP2E1/KKmut in the presence or absence of
added db-cAMP. Results show that CyHsp70 was co-immunoprecipitated with
antibody to CYP2E1 only in cells transfected with WT CYP2E1 but not
with S129A mutant CYP2E1, suggesting the importance of phosphorylation
for in vivo interaction. Mutations at the N-terminal positively charged residues (KKmut) had no effect on CYP2E1 interaction with CyHsp70 protein. As expected, addition of db-cAMP markedly increased the binding further confirming the role of PKA-mediated phosphorylation for CYP2E1 binding to CyHsp70. Results in Fig. 6B also show that the levels of CYP2E1 were nearly similar
in different cell extracts, ensuring that varying levels of
co-immunoprecipitation of CyHsp70 were not the result of limiting
CYP2E1 levels. Additionally, pre-immune antibody did not
immunoprecipitate significant CYP2E1 or CyHsp70 proteins (Fig.
6B, lane 1).
Next, we assessed the extent of binding of phosphorylated and
unphosphorylated CYP2E1 to liposome-reconstituted TOM40 protein. We
used +33/1A1, whose mitochondrial targeting does not require phosphorylation, S129A mutant CYP2E1, and cytosolic protein DHFR as
controls. As shown in Fig. 6C, the wild-type CYP2E1, S129A mutant, and +33/1A1 proteins did not bind significantly to liposomes without added TOM40 protein. The phosphorylated wild-type CYP2E1 protein bound to reconstituted TOM40 protein in a
concentration-dependent manner (7.5-12%). The S129A
mutant protein under these conditions bound at 0.8-2% level. The
+33/1A1 protein also bound efficiently (10%) to reconstituted TOM40.
As expected, DHFR, a cytosolic protein, bound at a very low level.
Although these results suggest that phosphorylation increases the
affinity of CYP2E1 for TOM40, use of total translation mix containing
CyHsp70 leaves open the possibility that increased binding is because
of conformational changes induced by the latter. This question was
addressed using reaction mixture treated with urea (followed by
dialysis) to disrupt the complex or translation mix depleted of CyHsp70
by immunoadsorption following urea treatment. As shown in Fig.
6D, control dialyzed reaction mixture without urea treatment
(lane 2) bound to reconstituted TOM40 at
approximately the same level (9-10%) as non-dialyzed and untreated
control (lane 1). The translation product from urea-treated and dialyzed sample, however, showed a markedly reduced binding of
~2.7% (lane 3). Furthermore, the translation
mix depleted of CyHsp70 (see Fig. 7)
showed no detectable binding (lane 4). These results suggest that CyHsp70 binding directly or indirectly facilitates the binding of nascent CYP2E1 to reconstituted TOM40.
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Fig. 7.
Role of CyHsp70 on mitochondrial import of
CYP2E1. 35S-Labeled translation products (CYP2E1 WT
and 1-160 CYP2E1/DHFR) were generated in the presence of added PKA.
Untreated (lanes 1-4) translation products
(T.P.) or CyHsp70-depleted translation products
(lanes 5-8) were used for mitochondrial import.
For each sample, a companion set of mitochondria was treated with 300 µg/ml trypsin. In some cases, purified CyHsp70 was added before the
import reaction (lanes 3, 4,
7, and 8).
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Requirement for CyHsp70 in Mitochondrial Import of CYP2E1--
The
role of CyHsp70 in the mitochondrial import of CYP2E1 in
vitro was directly tested by selective depletion of, and
supplementation with, purified CyHsp70. Initial experiments (not
presented) showed that immunodepletion of CyHsp70 drastically inhibited
in vitro translation of CYP2E1 nascent protein. We therefore
translated CYP2E1 and 1-160 CYP2E1/DHFR in RRL in presence of added
PKA, and the RRL was then depleted of CyHsp70 by immunoadsorption. ~20% of labeled CYP2E1 chains were lost during the depletion steps, which include treatment with urea, followed by dialysis and
immunoadsorption. As seen in Fig. 7, in vitro translation
products (CYP2E1, top panel; CYP2E1-DHFR fusion
protein, bottom panel) in control and depleted-RRL were used for in vitro mitochondrial import
assay. Mitochondrial binding was reduced by 50-80% (lane
5) with in vitro translation products depleted of
CyHsp70, and the mitochondrial import was nearly completely abolished
(lane 6). However, both binding to mitochondria
(lane 7) and import (lane
8) were nearly completely restored by supplementing the
reaction mixture with purified CyHsp70. However, addition of purified
CyHsp70 to control RRL did not have any effect on either the binding or
import of CYP2E1 and CYP2E1-DHFR proteins (lanes
3 and 4). The translation products as well as
imported products in CyHsp70-deleted extracts appear as doubles, which
may represent translation products with alternate start sites or minor
degradation. These results further support the view that CYP2E1 nascent
chain binding to CyHsp70 is important for its mitochondrial transport.
Role of Phosphorylation on the Extent of CYP2E1 Interaction with ER
Translocator Protein Sec61 in Transfected COS Cells--
The
significance of in vitro data on the binding of
phosphorylated and unphosphorylated CYP2E1 nascent protein to SRP and CyHsp70 (Figs. 4 and 6) was further assessed in a whole cell setting by
using chemical cross-linking. COS cells co-transfected with CYP2E1 and
CYP2B1 cDNA constructs (for 8 h), and treated with or without
db-cAMP, were subjected to cross-linking with membrane-permeable cross-linker MBS. The CYP proteins in association with the ER-specific protein Sec61 or the cytosolic fraction were detected by
immunoprecipitation. As an initial first step of protein targeting to
ER, the SRP-bound nascent chains associate with the ER translocator
protein Sec61, and remain bound to this protein until polypeptide
chains are completed (32). We have therefore used association with
Sec61 as a criterion for ER association. As shown in Fig.
6A, the microsomal proteins from cells incubated with
cross-linker were subjected to immunoprecipitation with various
antibodies and the immunoprecipitates were further probed with Sec61
antibody by immunoblot analysis. Results show that a significant
amounts of CYP2E1, CYP2B1, and calreticulin exist in association with
Sec61, as indicated by the level of ~93-kDa cross-linked product.
Although not shown, immunoblot analysis failed to detect 52-kDa CYP2E1
and CYP2B1 proteins in this fraction. Results also show that cAMP did
not affect the level of CYP2E1 cross-linking with Sec61, whereas this treatment markedly reduced the level of CYP2B1 cross-linking with Sec61. The latter results are consistent with our previous observation that PKA-mediated phosphorylation reduces the level of microsomal targeting by way of reduced affinity of the apoprotein for SRP (10).
Finally, treatment with cAMP marginally increased the level of
cross-linked product with calreticulin, which is likely the result of
increased translation rates. As expected, pre-immune serum did not
yield any antibody reactive protein species.
The steady state levels of proteins in the soluble fraction of
cAMP-treated and untreated control cells were assessed by
immunoprecipitation followed by immunoblot analysis (Fig.
6B). In the case of CYP2E1 and CYP2B1 proteins, most of the
antibody reactive proteins were detected in higher molecular forms of
~120-210 kDa. Low levels of 52-kDa species were also detected in
both cases. As expected of a true ER-targeted protein, no significant
calreticulin was detected in the soluble cytoplasmic fraction. Results
in Fig. 6B also show that the level of both CYP2E1 and
CYP2B1 in the putative cross-linked species (120-210 kDa) markedly
increased in cells treated with cAMP. Because the in vitro
studies in Fig. 6 showed that phosphorylated CYP2E1 binds to CyHsp70
with increased efficiency, we reprobed the blots with Hsp70 antibody.
Immunoblot in Fig. 6C shows that the slow migrating
complexes indeed represent CYP2E1 and CYP2B1 cross-linked to CyHsp70
protein. The nature of the ~210-kDa species remains unknown, although
it might represent a dimer of CYP2E1-CyHsp70 cross-linked product.
These results provide confirmatory evidence that phosphorylation does
not alter the rate of CYP2E1 targeting to the ER, but significantly
increases the CyHsp70-bound pool in the cytosol.
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DISCUSSION |
A recent study from our laboratory showed that
hepatic mitochondrial P450MT5 from pyrazole-treated rats exhibits
primary sequence identical to that of similarly induced microsomal
CYP2E1, except that it has a higher level of Ser-129 phosphorylation
(12). In support of these results, here we show that Ser-129
phosphorylated CYP2E1 protein is efficiently targeted to mitochondrial
compartment both in vitro and in vivo. It was
recently shown that the N-terminal 29 amino acid residues, the putative
transmembrane anchor domain of CYP2E1, contain the signal for ER
targeting although it is inefficient for retaining the protein in the
ER (26). This study demonstrates that the sequence region between
residues 21 and 31 also contains a cryptic signal for mitochondrial
targeting, which is activated by phosphorylation at Ser-129. In this
study, we show that Ser-129-phosphorylated CYP2E1 with intact N
terminus is efficiently targeted to mitochondrial compartment both
in vitro and in vivo. We demonstrate that the
sequence region between 21 and 31 also contains a cryptic mitochondrial
targeting signal, which is activated by phosphorylation at Ser-129.
Previously we showed that cAMP-mediated phosphorylation markedly
increases the efficiency of CYP2B1 targeting to the mitochondrial
compartment (10). In extension of these results, we now show that
phosphorylation at Ser-129 markedly increases the extent of CYP2E1
protein binding to cytoplasmic chaperone Hsp70 under both in
vitro and in vivo conditions, which in turn increases
the affinity of the nascent protein for mitochondrial translocase
TOM40. It is likely that CyHsp70 binding induces a conformational
change in CYP2E1 protein, thus facilitating its interaction with TOM40
and other mitochondrial translocases. The phosphorylation-mediated
signal activation, therefore, appears to be related to
pre-translocation steps of chaperone binding and presentation to
translocase complexes.
CYP2E1 is believed to play important roles in modulating oxidative
stress-related cellular pathologies and toxicity. Its expression at
both mRNA and protein levels is induced under various
pathophysiological conditions, including diabetes, obesity, starvation
(13), and alcohol ingestion (33). CYP2E1 metabolizes endogenous
compounds of physiological importance, such as lipid hydroperoxides and ketone bodies (34, 35), in addition to various exogenous chemicals (36,
37). Recent studies show that CYP2E1 has a direct role in the
production of reactive O2 species, induction of oxidative stress, and apoptosis (38, 14). Because of the expanding role of
mitochondrial genetic and membrane systems in diverse metabolic processes, integration and execution of apoptotic signal (39), and
generation of stress signaling that affects nuclear gene expression (40, 41), it is important to understand the mechanism of CYP2E1 targeting and its regulation.
The present study demonstrates that the two positively charged residues
at positions 24 and 25 are critical for mitochondrial targeting. The
chimeric N-terminal signal (residues 1-31) of CYP2E1 appears to
resemble the bimodal targeting signal of CYP2B1 with subtle
differences. A recent study using transient transfection of CYP2E1
lacking the N-terminal 1-29 residues of the protein concluded that the
mitochondrially targeted component is a 40-kDa soluble protein, which
is generated by the MPP-mediated cleavage at an undetermined N-terminal
site (42, 43). Using a full-length protein, however, we were unable to
demonstrate any detectable processing either under conditions when MPP
activity was induced by Mg2+ and Mn2+ or by
incubation with purified yeast MPP protein (results not presented).
Furthermore, in our hands any deletion beyond the positively charged
residue at position 25 abolished mitochondrial targeting under both
in vivo and in vitro conditions. The
physiological significance of the earlier study (43) using a truncated
protein that lacked the critical components of the mitochondrial
targeting signal remains unclear.
Protein phosphorylation plays a critical role in many cellular
processes, including signal amplification, modulation of
structure/function of proteins, and protein ubiquitination (44). In
extension of our recent observation with CYP2B1, the present study
demonstrates yet another function for protein phosphorylation in the
activation of an otherwise inactive mitochondrial targeting signal.
Specifically, our results show that phosphorylation at an internal site
results in increased affinity of nascent CYP2E1 for binding to CyHsp70 in vitro as well as in vivo, and for a major
mitochondrial outer membrane translocase, TOM40. In this respect, our
present and previous studies (10) together provide an important insight into a novel cAMP-dependent regulation of mitochondrial
biogenesis. A notable difference from the previously published results
with CYP2B1 is that phosphorylation at Ser-129 did not alter the
efficiency of nascent CYP2E1 protein binding to SRP. In contrast, the
phosphorylated nascent CYP2B1 showed a markedly lower affinity for
binding to SRP. We believe that phosphorylation of CYP2E1
translated on free ribosomes for mitochondrial targeting is
a distinct event from the phosphorylation of microsome-associated
CYP2E1, which has been implicated in the regulation of catalytic
function of the microsomal CYP (45) and its ubiquitination (46).
Another major difference between the bimodal targeting of CYP2E1,
CYP2B1, and CYP1A1 is their mode of translation. Our results show that
in vitro translation of CYP2B1 is nearly completely inhibited by saturating levels of SRP, whereas the translation of
CYP2E1 was inhibited by only ~40%. Inhibition of CYP1A1 followed a
pattern intermediary to these two proteins
(9).3 Extent of membrane
insertion as tested by alkaline Na2CO3
extraction and binding to SRP by chemical cross-linking essentially
follow this pattern. CYP2E1 showed a generally low affinity for binding to SRP, as indicated by cross-linking, and a large fraction (>50%) of
CYP2E1 was not inserted into added ER membrane, further suggesting that
part of the nascent chains is translated in membrane-free form. On the
other hand, under low cAMP conditions, CYP2B1 was translated as a
bona fide ER-targeted protein (10), with a markedly higher efficiency for binding to SRP and >90% of nascent
chains associating with ER. CYP1A1 behaved in an intermediary manner in
that ~25-30% of the nascent chains escaped ER targeting
irrespective of the phosphorylation status (9). The contrasting
efficiencies of phosphorylated CYP2E1 and CYP2B1 for binding to SRP is
further supported by cross-linking studies under in vivo
conditions (Fig. 8), which show no change
in interaction of CYP2E1 with Sec61 under induced cAMP conditions, when
interaction of CYP2B1 with Sec61 is markedly decreased. A comparison of
the structural properties of sequence 1-36, representing the chimeric
signals of those three CYP proteins is presented in Table
I. The PROT-SCALE-based analysis shows
that the chimeric signal of CYP2E1 is least hydrophobic and has least
-helical content, and is conformationally most unstable as compared
with that of CYP2B1, which is at the other end of the spectrum with
respect to these properties. CYP1A1 exhibits intermediary
properties.
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Fig. 8.
Effects of cAMP on the extent of interaction
of CYP2E1 with Sec61 and CyHsp70 proteins in intact cells. COS
cells were co-transfected with CYP2E1 and CYP2B1 cDNA constructs
and incubated for 8 h in the absence or presence of added db-cAMP
(10 µM). Cells were then incubated for 1 h with 500 µM membrane-permeable cross-linker; MBS and subcellular
fractions were isolated as described under "Experimental
Procedures." In A, microsomal proteins (500 µg each)
were immunoprecipitated (IP) with indicated antibodies
(ab) and the immunoprecipitates were probed with antibody to
Sec61 protein by immunoblot analysis. In B, cytosolic
proteins (500 µg each) were immunoprecipitated with antibodies to
CYP2E1, CYP2B1, or calreticulin and the immunoprecipitates were probed
the same antibody by immunoblot analysis. In C,
immunoprecipitates, as in B, were probed with antibody to
CyHsp70. Details of transfection, chemical cross-linking, cell
fractionation, and immunoprecipitation were as described under
"Experimental Procedures."
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Table I
Comparison of the N-terminal regions of CYPs that contain chimeric
signals for bimodal targeting
The N-terminal amino acid 1-36 sequence regions were analyzed using
ExPaSy (Expert Protein Analysis System) molecular biology server of the
Swiss Institute of Bioinformatics. The number of + indicates the
degree of -helicity or hydrophobicity.
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A common property among the three CYPs (2E1, 2B1, and 1A1) with
chimeric signals is their inability to undergo elongation arrest during
in vitro translation in the presence of added SRP (10, 47).
Elongation arrest is thought to increase the translocation efficiency
of nascent chains possibly by extending the time of its interaction
with the translocation machinery (2, 29). In the case of CYP2B1, a high
affinity of the ER targeting signal for SRP under non-phosphorylated
state probably enables its efficient targeting to the ER. In the case
of CYP1A1 and CYP2E1, the relatively low efficiency of the N-terminal
signal for SRP binding, coupled with inability to undergo elongation
arrest may be the reason why ~25-50% of the nascent chains escape
ER targeting and undergo translation in a membrane-free state.
Phosphorylated CYP2B1, on the other hand, behaves similarly to the
chimeric signal of CYP2E1, in that it shows lower affinity for SRP
binding, thus showing higher propensity for translation in a
membrane-free form. Consistent with this, results of in
vitro and in vivo studies together show that
phosphorylated CYP2E1 binds to CyHsp70 with increased efficiency, which
in turn increases the efficiency with which the nascent protein binds
to TOM40. Based on these observations, we propose a model for
mitochondrial CYP2E1 targeting (Fig. 9),
which implies that lower affinity for SRP binding under both high cAMP
or low cAMP conditions causes >50% of the nascent chains to escape ER targeting and undergo translation as membrane free protein.
cAMP-dependent phosphorylation at Ser-129 of the protein
results in the activation of cryptic mitochondrial signal, which
includes more efficient association with cytoplasmic chaperones and
more efficient binding to mitochondrial translocases. We postulate that
these latter two events enable preferential targeting of complete,
unprocessed, but phosphorylated apoprotein to mitochondria (Fig. 8). In
variance from the mechanism described for CYP2B1, cAMP regulates only
the mitochondrial targeting but not the ER targeting of CYP2E1.
In summary, we provide confirmatory evidence for a novel class of
chimeric signals with dual targeting property. Results also provide
valuable new insight as to how cellular cAMP levels regulate the
biogenesis of mitochondrial enzymes associated with drug and alcohol
toxicity by modulating the rate of mitochondrial targeting of CYP2E1
and also other CYPs.
| |
ACKNOWLEDGEMENTS |
We thank Drs. Reid Gilmore and David Andrews
for generously providing some of the reagents and antibodies used in
this study. We are thankful to Haider Raza and members of the Avadhani
laboratory for comments and suggestions on the manuscript and to Molly
Higgins for editorial help on the manuscript.
| |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant GM-34883 (to N. G. A.) and GM-57067 (to D. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
215-898-8819; E-mail: narayan@vet.upenn.edu.
Published, JBC Papers in Press, August 20, 2002, DOI 10.1074/jbc.M203292200
2
H. K. Anandatheerthavarada, M.-A. Robin, G. Biswas, N. B. V. Sepuri, D. M. Gordon, D. Pain, and
N. G. Avadhani, unpublished results.
3
M.-A. Robin, H. K. Anandatheerthavarada, G. Biswas, N. B. V. Sepuri, D. M. Gordon, D. Pain, and
N. G. Avadhani, unpublished results.
| |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic reticulum;
CYP, cytochrome P450;
db-cAMP, dibutyryl cAMP;
DHFR, dihydrofolate reductase;
DTT, dithiothreitol;
Hsp, heat shock
protein;
CyHsp70, cytoplasmic Hsp70;
MtHsp70, mitochondrial Hsp70;
MBS, m-maleimidobenzoyl- N-hydroxysuccinimidate
ester;
S-MBS, m-maleimidobenzoyl-N-hydroxysulfosuccinimidate
ester;
MOPS, 4-morpholinepropanesulfonic acid;
MPP, matrix processing
peptidase;
PPL, preprolactin;
PKA, protein kinase A;
PKI, protein
kinase A inhibitor myristoylated peptide;
RRL, rabbit reticulocyte
lysate;
SRP, signal recognition particle;
TIM, translocase of the inner
membrane;
TOM, translocase of the outer membrane;
WGL, wheat germ
lysate;
WT, wild-type.
| |
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ABSTRACT
INTRODUCTION
