Mitochondrial localization of N-terminal truncated CYP1A1 The analysis of the microsomal fractions from yeast strains expressing full-length CYP1A1, + 5⁄ 1A1 and + 331A1 showed significan
Trang 1N-terminal truncated CYP1A1 proteins in Saccharomyces
targeting of CYP2E1
Naresh B V Sepuri, Sanjay Yadav, Hindupur K Anandatheerthavarada and Narayan G Avadhani Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA
Cytochrome P450s (CYPs) belong to a superfamily of
heme-thiolate enzymes that catalyze the oxidation of
xenobiotic as well as endogenous compounds [1–3] A
majority of the constitutively expressed and inducible
CYPs belonging to families 1–4 are primarily localized
in the endoplasmic reticulum (ER), hereafter referred to
as microsomes However, there is increasing evidence
suggesting that some of the inducible CYPs are also bimodally targeted to the mitochondrial compartment [4–7] Studies from our laboratory and others demon-strated that b-naphthoflavone-inducible CYP1A1, pyrazole-inducible CYP2E1, and phenobarbital-induci-ble CYP2B1, known to be bona fide microsomal forms, are also targeted to mitochondria [5,6,8–10] These
Keywords
chimeric targeting signals; CYP2E1;
evolutionary conservations; mitochondrial
protein targeting; xenobiotic metabolism
Correspondence
N G Avadhani, Department of Animal
Biology, School of Veterinary Medicine,
University of Pennsylvania, 3800 Spruce
Street, Philadelphia, PA 19104, USA
Fax: +1 215 573 6651
Tel: +1 215 898 8819
E-mail: narayan@vet.upenn.edu
(Received 30 April 2007, revised 6 July
2007, accepted 13 July 2007)
doi:10.1111/j.1742-4658.2007.05990.x
Previously we showed that intact rat cytochrome P450 2E1, cytochrome P450 2B1 and truncated cytochrome P450 1A1 are targeted to mito-chondria in rat tissues and COS cells However, some reports suggest that truncated cytochrome P450 2E1 is targeted to mitochondria In this study,
we used a heterologous yeast system to ascertain the conservation of targeting mechanisms and the nature of mitochondria-targeted proteins Mitochondrial integrity and purity were established using electron microscopy, and treatment with digitonin and protease Full-length cyto-chrome P450 2E1 and cytocyto-chrome P450 2B1 were targeted both to micro-somes and mitochondria, whereas truncated cytochrome P450 1A1 (+ 5 and + 33⁄ cytochrome P450 1A1) were targeted to mitochondria Inability
to target intact cytochrome P450 1A1 was probably due to lack of solic endoprotease activity in yeast cells Mitochondrial targeting of cyto-chrome P450 2E1 was severely impaired in protein kinase A-deficient cells Similarly, a phosphorylation site mutant cytochrome P450 2E1 (Ser129A) was poorly targeted to the mitochondria, thus confirming the importance
of protein kinase A-mediated protein phosphorylation in mitochondrial targeting Mitochondria-targeted proteins were localized in the matrix compartment peripherally associated with the inner membrane and their ethoxyresorufin O-dealkylation, erythromycin N-demethylase, benzoxyres-orufin O-dealkylation and nitrosodimethylamine N-demethylase activities were fully supported by yeast mitochondrial ferredoxin and ferredoxin reductase
Abbreviations
BROD, benzoxyresorufin O-dealkylation; CCPO, cytochrome c peroxidase; CYP, cytochrome P450; DHFR, dihydrofolate reductase; DMPS, dolichol mannose phosphate synthase; ERND, erythromycin N-demethylase; ER, endoplasmic reticulum; FDX, ferredoxin 1; FDXR, ferredoxin reductase; NDMA, nitrosodimethylamine; NDMA-d, nitrosodimethylamine N-demethylase; PKA, protein kinase A; Put2, D1-pyrroline-5-carboxylate dehydrogenase; TIM, translocase of inner membrane; TOM, translocase of outer membrane.
Trang 2studies led us to propose the concept of chimeric
pro-tein-targeting signals that drive the bimodal targeting of
the same primary translation product to more than one
subcellular compartment [10–12]
Protein targeting to the microsomes requires the
co-translational insertion of the newly synthesized protein
into the microsomal membrane, where the N-terminal
hydrophobic signal sequence of the protein interacts
with a signal recognition particle This interaction
sub-sequently results in the association of the translational
complex with the microsomal membrane [13,14] Thus,
the N-terminal hydrophobic sequences of CYPs are
important for their targeting to and retention in the
ER [15–17] Protein translocation into the
mitochon-dria requires a cytosolic chaperone-mediated
associa-tion of precursor protein with peripheral translocase of
outer membrane (TOM) receptors (TOM20, TOM22
and TOM70), which enables the translocation of
proteins through the outer membrane and the inner
membrane channel-forming proteins, TOM40 and
translocase of inner membrane 23 (TIM23) [18–20]
Our studies defined two distinct mechanisms of
acti-vation of cryptic mitochondria-targeting signals at the
N-terminus of mammalian CYP proteins [4,10] We
found that post-translational processing of CYP1A1
at the 4th and 32nd amino acid residues by a cytosolic
endoprotease is critical for the activation of cryptic
mitochondria-targeting signal at the 33–44 positions
of CYP1A1 [4,8,9] This endoprotease was unable to
cleave the N-termini of CYP2E1 and CYP2B1 [6,12]
In the case of CYP2B1 and CYP2E1, uncleaved intact
proteins were targeted to the mitochondria in both
inducer-treated rat liver and transiently transfected
COS cells [6,10] In both of these cases, protein
kinase A (PKA)-mediated phosphorylation at Ser128
or Ser129 [12] was necessary for the activation of a
cryptic mitochondria-targeting signal at positions
21–36 of the protein [6,12] In contrast to our
obser-vations on the targeting of intact CYP2E1 to
mito-chondria, Neve & Ingelman-Sundberg [21] showed
that an N-terminally truncated CYP2E1 was targeted
to mitochondria in transiently transfected hepatoma
cells [22] The nature and specificity of the
endopro-tease and the site of proteolytic cleavage remain
unknown The same investigators were unable to see
any significant intramitochondrial localization of
intact or N-terminal truncated CYP2E1 in yeast cells
[23] The precise nature of CYP proteins targeted to
mitochondria and the conservation of targeting
mech-anism is important for understanding the evolution
and regulation of bimodal targeting Our primary
objective here was to address this important question
using rigorous approaches
Yeast provides an ideal system for the heterologous expression of genes to study both gene function and metabolism The protein translocation machineries of both the mitochondria and microsomes are highly con-served among mammals and yeast [24,25] As protein trafficking has been very well characterized in budding yeast and is thought to involve a similar translocation mechanism as that in mammalian cells, the yeast expression system is well suited for the study of the bimodal targeting mechanism described mostly in tran-siently transfected mammalian cells As targeting of intact CYP2E1 and the requirement for PKA-mediated phosphorylation for mitochondrial targeting are con-tradicted by other studies [22,23,26], we sought cell systems lacking specific PKA subunits to address this important question The availability of PKA gene dele-tion yeast strains provided another advantage for the present study
We show here that mammalian CYPs are targeted efficiently to both the microsomes and mitochondria in yeast cells, depending on the nature of the chimeric signals that they carry In transformed yeast cells, + 33⁄ 1A1 was exclusively localized to the mito-chondria, whereas + 5⁄ 1A1 was localized in both the mitochondria and microsomes Also, we found that full-length CYP2E1 and CYP2B1 were targeted to the mito-chondria as well as microsomes By using PKA-deficient cells, we further show the importance of PKA-mediated phosphorylation in the mitochondrial targeting of CYP2E1 Most importantly, substrate conversion by mitochondria-targeted CYPs was fully supported by yeast mitochondrial ferredoxin (FDX) + ferredoxin reductase (FDXR), homologs of mammalian ferredoxin and ferrodoxin reductase [27,28]
Results
Expression of intact and truncated rat CYPs in Saccharomyces cerevisiae
The levels of expression of various CYP cDNAs were measured by resolving whole cell extracts on SDS⁄ PAGE and western blot analysis using CYP-specific antibodies As shown in Fig 1A,B, the western blot patterns of CYP proteins in cells transformed with plasmids dihydrofolate reductase (DHFR)-1A1, intact CYP1A1, + 5⁄ 1A1, + 33 ⁄ 1A1 and CYP2E1 were con-sistent with their predicted molecular masses Yeast strain BY4741, transformed with plasmid CYP2B1 cDNA, showed extensive degradation of the CYP2B1 protein (Fig 1B) This result was consistent with a pre-vious report showing similar degradation of CYP2B1
in yeast cells [29] Use of the protease-deficient strain
Trang 3pepD as suggested by Liao et al [29] yielded more
intact CYP2B1 protein (Fig 1B) Consistent with the
reported size of CYP2E1 protein, cells transformed
with CYP2E1 cDNA plasmid yielded a 52 kDa band
This extract also yielded additional antibody-reactive
bands of about 28–32 kDa, which probably represent
degradation products
Mitochondrial localization of N-terminal
truncated CYP1A1
The analysis of the microsomal fractions from yeast
strains expressing full-length CYP1A1, + 5⁄ 1A1 and
+ 331A1 showed significant levels of full-length
CYP1A1 protein, reduced levels of + 5⁄ 1A1 protein,
and vastly reduced levels of + 33⁄ 1A1 protein
(Fig 2A, first four lanes) We also found nearly
un-detectable full-length CYP1A1 and clearly visible
+ 5⁄ 1A1 and + 33 ⁄ 1A1 in the mitochondrial fraction
(Fig 2A, last four lanes) As expected, full-length
CYP1A1 and + 5⁄ 1A1 from the microsomal
mem-brane fraction were degraded by trypsin treatment
(Fig 2A, first four lanes) This is consistent with the
model suggesting a transmembrane topology of CYPs
with a single N-terminal membrane anchor and most
of the remaining protein exposed to the cytosolic side
[15,17,30,31] The intramitochondrial localization
of CYPs and their topologies were studied using a
combination of treatment with trypsin, treatment with digitonin plus trypsin, and extraction with alkaline
Na2CO3 + 5⁄ 1A1, + 33 ⁄ 1A1, and TIM23, which was used as an internal control, were protected fully against trypsin up to 100 lgÆmL)1, whereas full-length CYP1A1 was completely digested (Fig 2A, last four lanes) These results suggest that full-length CYP1A1
is peripherally associated with the mitochondria We found that both + 5⁄ 1A1 and + 33 ⁄ 1A1 were resistant
to protease digestion even after selective removal of the outer membrane by digitonin treatment (Fig 2B) Under these conditions, TIM23, with a significant pro-portion of its sequence exposed outside the inner mem-brane, facing the intermembrane space, was degraded significantly These results suggested that the imported + 5⁄ 1A1 and + 33 ⁄ 1A1 proteins were localized inside
of the inner membrane Moreover, the imported + 5⁄ 1A1 and + 33 ⁄ 1A1 proteins were extractable with alkaline Na2CO3 as shown in Fig 2C, suggesting that both proteins are localized in the matrix compartment
in a membrane-extrinsic topology
We found that DHFR-1A1 was peripherally associ-ated with the mitochondria and microsomes, as it was vulnerable to very low concentrations of trypsin, sug-gesting that the intracellular distribution of CYPs was not due to random insertion into the microsomal or mitochondrial compartments (Fig 2D) As expected, TIM23 used as an internal control for the mitochon-drial fraction was protected from protease, whereas dolichol phosphate mannose synthase (DMPS) used as internal control for the microsomes was not protected from the externally added trypsin (Fig 2D) In previ-ous studies, we showed that the positively charged amino acids at positions 34 and 39 were important for targeting of + 5⁄ 1A1 and + 33 ⁄ 1A1 proteins to the mitochondrial compartment In keeping with these observations, the results in Fig 2E show that the asso-ciation of a single mutant (R34D) or double mutants (R34D and K39I) of + 331A1 with the mitochondrial membrane was sensitive to protease treatment (Fig 2E) These results suggest that, as in the mamma-lian cell system, the cryptic signal sequence at amino acids 33–44 serves as a mitochondria-targeting signal
in the yeast system
Mitochondrial localization of intact CYP2E1
in yeast cells Because of the existing ambiguity in the literature on the nature and extent of CYP2E1 import into mito-chondria, we first established the relative purity of mitochondrial preparations by biochemical and elec-tron microscopy techniques Figure 3A (top left panel)
+33/1A1+5/1A1DHFR1A1 1A1
35
47
62
81
kDa
2B1/BY4746
2E1 2B1/pep4
Δ'
25 35 47 62 81 kDa
16
25 35 47 62 81 kDa
16
Fig 1 The levels of expression of CYP1A1, CYP2B1 and CYP2E1
proteins in yeast cells Yeast strains BY4741 or PeP4D,
trans-formed with cDNA constructs, were grown to log phase, and cell
lysates were analyzed by SDS ⁄ PAGE and western blotting (A)
Extracts from cells transformed with the indicated CYP1A1
con-structs were probed with antibody to CYP1A1 (B) Extracts from
cells transformed with CYP2B1 (lanes 5 and 6) and CYP2E1
con-struct were probed with either CYP2B1 antibody (left panel, lanes
5 and 6) or CYP2E1 antibody (right-most panel) All transformations
were done in the BY4741 strain, except for the middle panel of (B),
where the protease-deficient strain PeP4D was used In each case,
50 lg of protein was used for immunoblot analysis.
Trang 4shows the transmission electron microscopy pattern of a
representative mitochondrial preparation A
representa-tive field shows several well-defined mitochondrial
parti-cles with minor membrane contamination As shown in
the insets, a large majority of mitochondrial
prepara-tions showed intact inner and outer membrane
compo-nents, confirming the structural integrity of mito-chondrial isolates
As shown in Fig 3B, mitochondrial preparations from cells transfected with plasmid pNS61(CYP2E1 cDNA) lacked significant levels of the microsomal marker protein DMPS, and also the cytosolic protein
Trypsin (µg/mL)
Microsomes
1A1
25 50 100
25
A
C
B
TIM23
+33/1A1
TIM23 +5/1A1
TIM23
Digitonin
Mitochondria Mitochondria
1A1 TIM23 +33/1A1
TIM23 +5/1A1 TIM23
Alk insol Alk Sol.
+5/1A1 mito
TIM23ab 1A1ab
+33/1A1 mito
Alk insol Alk Sol.
+
TIM23
Micro Mito.
Trypsin:
DPMS
+
DHFR/1A1
Micro Mito.
+33(R34D)+33(R34D&K39I) +33(R34D) +33(R34D&K39I)
+33/1A1 +33/1A1
– + – + – +33/1A1
TIM23
Fig 2 Mitochondrial targeting of truncated CYP1A1 in yeast cells Mitochondrial and microsomal fractions of yeast cells expressing CYP1A1, + 5 ⁄ 1A1, DHFR-1A1 and + 33 ⁄ 1A1 were separated by SDS ⁄ PAGE and subjected to western blotting Membrane topologies of mitochondria-associated + 5 ⁄ 1A1, + 33 ⁄ 1A1, CYP1A1 (A, B, C), DHFR-1A1 (D) + 33 ⁄ 1A1, + 33 ⁄ 1A1(R34D) and + 331A1(R34D and K39I) (E) were determined by protease treatment of microsomal and mitochondrial isolates before (A, D, E) or after (B) digitonin treatment In (C), digitonin-treated mitochondria were subjected to alkaline Na 2 CO 3 extraction In (A), increasing concentrations of trypsin (0–100 lgÆmL)1) were used, and in (B), (D) and (E), a fixed concentration (50 lgÆmg)1) of trypsin were used Fifty micrograms of protein in each case was subjected to immunoblot analysis Stripped blots were redeveloped with antibodies to marker proteins, TIM23 (mitochondrial marker) or DMPS, a micro-somal marker.
Trang 53-phospho glycerate kinase (3-PGK), but contained
mitochondria-specific TIM23 protein Additionally, both
the ER and mitochondrial fractions showed CYP2E1
antibody crossreactivity, although the latter fraction
showed 60% lower band intensity The preparations
lacked significant levels of oligomycin-insensitive
NADPH cytochrome c reductase, a microsome-specific
marker enzyme (results not shown) Furthermore, the
mitochondrial preparations contained < 90% of total
cellular cytochrome c oxidase activity (results not
shown) The results in Fig 3C show that both FDXR
(matrix protein) and TIM23 (inner membrane) were
resistant to protease treatment However, addition of
trypsin to digitonin-treated fraction reduced the level
of TIM23 but not that of FDXR The antibody to
mouse FDXR used in this study weakly crossreacts
with the yeast homolog Addition of trypsin to
Triton-solubilized samples completely degraded TIM23 and
FDXR Figure 3D represents a full view of an
immu-noblot of mitochondrial and microsomal proteins
developed with a combination of CYP2E1 and TIM23
antibodies It is seen that both microsomal and mito-chondria-associated CYP2E1 consisted of a major antibody-reactive full-length protein and minor faster-migrating bands Furthermore, the mitochondrial frac-tion crossreacted with TIM23 antibody, whereas the microsomal fraction lacked detectable TIM23 These results suggested that the full-length CYP2E1 is tar-geted to mitochondria in yeast cells
Localization of CYP2E1 in the mitochondrial inner membrane–matrix compartment
We used multiple approaches to determine the precise intramitochondrial localization of CYP2E1 in trans-formed yeast cells In the first approach, we assessed the effects of treatment of mitochondria and mitoplasts with trypsin As shown in Fig 4A, we found that mito-chondria-associated P4502E1 was relatively resistant to trypsin treatment, whereas the outer membrane protein TOM20 was completely degraded Additionally, CYP2E1 was resistant to trypsin when the outer
Micro Mito
2E1
TIM23
TIM23
B
D A
C
PGK 2E1 Cyto Micro Mito.
DPMS
Trypsin:
Digitonin:
Tx-100:
TIM23
+ +
+ + +
+
-Adx-red
Fig 3 The nature of mitochondria-associated CYP2E1 in yeast cells (A) Assessment of the integrity of the isolated mitochondria Mitochon-drial isolates were subjected to scanning electron microscopy as described in Experimental procedures (magnification: · 30 000) (B) The rel-ative purity of the isolated mitochondrial, microsomal and cytosolic fractions Mitochondria, microsomes and cytosol were isolated by differential centrifugation as described in Experimental procedures Fifty micrograms of protein from each fraction was subjected to SDS ⁄ PAGE and probed with antibodies specific for microsomes (DMPS), mitochondria (TIM23), and cytosol (3-phosphoglycerokinase), as indicated (C) Isolated mitochondrial fractions were treated with or without digitonin, trypsin or Triton X-100 (Tx-100; 0.2%) and probed with antibodies against human FDXR and TIM23 as indicated (D) A full gel pattern of the microsomal and mitochondrial fractions of cells express-ing full-length P4502E1.
Trang 6membrane was stripped by digitonin Under these
treat-ment conditions, inner membrane-associated TIM23,
which is exposed out of the membrane lipid bilayer
towards the intermembrane space, was degraded in a
concentration-dependent manner (Fig 4A) The case
was similar with the intermembrane space protein,
cytochrome c peroxidase (CCPO) By contrast,
D1-pyrr-oline-5-carboxylate dehydrogenase (Put2), a
matrix-localized protein, was protected against protease
treatment even after stripping of the outer membrane
These results suggest that mitochondria-associated
CYP2E1 is localized inside the inner membrane
In the second series of experiments, we treated intact
mitochondria with various concentrations of digitonin
It is known that low concentrations of digitonin (about
0.05%) selectively damage the outer membrane, and
higher concentrations (about 0.1%) damage the inner
membrane In this experiment, we determined the
con-centration of digitonin required to release
mitochon-dria-associated CYP2E1 into the soluble fraction, and
compare it with the amounts needed to release the
outer membrane-specific marker protein porin and the
mitochondrial matrix protein Put2 Figure 4B shows
that significant CYP2E1 release occurred at digitonin concentrations between 0.05% and 0.1% (w⁄ v), at which concentrations Put2 was also released to the sol-uble fraction to a large extent The release of porin started at a much lower concentration of 0.025% These results further support the possibility that mito-chondria-associated CYP2E1 is located inside the innermembrane compartment
In the third approach, we used alkaline Na2CO3 extraction to determine whether mitochondrial CYP2E1
is a membrane-intrinsic or membrane-extrinsic protein The results showed that most part of the microsomal-associated CYP2E1 resisted Na2CO3 extraction, suggesting a transmembrane topology The mitochon-dria-associated CYP2E1, on the other hand, was mostly partitioned in the soluble fraction, indicating a mem-brane-extrinsic topology (Fig 4C) TIM23, a bona fide inner membrane protein partitioned mostly in the
Na2CO3-insoluble fraction, whereas Put2, a bona fide matrix protein, was nearly completely extracted in the soluble phase (Fig 4C) These results suggest that mito-chondrial CYP2E1 is mostly a membrane-extrinsic protein localized in the matrix compartment However,
Trypsin (µg/mL):
A
B
C
-2E1
25 50
CCPO PUT2 TIM23
Mito
Mito
Mitoplast
Mito
Mitoplast
Mito
Mitoplast
Mitoplast
TOM20 Mitoplast
Mito
Pellet
pPut2 Porin
2E1 Digitonin%:
Supernatant
0.01 0.025 0.05 0.1 0.2 0.4 0.01 0.025 0.05 0.1 0.2 0.4
pPut2 Porin 2E1
Micro Mito Mito
2E1 2E1 TIM23 pPut2
Mito
Fig 4 Intramitochondrial localization of CYP2E1 in transformed yeast cells (A) Mitochondria and mitoplasts from cells expressing CYP2E1 were treated with 0–50 lgÆmL)1trypsin as indicated Mitochondria reisolated by banding through a sucrose layer were analyzed by immuno-blotting (50 lg protein each) with antibody to CYP2E1 Put2, TIM23 and CCPO and TOM20 were used as matrix, inner membrane, inter-membrane space and outer inter-membrane markers, respectively (B) Isolated mitochondria were incubated with increasing concentrations of digitonin (0–0.4%; 0–400 lgÆmg)1protein) for 30 min as described in the text The digitonin-insoluble (pellet, left panel) and digitonin-soluble (supernatant, right panel) fractions were separated by centrifugation (14 000 g for 10 min), analyzed by immunoblot, and probed with anti-bodies to porin (outer membrane marker protein), pPut2 (matrix marker protein), and CYP2E1 (C) Mitochondria of yeast cells expressing wild-type CYP2E1 were treated with bicarbonate, and both the soluble and insoluble fractions were separated and analyzed by immunoblot analysis with CYP2E1, TIM23 and Put2 antibodies as indicated.
Trang 7the protein was peripherally associated with the inner
membrane and required washing with a high salt
con-centration (0.2 m NaCl) to be released from the
mem-brane (data not shown)
Role of PKA in the mitochondrial targeting of
CYP2E1
The role of PKA in mitochondrial targeting of
CYP2E1 was investigated using two approaches The
first approach involved measuring the level of
mito-chondrial targeting of wild-type CYP2E1 in
PKA-defi-cient (a⁄ c deleted or a ⁄ b deleted) yeast strains The
western blot in Fig 5A shows that in control yeast
cells, the microsomal CYP2E1 content was
approxi-mately 4–6-fold higher than the mitochondrial content,
and the microsome-localized CYP2E1 was highly
sensi-tive to trypsin (Fig 5A, compare lanes 1 and 3) The
mitochondrial CYP2E1 was resistant to externally
added trypsin, and in this regard was similar to the
inner membrane protein TIM23 (Fig 5A, compare
lanes 2 and 4) The microsome-associated CYP2E1 levels in both the PKA subunit a⁄ c and a ⁄ b deleted strains were similar to that in the control yeast strain (Fig 5A, lane 1) As observed with the control yeast, the microsome-associated CYP2E1 in PKA mutant strains was sensitive to trypsin treatment Quantitation
of the gel pattern presented in Fig 5B showed that the mitochondrial CYP2E1 levels were reduced to < 10%
in the a⁄ c mutant and < 3% in the a ⁄ b mutant, as compared to about 25% in the control strain We also tested the targeting to mitochondria of Su9-DHFR, in which the presequence of ATPase subunit 9 of Neuros-pora crassa was fused to a passenger protein, DHFR
As seen in Fig 5A, the level of mitochondrial targeting
of Su9-DHFR, which lacks a canonical PKA phos-phorylation site, was similar in all three cell lines tested CYP2E1 contained a single PKA target site at Ser129, which was shown to be important in mito-chondrial targeting of the protein in COS cells In the second approach, we tested the level of mitochondrial targeting of S129A mutant CYP2E1 in transformed
0 10 20 30 40 50 60 70 80 90 100
WT
Micro Mito
PKA
Δ α/γ
Δ α/β
Δ α/γ
PKA
Δ α/β
Strain background
TIM23
TIM23
2E1
2E1
Su9-DHFR
Su9-DHFR
*
TIM23
Micro
A
B
C
Mito Micro Mito
2E1
WT
Su9-DHFR
+
*
Micro Mito Micro Mito
2E1(S129A)
TIM23
100 72 2 10 % distribution
% distribution 100
Fig 5 Role of PKA-mediated phosphorylation in mitochondrial targeting of CYP2E1 (A) Isolated mitochondrial and microsomal fractions from the wild-type and PKA deletion strains were transformed with CYP2E1 or SU9-DHFR expression cDNA constructs or empty vectors Subcel-lular fractions were subjected to immunoblot analysis following treatment with or without trypsin as indicated TIM23 was used as a mito-chondrial marker (B) The amount of CYP present in the microsomal or mitomito-chondrial fractions obtained from various PKA mutants were quantified and represented as a bar graph (C) Reduced mitochondrial targeting of phosphorylation site mutated CYP2E1 (S129A) Mitochon-dria and microsomal fractions of yeast cells expressing S129A mutant CYP2E1 were treated with or without trypsin and subjected to immu-noblot analysis with CYP2E1 and TIM23 antibodies.
Trang 8yeast cells The results in Fig 5C show that, as with
the wild-type protein, full-length mutant protein was
targeted to mitochondria, although at a markedly
reduced level (30–40% as compared to the wild-type
construct) These results collectively show that
PKA-mediated phosphorylation, most likely targeted to the
S129 consensus site, is very important for
mitochon-drial targeting of CYP2E1 protein
Mitochondrial targeting of CYP2B1 in yeast cells
We used the protease-deficient strain pep4D for testing
the mitochondrial targeting of rat CYP2B1 The
western blot in Fig 6A shows that both ER and
mitochondrial preparations from transformed yeast
cells contained CYP2B1 protein, with about 25% of
protein in the mitochondrial fraction As in previous
experiments, the mitochondrial preparations contained
TIM23 protein, whereas the microsomes lacked signifi-cant levels of TIM23 The western blot in Fig 6B also shows that the microsome-targeted CYP2B1 was sensi-tive to trypsin treatment, whereas the mitochondrial protein showed significant resistance, suggesting an intramitochondrial location
CYP contents and catalytic activities With the aim of correlating the levels of expression of various apoproteins in yeast with CYP contents, we measured the P450-heme contents by CO-reduced spec-tra As shown in Fig 7, mitochondrial isolates from + 33⁄ 1A1-expressing yeast cells yielded a CO reduced and dithionite reduced spectrum with a peak at
448 nm No peak was observed with mitochondria from cells transformed with empty vector (data not shown) Additionally, we did not detect any character-istic spectrum with mitochondria from cells expressing + 33⁄ 1A1 mutant constructs (data not shown) Figure 7B shows the P450-heme contents of mito-chondria and microsomal fractions from yeast strains expressing various CYP constructs based on CO differ-ence spectral analysis Consistent with the negligible mitochondrial localization of full-length CYP1A1, we detected no significant CYP in the mitochondrial isolates However, the microsomal fraction showed a high (6.5 nmolÆmg)1) CYP content Cells expressing + 5⁄ 1A1 showed nearly equal CYP contents in the mitochondrial and microsomal fraction Cells express-ing + 33⁄ 1A1 showed no detectable CYP in the microsomal fraction, but a high (3.5 nmolÆmg)1) level
of CYP in mitochondria Cells expressing full-length CYP2E1 showed about 300 pmolÆmg)1 CYP in the microsomes and 55 pmolÆmg)1 in mitochondria The mitochondrial fraction from CYP2B1-expressing cells showed about 50 pmolÆmg)1CYP
As shown in Fig 8A, the microsomal fraction from full-length CYP1A1- and + 5⁄ 1A1-expressing cells
0.005
0.055
0.105
0.155
0.205
Wave length (nm)
+331A1 mito
CYP450
2B1
B A
ND
pmole/mg microsomal protein
pmole/mg mitochondrial protein Fig 7 Mitochondrial CYP contents in yeast
cells expressing CYP1A1, CYP2E1 and CYP2B1 proteins (A) The reduced CO spec-tra of the mitochondrial fraction expressing + 331A1 The reduced CO spectrum was performed essentially as described by Anandatheerthavarada et al [5] (B) Relative levels of CYP in mitochondria and micro-somes from cells expressing different CYP proteins CYP content was measured by CO difference spectra as described in (A) The values are the mean of three experiments.
Micro Mito
25
35
47
62
81
kDa
TIM23
2B1
Micro Mito
Trypsin +
+
Fig 6 Immunoblot analysis of PeP4D strain expressing full-length
CYP2B1 Isolated mitochondrial or microsomal fractions were
trea-ted with (B) or without (A) trypsin, and subjectrea-ted to SDS ⁄ PAGE
and immunoblot analysis with CYP2B1 and TIM23 antibodies.
Trang 9showed high activity of ethoxyresorufin O-dealkylation
(EROD), which is a specific marker enzyme for
ER-associated CYP1A1 +33⁄ 1A1-expressing cells,
how-ever, showed very low ERND activity The latter is
consistent with the microsomal CYP content of cells
expressing + 33⁄ CYP1A1 (Fig 7) The mitochondrial
isolates from all these three cell types showed very low
EROD activity The erythromycin N-demethylase
(ERND) activity pattern (Fig 8B) was significantly
different from the EROD activity pattern (Fig 8A)
The microsomal fraction of cells expressing full-length
CYP1A1 and + 5⁄ 1A1 showed relatively low ERND
activity Similarly, consistent with the low or
non-significant mitochondrial localization of full-length
CYP1A1, mitochondria from these cells also showed
very low activity The mitochondrial isolates from
+ 5⁄ 1A1- and + 33 ⁄ 1A1-expressing cells, however,
showed high ERND activity (2.0–2.5 nmolÆmg)1) with
the endogenous yeast FDX + FDXR Expression of
mutant 33⁄ 1A1 with impaired mitochondrial targeting
showed vastly reduced mitochondrial ERND activity
Our results on ERND activity of
mitochondria-targeted CYP1A1 supported by mitochondrial
FDX1 + FDXR are consistent with previous studies
from our laboratory [32,33] showing altered catalytic
property of mitochondria-targeted rat and mouse
CYP1A1
As shown in Fig 8C, both microsomal and
mito-chondrial fractions from CYP2B1 cDNA-transformed
cells show benzoxyresorufin O-dealkylation (BROD) activity The BROD was reduced by about 60% when the mitochondrial or microsomal fractions were prein-cubated with CYP2B1 antibody, indicating the specific-ity of the assay
As shown in Fig 9A, both microsomal and mitochon-drial fractions from wild-type yeast cells transformed with CYP2E1 cDNA showed nitrosodimethylamine N-demethylase (NDMA-d) activity The activities of both the microsomal CYP2E1 and mitochondrial CYP2E1 were dependent on the addition of NADPH (Fig 9A) The catalytic activities were reduced when the mitochondrial or microsomal enzymes were prein-cubated with CYP2E1 antibody or SKF-525, a general inhibitor of CYPs These results suggest the specificity
of the assay We did not observe any significant increases in the activity of mitochondrial CYP2E1 after supplementing the reaction with purified bovine FDX1 + FDXR, possibly because of adequate endo-genous FDX1 + FDXR in these mitochondrial prepa-rations (Fig 9A) Additionally, the activity with the mitochondrial fraction was inhibited by 50% after addi-tion of polyclonal antibody to human FDX1 (Fig 9A), confirming the role of endogenous FDX + FDXR in supporting the activity
To further confirm the role of endogenous FDX + FDXR in supporting the catalytic activity, we transformed the FDX (Yah1)- and FDXR (Arh1)-depleted yeast cells with CYP2E1 Expression of
0 0.05 0.1 0.15 0.2
0.25
Micro Mito
CYP2B1: -
- -
-+ -+ + + Anti-2B1: + +
BROD ACTIVITY
ERND ACTIVITY
0 0.5
1 1.5
2 2.5
3
1A1 +5 +33
Micro Mito
EROD ACTIVITY
A
C
B
0 0.2 0.4 0.6 0.8
1 1.2 1.4 1.6 1.8
2
Micro Mito
Fig 8 Metabolic activities of the
micro-somal and mitochondrial CYPs in
trans-formed yeast cells Microsomes and
digitonin-treated mitoplasts from cells
expressing various CYP variants were
assayed for their enzyme activities as
indi-cated in Experimental procedures (A) and
(B) represent the EROD and ERND activities
of cells transformed with various CYP1A1s,
and (C) represents the BROD activity of
cells transformed with CYP2B1 constructs
as indicated.
Trang 10CYP2E1 in Yah1-depleted cells turned out to be lethal.
We therefore analyzed the Arh1-depleted cells
express-ing CYP2E1 The catalytic activity of the mitochondrial
fraction was reduced by about 60% in FDXR-depleted
cells (Fig 9B) However, the activity with the
mito-chondrial fraction was restored by the addition of
puri-fied bovine FDX + Fdr These results confirm that
yeast mitochondrial FDX + FDXR is capable of
sup-porting the catalytic activity of mammalian CYPs
Discussion
A large majority of mitochondrial proteins are
encoded by nuclear genes, synthesized in the cytoplasm
and post-translationally transported to mitochondria
The mitochondrial proteome in mammalian cells is
estimated to consist of well over 1500 proteins
imported from the cytoplasm [34] Several
mitochon-drial matrix-targeted proteins contain an N-terminal
extension or ‘presequence’ that is cleaved upon import
into mitochondria [35] However, the current estimates
are that more than 50% of the
mitochondrial-associ-ated proteins lack the canonical mitochondria-targeting
signals, and the precise mechanisms by which these
proteins are translocated to the mitochondrial
com-partment remain unclear [35,36] The mitochondrial
inner membrane-associated carrier protein, uncoupler
proteins and outer membrane proteins belong to this
latter class [37,38] Additionally, the bimodal targeting
of CYPs to the ER and mitochondria, Alzheimer’s amyloid precursor protein to the plasma membrane and mitochondria, and translocation of the cytosolic glutathione S-transferases to the mitochondrial matrix compartment, probably represent the targeting of non-canonical signal-containing proteins to the mitochon-drial compartment [4–6,12,39] We have shown that xenobiotic-inducible CYPs such as rat CYP1A1, CYP2E1 and CYP2B1, and mouse CYP1A1, contain chimeric noncanonical-targeting signals that are capa-ble of targeting proteins to both the ER and mitochon-dria [5,11,12] Our results showed that the cryptic mitochondria-targeting signals present at residues 29–40 in various CYP proteins are activated by two different mechanisms: (a) proteolytic processing at the N-terminus by a cytosolic endoprotease, resulting in the exposure of cryptic mitochondrial targeting signal,
as in the case of CYP1A1 [4]; and (b) PKA- or protein kinase C-mediated phosphorylation of nascent chains either at the N-terminus (Ser128 or Ser129) or the C-terminus, which promotes the mitochondrial target-ing of CYP2E1, CYP2B1, and GSTA4-4 [6,12,40] In this study, we show that bimodal targeting of CYP1A1, CYP2E1 and CYP2B1 are conserved in the heterologous cell system Saccharomyces cerevisiae The N-terminal four or 32 amino acid sequence (+ 5⁄ 1A1 or + 33 ⁄ 1A1) of CYP1A1 exposes the cryp-tic mitochondria-targeting sequence at positions 33–39
of the protein, thus targeting the + 331A1 protein
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Cell Fractions
NADPH
FDR& FDX
Ant-FDX
SKF-525
Anti-2E1
Anti-IgG
Wt cells expressing CYP2E1
0 0.5 1 1.5 2 2.5 3 3.5 4
Mito
Cell Fractions NADPH FDR& FDX FDXR depleted cells expressing CYP2E1
Fig 9 Mitochondrial CYP2E1 activity in wild-type and FDXR-depleted yeast strains Mitochondrial and microsomal NMDA-d activity in (A) wild-type cells expressing CYP2E1 and (B) FDXR-depleted cells expressing CYP2E1 Reactions were carried out as described in Experi-mental procedures, using 50 lg of mitochondria or microsomal proteins NADPH (1 m M ), FDX + FDXR (1 lg each), antibody to FDX (2 lg), antibody to CYP2E1 (1 lg), preimmune IgG (anti-IgG) and SKF-525 (0.1 m M ) were added to the reaction before initiating the enzyme activity
by adding the dimethylnitrosomine (4 m M ) Depletion of FDXR was carried out as described in Experimental procedures Details of enzyme assays are given in Experimental procedures.