Mitochondrial targeting of intact CYP2B1 and CYP2E1 and
N-terminal truncated CYP1A1 proteins in Saccharomyces
cerevisiae
)
role of protein kinase A in the mitochondrial
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

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.
FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania. Journal compilation ª 2007 FEBS 4615
studies 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

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

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
Na
2
CO
3
.+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

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
+5/1A1
DHFR1A1
1A1
35
47
62
81
kDa
AB
1234
2B1/BY4746
2E1

N. B. V. Sepuri et al. Mitochondrial targeting of rat CYPs in yeast cells
FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania. Journal compilation ª 2007 FEBS 4617
shows 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 5010025
A
C
DE
B
––50 100
TIM23
+33/1A1
TIM23
+5/1A1
TIM23
Digitonin
+Trypsin:

+33(R34D&K39I)
+33(R34D)
+33(R34D&K39I)
Trypsin:
+
+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.
Mitochondrial targeting of rat CYPs in yeast cells N. B. V. Sepuri et al.
4618 FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania. Journal compilation ª 2007 FEBS
3-phospho glycerate kinase (3-PGK), but contained
mitochondria-specific TIM23 protein. Additionally, both

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
++
+
+
+
+

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 Na
2
CO
3
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 Na
2
CO

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

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

Su9-DHFR
Su9-DHFR
*
TIM23
Micro
A
B
C
Mito Micro Mito
Trypsin:
+
2E1
WT
Su9-DHFR
+
*
Micro Mito
Micro
Mito
Trypsin:
++
2E1(S129A)
TIM23
100 72 2 10
% distribution
% distribution
10041
96
12
3

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 nmolÆ mg
)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

DHFR 1A1 ND ND
+331A1 (m) ND ND
Not determined 50
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
AB
TIM23
2B1

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-

0.5
1
1.5
2
2.5
3
1A1 +5 +33 1A1 +5 +33 33(M)
Micro
Mito
nmoles/mg/min
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

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

0.5
1
1.5
2
2.5
3
3.5
4
4.5
Micro
Mito
nmoles/HCHO/min/mg
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.
Mitochondrial targeting of rat CYPs in yeast cells N. B. V. Sepuri et al.
4624 FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania. Journal compilation ª 2007 FEBS
exclusively to the mitochondria. In the present study,
we observed no significant targeting of intact CYP1A1
to mitochondria, possibly due to the absence of cyto-

Our results from the mutational analysis also showed
that the two positively charged residues at positions 24
and 25 of CYP2E1 formed a critical part of the mito-
chondria-targeting signal [12]. Accordingly, N-terminal
truncated proteins (+ 29 ⁄ 2E1 and + 36 ⁄ 2E1) failed to
show significant mitochondrial targeting under in vitro
and in vivo conditions [10]. In sharp contrast to our
results, Ingelman-Sunderberg’s group first reported
that N-terminal truncated + 29 ⁄ 2E1 expressed in hepa-
toma cells is targeted to mitochondria as a 50 kDa
soluble protein [21], suggesting proteolytic processing
at an uncharacterized internal site. These same investi-
gators reported that N-terminal truncated + 29 ⁄ 2E1
and + 82 ⁄ 2E1 expressed in yeast cells failed to enter
mitochondria, but existed as outer membrane-bound
forms [23]. In the present study, using rigorous con-
trols on mitochondrial integrity and selective markers
for the outer membrane, intermembrane space, inner
membrane and matrix compartment, we demonstrated
that CYP2E1 is targeted to yeast mitochondria as an
intact protein, and that PKA-mediated phosphoryla-
tion is critical for mitochondrial targeting. These find-
ings are consistent with results from three different
groups showing nearly identical gel migration of mito-
chondrial and microsomal CYP2E1 in the mouse and
rat liver under different pathophysiologic conditions
[5,10,26,42,43].
Although the precise reasons for this sharp differ-
ence in the targeting patterns of CYP2E1 remains
unclear, it is likely that the hepatoma cells used by

when the matrix protein Put2 was released (Fig. 4B).
These results provide rigorous proof for the intra-
mitochondrial location of CYP2E1. Furthermore, as
shown for rat liver mitochondrial and COS cell mito-
chondrial CYP2E1, the yeast mitochondrial CYP2E1
is readily extracted by alkaline Na
2
CO
3
, suggesting its
membrane-extrinsic orientation (Fig. 4C).
PKA is known to play important roles in cellular
regulation, cell growth, metabolism, stress resistance,
and filamentous invasive growth [45,46]. Studies have
also shown that in yeast, PKA controls the nuclear
localization of certain transcription factors, such as
Msn2 and Msn4, and of snf1 kinase in the cytosol in
N. B. V. Sepuri et al. Mitochondrial targeting of rat CYPs in yeast cells
FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania. Journal compilation ª 2007 FEBS 4625
response to glucose, suggesting the importance of PKA
in protein trafficking [47,48]. In this study, we used
PKA knockout mutants to determine the role of PKA
in mitochondrial targeting of CYP2E1. In mutants
lacking PKA, CYP2E1 was exclusively localized to the
microsomal fraction, indicating that PKA is required
for CYP2E1 targeting to the mitochondria (Fig. 5A).
The present findings suggest that a conserved PKA-
mediated pathway is required for targeting of a subset
of precursor proteins to mitochondria. It is likely that
PKA phosphorylation is a more general pathway for

lacking. Our results provide rigorous documentation of
the import of intact CYP2E1 into mitochondria, which
is also dependent on PKA-mediated phosphorylation
at Ser129. The precise physiologic significance of mito-
chondria-targeted CYPs remains unknown, although
several studies, including ours, suggest roles in drug
metabolism and reactive oxygen species production
[26,50]. Increased levels of mitochondrial CYP2E1 in
streptozotocin-induced diabetes have been implicated
in reactive oxygen species production and depletion of
the mitochondrial glutathione pool, thus contributing
to oxidative stress [26,50].
Experimental procedures
Materials
Mata his3D1 leu2D0 met15D0 ura3D0 strain BY4741 was
obtained from Research Genetics Inc. (Huntsville, AL,
USA). The protease-deficient strain (pep4D) ade2–101
met2 his3D200 lys2–801 ura3–52 was a kind gift from E
Johnson (Thomas Jefferson University). BY4741 and pep4D
strains were used to express rat CYP cDNAs driven by the
elongation factor promoter on either a 2 lm or centromeric
URA3 or Leu2 plasmids. W303 strain MATa ade2-1 trp1-1
his3-11,15 can1-100 ura3-1 leu2-3,112, PKA a ⁄ cDstrain
MATa tpk1::URA3 tpk2::HIS3 leu2-3112 ura3-1 trp1-1 his3-
11,15 ade2-1 can1-100, PKA a ⁄ bDstrain MAa tpk1::URA3
tpk3::TRP1 leu2-3112 ura3-1 trp1-1 his3-11,15 ade2-1 can1-
100 and PKA b ⁄ cDstrain MATa tpk2::HIS3 tpk3::TRP1
leu2-3112 ura3-1 trp1-1 his3-11,15 ade2-1 can1-100 were kind
gifts from M Carlson (Columbia University, New York).
MATa ura3-52 lys2-80(amber) his3-D200 trp1-D63 leu2-D1

were grown on selective synthetic medium in the presence
Mitochondrial targeting of rat CYPs in yeast cells N. B. V. Sepuri et al.
4626 FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania. Journal compilation ª 2007 FEBS
of 2% glucose or raffinose and were harvested during log
phase and treated with zymolyase to produce spheroplasts.
Spheroplasts were homogenized in SEM buffer (250 mm
sucrose, 1 mm EDTA, 20 mm Mops, pH 7.2) containing
protease inhibitors and 0.4% BSA. The homogenate was
centrifuged twice for 5 min at 2500 g (Sorvall RC5B,
SA600) to remove unbroken cells and nuclei; the superna-
tant was then centrifuged at 12 000 g for 10 min (Sorvall
RC5B, SA600). The pellet was washed twice with SEM buf-
fer, and the final pellet was resuspended in SEM buffer and
passed through a 0.65 m sucrose cushion. The pellet was
again resuspended in either SEM buffer or CYP buffer
(50 mm potassium phosphate, pH 7.4, 20% glycerol,
0.5 mm dithiothreitol, 1 mm EDTA, 0.1 mm phenyl-
methanesulfonyl fluoride). The 12 000 g supernatant was
spun at 100 000 g to isolate microsomes (Beckman L7
ultracentrifuge, SW 50.1 rotor), which were then suspended
in the CYP buffer. The purity of the mitochondria was rou-
tinely assessed by immunoblotting the subcellular fractions
with antibodies specific for mitochondria (TIM23), micro-
somes (DMPS), and cytosol (3-phosphoglycerate kinase).
To remove proteins that are peripherally associated with
the mitochondrial fraction, isolated mitochondria were trea-
ted with trypsin in SEM buffer for 20 min on ice. Trypsin
was then inhibited by adding a 10-fold excess of soybean
trypsin inhibitor followed by washing the mitochondria
with SEM buffer containing phenylmethanesulfonyl fluo-

were used to amplify the DHFR ⁄ 1A1 and cloned
into the same sites of yeast vector
[5]
pNS48, + 331A1
point mutation (R34D)
pTEF 2l-Ura3 The point mutant generated by incorporating
appropriate base substitutions in the forward primer
and cloned into yeast vector as above
This study
pNS49, + 331A1 pTEF2l-Ura3 Cloned as above [1]
pNS56, + 51A1 pTEF2l-Ura3 Cloned as above [1]
pNS58, + 331A1
(double mutant, R34D; K39I)
pTEF2l-Ura3 The double mutant was generated as pNS48 This study
pNS59, 2B1 pTEF2l-Ura3 5¢ EcoRI sense and 3¢ XhoI antisense primers
were used to clone into yeast vector as above
[3]
pNS61, 2E1 pTEF2l-Ura3 5¢ EcoRI sense and 3¢ XhoI antisense primers
were used to clone into yeast vector as above
[7]
pNS62, 2E1
(point mutation S128A)
pTEF2l-Ura3 5¢ EcoRI sense and 3¢ XhoI antisense primers
were used to clone the phosphomutant into
yeast vector as above
[7]
pNS69, su9-DHFR pTEF2l-Ura3 5¢ BamH1 sense and 3¢ HindIII antisense primers
were used to amplify the su9-DHFR and cloned
into the same sites of yeast vector
This study

addition of 3 mm NADPH, and incubation was continued
for 30 min at 37 °C in a shaking water bath. The reactions
were terminated by adding 2 mL of ice-cold methanol, and
insoluble particles were sedimented by centrifugation at
10 000 g for 10 min at room temperature (Labnet, Hermlez-
233M, 220.59 rotor). Spectrophotometric determinations of
the supernatant containing resorufin were made at the exci-
tation and emission wavelengths of 528 nm and 590 nm,
respectively. The ERND activities of mitochondria and
mitoplast fractions expressing yeast CYP1A1 were measured
as described by Anandatheerthavarada et al. [32]. The assay
mixture, containing 50 mm Tris ⁄ HCl (pH 7.4), 20 mm
MgCl
2
, 500 lg of protein, and 1 mm erythromycin, was
preincubated for 3 min. The reaction was initiated by the
addition of 3 mm NADPH, and continued for 30 min at
37 °C in a shaking water bath. The reaction was terminated
by the addition of 250 lL of ice-cold 10% trichloroacetic
acid. The reaction product, formaldehyde, was measured as
previously described [56]. In all cases, both enzyme and zero-
time blanks were also analyzed.
NDMA-d activity was assayed by the Anderson & Angel
method [57] as modified by Yadav et al. [58]. The assay
mixture contained 50 lg of protein, 70 mm Tris ⁄ HCl
(pH 7.4), 10 mm semicarbazide, 14 mm MgCl
2
, 215 mm
KCl, 1 mm NADPH, and 4 mm NDMA in a 1.0 mL final
volume. The reaction mixture was incubated at 37 °C for

6, 947–960.
4 Addya S, Anandatheerthavarada HK, Biswas G,
Bhagwat SV, Mullick J & Avadhani NG (1997) Target-
ing of NH2-terminal-processed microsomal protein to
mitochondria: a novel pathway for the biogenesis of
hepatic mitochondrial P450MT2. J Cell Biol 139,
589–599.
5 Anandatheerthavarada HK, Addya S, Dwivedi RS,
Biswas G, Mullick J & Avadhani NG (1997) Locali-
zation of multiple forms of inducible cytochromes P450
in rat liver mitochondria: immunological characteristics
and patterns of xenobiotic substrate metabolism. Arch
Biochem Biophys 339, 136–150.
6 Anandatheerthavarada HK, Biswas G, Mullick J, Sepu-
ri NB, Otvos L, Pain D & Avadhani NG (1999) Dual
targeting of cytochrome P4502B1 to endoplasmic reticu-
lum and mitochondria involves a novel signal activation
by cyclic AMP-dependent phosphorylation at ser128.
EMBO J 18, 5494–5504.
7 Omura T (2006) Mitochondrial P450s. Chem Biol Inter-
act 163, 86–93.
8 Bhagwat SV, Biswas G, Anandatheerthavarada HK,
Addya S, Pandak W & Avadhani NG (1999) Dual
targeting property of the N-terminal signal sequence
of P4501A1. Targeting of heterologous proteins to
endoplasmic reticulum and mitochondria. J Biol Chem
274, 24014–24022.
Mitochondrial targeting of rat CYPs in yeast cells N. B. V. Sepuri et al.
4628 FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania. Journal compilation ª 2007 FEBS
9 Boopathi E, Anandatheerthavarada HK, Bhagwat SV,

protein (SRP) mediates the selective binding to
microsomal membranes of in-vitro-assembled poly-
somes synthesizing secretory protein. J Cell Biol 91,
551–556.
15 Black SD & Coon MJ (1982) Structural features of liver
microsomal NADPH-cytochrome P-450 reductase.
Hydrophobic domain, hydrophilic domain, and connect-
ing region. J Biol Chem 257, 5929–5938.
16 Fujii-Kuriyama Y, Negishi M, Mikawa R & Tashiro Y
(1979) Biosynthesis of cytochrome P-450 on membrane-
bound ribosomes and its subsequent incorporation into
rough and smooth microsomes in rat hepatocytes. J Cell
Biol 81, 510–519.
17 Sakaguchi M, Mihara K & Sato R (1987) A short
amino-terminal segment of microsomal cytochrome
P-450 functions both as an insertion signal and as a
stop-transfer sequence. EMBO J 6, 2425–2431.
18 Davis AJ, Sepuri NB, Holder J, Johnson AE & Jensen
RE (2000) Two intermembrane space TIM complexes
interact with different domains of Tim23p during its
import into mitochondria. J Cell Biol 150, 1271–1282.
19 Sepuri NB, Gordon DM & Pain D (1998) A GTP-
dependent ‘push’ is generally required for efficient
protein translocation across the mitochondrial inner
membrane into the matrix. J Biol Chem 273, 20941–
20950.
20 Sepuri NB, Schulke N & Pain D (1998) GTP hydrolysis
is essential for protein import into the mitochondrial
matrix. J Biol Chem 273, 1420–1424.
21 Neve EP & Ingelman-Sundberg M (1999) A soluble

doxin reductase. Yeast 14, 839–846.
29 Liao M, Zgoda VG, Murray BP & Correia MA (2005)
Vacuolar degradation of rat liver CYP2B1 in Saccharo-
myces cerevisiae: further validation of the yeast model
and structural implications for the degradation of
mammalian endoplasmic reticulum P450 proteins. Mol
Pharmacol 67, 1460–1469.
30 Bar-Nun S, Kreibich G, Adesnik M, Alterman L,
Negishi M & Sabatini DD (1980) Synthesis and
insertion of cytochrome P-450 into endoplasmic
reticulum membranes. Proc Natl Acad Sci USA 77,
965–969.
31 Monier SLP, Kreibich G, Sabatini DD & Adesnik M
(1988) Signals for the incorporation and orientation of
cytochrome P450 in the endoplasmic reticulum mem-
brane. J Cell Biol 107, 457–470.
32 Anandatheerthavarada HK, Addya S, Mullick J &
Avadhani NG (1998) Interaction of adrenodoxin with
P4501A1 and its truncated form P450MT2 through
N. B. V. Sepuri et al. Mitochondrial targeting of rat CYPs in yeast cells
FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania. Journal compilation ª 2007 FEBS 4629
different domains: differential modulation of enzyme
activities. Biochemistry 37, 1150–1160.
33 Anandatheerthavarada HK, Vijayasarathy C, Bhagwat
SV, Biswas G, Mullick J & Avadhani NG (1999) Physio-
logical role of the N-terminal processed P4501A1 tar-
geted to mitochondria in erythromycin metabolism and
reversal of erythromycin-mediated inhibition of mito-
chondrial protein synthesis. J Biol Chem 274, 6617–6625.
34 Taylor SW, Fahy E & Ghosh SS (2003) Global organel-

cleavable preprotein. J Biol Chem 274, 16522–16530.
42 Raza H, Ahmed I, John A & Sharma AK (2000) Modu-
lation of xenobiotic metabolism and oxidative stress in
chronic streptozotocin-induced diabetic rats fed with
Momordica charantia fruit extract. J Biochem Mol
Toxicol 14, 131–139.
43 Robin MA, Sauvage I, Grandperret T, Descatoire V,
Pessayre D & Fromenty B (2005) Ethanol increases
mitochondrial cytochrome P450 2E1 in mouse liver and
rat hepatocytes. FEBS Lett 579, 6895–6902.
44 Lemire BD, Fankhauser C, Baker A & Schatz G
(1989) The mitochondrial targeting function of ran-
domly generated peptide sequences correlates with
predicted helical amphiphilicity. J Biol Chem 264,
20206–20215.
45 Thevelein JM & de Winde JH (1999) Novel sensing
mechanisms and targets for the cAMP-protein kinase A
pathway in the yeast Saccharomyces cerevisiae. Mol
Microbiol 33, 904–918.
46 Pagliarini DJ & Dixon JE (2006) Mitochondrial modu-
lation: reversible phosphorylation takes center stage?
Trends Biochem Sci 31, 26–34.
47 Hedbacker K, Townley R & Carlson M (2004) Cyclic
AMP-dependent protein kinase regulates the subcellular
localization of Snf1-Sip1 protein kinase. Mol Cell Biol
24, 1836–1843.
48 Wiatrowski HA & Carlson M (2003) Yap1 accumulates
in the nucleus in response to carbon stress in Saccharo-
myces cerevisiae. Eukaryot Cell 2, 19–26.
49 Alves R, Herrero E & Sorribas A (2004) Predictive

ible by ethanol. Drug Metab Dispos 13, 548–552.
57 Anderson LM & Angel M (1980) Induction of dimethyl-
nitrosamine demethylase activity in mouse liver by poly-
chlorinated biphenyls and 3-methylcholanthrene.
Biochem Pharmacol 29, 1375–1383.
58 Yadav S, Dhawan A, Singh RL, Seth PK & Parmar D
(2006) Expression of constitutive and inducible cyto-
chrome P450 2E1 in rat brain. Mol Cell Biochem 286,
171–180.
Mitochondrial targeting of rat CYPs in yeast cells N. B. V. Sepuri et al.
4630 FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania. Journal compilation ª 2007 FEBS