Tài liệu Báo cáo khoa học: 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 pdf

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 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 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 mitochondria 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 cytochrome P450 2E1 and cytochrome P450 2B1 were targeted both to microsomes and mitochondria, whereas truncated cytochrome P450 1A1 (+ and + 33 ⁄ cytochrome P450 1A1) were targeted to mitochondria Inability to target intact cytochrome P450 1A1 was probably due to lack of cytosolic endoprotease activity in yeast cells Mitochondrial targeting of cytochrome 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, benzoxyresorufin O-dealkylation and nitrosodimethylamine N-demethylase activities were fully supported by yeast mitochondrial ferredoxin and ferredoxin reductase 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 demonstrated that b-naphthoflavone-inducible CYP1A1, pyrazole-inducible CYP2E1, and phenobarbital-inducible CYP2B1, known to be bona fide microsomal forms, are also targeted to mitochondria [5,6,8–10] These 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-5carboxylate 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 Mitochondrial targeting of rat CYPs in yeast cells studies led us to propose the concept of chimeric protein-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 cotranslational 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 subsequently 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 mitochondria requires a cytosolic chaperone-mediated association 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 activation 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 observations on the targeting of intact CYP2E1 to mitochondria, 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 endoprotease 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 mechanism is important for understanding the evolution and regulation of bimodal targeting Our primary objective here was to address this important question using rigorous approaches 4616 N B V Sepuri et al 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 conserved 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 transiently transfected mammalian cells As targeting of intact CYP2E1 and the requirement for PKA-mediated phosphorylation for mitochondrial targeting are contradicted by other studies [22,23,26], we sought cell systems lacking specific PKA subunits to address this important question The availability of PKA gene deletion 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 mitochondria, whereas + ⁄ 1A1 was localized in both the mitochondria and microsomes Also, we found that fulllength CYP2E1 and CYP2B1 were targeted to the mitochondria 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, + ⁄ 1A1, + 33 ⁄ 1A1 and CYP2E1 were consistent 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 previous report showing similar degradation of CYP2B1 in yeast cells [29] Use of the protease-deficient strain FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS +3 kDa B /1 A1 +5 /1 A DH FR 1A 1A 1 A 81 62 kDa 81 62 47 kDa 81 62 47 35 35 35 25 16 47 2E Mitochondrial targeting of rat CYPs in yeast cells 2B 1/B Y4 74 2B 1/p ep 4Δ ' N B V Sepuri et al 25 16 Fig The levels of expression of CYP1A1, CYP2B1 and CYP2E1 proteins in yeast cells Yeast strains BY4741 or PeP4D, transformed 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 constructs were probed with antibody to CYP1A1 (B) Extracts from cells transformed with CYP2B1 (lanes and 6) and CYP2E1 construct were probed with either CYP2B1 antibody (left panel, lanes 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 pepD 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, + ⁄ 1A1 and + 331A1 showed significant levels of full-length CYP1A1 protein, reduced levels of + ⁄ 1A1 protein, and vastly reduced levels of + 33 ⁄ 1A1 protein (Fig 2A, first four lanes) We also found nearly undetectable full-length CYP1A1 and clearly visible + ⁄ 1A1 and + 33 ⁄ 1A1 in the mitochondrial fraction (Fig 2A, last four lanes) As expected, full-length CYP1A1 and + ⁄ 1A1 from the microsomal membrane 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 + ⁄ 1A1, + 33 ⁄ 1A1, and TIM23, which was used as an internal control, were protected fully against trypsin up to 100 lgỈmL)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 + ⁄ 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 proportion of its sequence exposed outside the inner membrane, facing the intermembrane space, was degraded significantly These results suggested that the imported + ⁄ 1A1 and + 33 ⁄ 1A1 proteins were localized inside of the inner membrane Moreover, the imported + ⁄ 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 associated with the mitochondria and microsomes, as it was vulnerable to very low concentrations of trypsin, suggesting 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 mitochondrial 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 previous studies, we showed that the positively charged amino acids at positions 34 and 39 were important for targeting of + ⁄ 1A1 and + 33 ⁄ 1A1 proteins to the mitochondrial compartment In keeping with these observations, the results in Fig 2E show that the association 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 mammalian 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 mitochondria, we first established the relative purity of mitochondrial preparations by biochemical and electron microscopy techniques Figure 3A (top left panel) FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS 4617 Mitochondrial targeting of rat CYPs in yeast cells A N B V Sepuri et al Microsomes Trypsin (µg/mL) – 50 100 – 25 B Mitochondria Mitochondria Digitonin +Trypsin: 50 100 25 – + 1A1 1A1 TIM23 TIM23 +33/1A1 +33/1A1 TIM23 TIM23 +5/1A1 +5/1A1 TIM23 Alk Sol Input Alk Sol Input Alk insol C Alk insol TIM23 1A1ab TIM23ab Trypsin: 3/ +3 DHFR/1A1 +3 3/ + 1A 1A E +3 + Mito – Micro – Mito Trypsin: Micro D +3 (R3 3( 4D +3 R34 ) 3( D R ) +3 34 3( D& R3 K 4D 39 &K I) 39 I) +33/1A1 mito +5/1A1 mito – + – + – + +33/1A1 TIM23 TIM23 DPMS Fig Mitochondrial targeting of truncated CYP1A1 in yeast cells Mitochondrial and microsomal fractions of yeast cells expressing CYP1A1, + ⁄ 1A1, DHFR-1A1 and + 33 ⁄ 1A1 were separated by SDS ⁄ PAGE and subjected to western blotting Membrane topologies of mitochondriaassociated + ⁄ 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), digitonintreated mitochondria were subjected to alkaline Na2CO3 extraction In (A), increasing concentrations of trypsin (0–100 lgỈmL)1) were used, and in (B), (D) and (E), a fixed concentration (50 lgỈmg)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 microsomal marker shows the transmission electron microscopy pattern of a representative mitochondrial preparation A representative field shows several well-defined mitochondrial particles with minor membrane contamination As shown in the insets, a large majority of mitochondrial preparations showed intact inner and outer membrane compo4618 nents, confirming the structural integrity of mitochondrial 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 FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS N B V Sepuri et al Mitochondrial targeting of rat CYPs in yeast cells A B Cyto Micro Mito TIM23 PGK 2E1 DPMS D C Tx-100: Digitonin: Trypsin: - - - + + - + + Micro Mito + - 2E1 + Adx-red TIM23 TIM23 Fig The nature of mitochondria-associated CYP2E1 in yeast cells (A) Assessment of the integrity of the isolated mitochondria Mitochondrial isolates were subjected to scanning electron microscopy as described in Experimental procedures (magnification: · 30 000) (B) The relative 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 expressing full-length P4502E1 3-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 Tritonsolubilized samples completely degraded TIM23 and FDXR Figure 3D represents a full view of an immunoblot of mitochondrial and microsomal proteins developed with a combination of CYP2E1 and TIM23 antibodies It is seen that both microsomal and mitochondria-associated CYP2E1 consisted of a major antibody-reactive full-length protein and minor fastermigrating bands Furthermore, the mitochondrial fraction crossreacted with TIM23 antibody, whereas the microsomal fraction lacked detectable TIM23 These results suggested that the full-length CYP2E1 is targeted 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 transformed 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 mitochondria-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 FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS 4619 Mitochondrial targeting of rat CYPs in yeast cells N B V Sepuri et al A 2E1 Porin pPut2 Porin Mito TIM23 2E1 pPut2 Mitoplast 2E1 0 0 25 0 Digitonin%: Mito B 50 25 0 25 0 - 0 Trypsin (µg/mL): Mitoplast Supernatant Pellet Mito PUT2 C Pellet Sup 2E1 CCPO Mito TIM23 Mito pPut2 Mito Micro 2E1 Mitoplast Mito Mitoplast Mito TOM20 Mitoplast Fig Intramitochondrial localization of CYP2E1 in transformed yeast cells (A) Mitochondria and mitoplasts from cells expressing CYP2E1 were treated with 0–50 lgỈmL)1 trypsin as indicated Mitochondria reisolated by banding through a sucrose layer were analyzed by immunoblotting (50 lg protein each) with antibody to CYP2E1 Put2, TIM23 and CCPO and TOM20 were used as matrix, inner membrane, intermembrane space and outer membrane markers, respectively (B) Isolated mitochondria were incubated with increasing concentrations of digitonin (0–0.4%; 0–400 lgỈmg)1 protein) for 30 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 antibodies 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 membrane was stripped by digitonin Under these treatment 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-pyrroline-5-carboxylate dehydrogenase (Put2), a matrixlocalized 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 concentration of digitonin required to release mitochondria-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 4620 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 soluble 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 mitochondria-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 microsomalassociated CYP2E1 resisted Na2CO3 extraction, suggesting a transmembrane topology The mitochondria-associated CYP2E1, on the other hand, was mostly partitioned in the soluble fraction, indicating a membrane-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 mitochondrial CYP2E1 is mostly a membrane-extrinsic protein localized in the matrix compartment However, FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS N B V Sepuri et al Mitochondrial targeting of rat CYPs in yeast cells the protein was peripherally associated with the inner membrane and required washing with a high salt concentration (0.2 m NaCl) to be released from the membrane (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 mitochondrial targeting of wild-type CYP2E1 in PKA-deficient (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 approximately 4–6-fold higher than the mitochondrial content, and the microsome-localized CYP2E1 was highly sensitive to trypsin (Fig 5A, compare lanes 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 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 of Neurospora 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 phosphorylation 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 mitochondrial targeting of the protein in COS cells In the second approach, we tested the level of mitochondrial targeting of S129A mutant CYP2E1 in transformed A 2E1 Micro * Mito Micro + - Mito + B * WT TIM23 Su9-DHFR % distribution of 2E1 Trypsin: 100 90 80 70 60 50 40 30 20 10 Micro Mito PKA PKA Δ α/γ Δ α/β Strain background WT 2E1 Δ α/γ TIM23 C Micro Mito Micro Mito + + Trypsin: - Su9-DHFR 2E1(S129A) 2E1 100 TIM23 72 10 Δ α/β % distribution TIM23 Su9-DHFR 4 100 96 % distribution Fig 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 Subcellular fractions were subjected to immunoblot analysis following treatment with or without trypsin as indicated TIM23 was used as a mitochondrial marker (B) The amount of CYP present in the microsomal or mitochondrial fractions obtained from various PKA mutants were quantified and represented as a bar graph (C) Reduced mitochondrial targeting of phosphorylation site mutated CYP2E1 (S129A) Mitochondria and microsomal fractions of yeast cells expressing S129A mutant CYP2E1 were treated with or without trypsin and subjected to immunoblot analysis with CYP2E1 and TIM23 antibodies FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS 4621 Mitochondrial targeting of rat CYPs in yeast cells N B V Sepuri et al yeast 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 PKAmediated phosphorylation, most likely targeted to the S129 consensus site, is very important for mitochondrial 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 A B kDa 81 Micro Mito + + Trypsin Micro Mito 62 2B1 47 35 25 TIM23 Fig Immunoblot analysis of PeP4D strain expressing full-length CYP2B1 Isolated mitochondrial or microsomal fractions were treated with (B) or without (A) trypsin, and subjected to SDS ⁄ PAGE and immunoblot analysis with CYP2B1 and TIM23 antibodies A TIM23 protein, whereas the microsomes lacked significant levels of TIM23 The western blot in Fig 6B also shows that the microsome-targeted CYP2B1 was sensitive 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 spectra 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 characteristic spectrum with mitochondria from cells expressing + 33 ⁄ 1A1 mutant constructs (data not shown) Figure 7B shows the P450-heme contents of mitochondria and microsomal fractions from yeast strains expressing various CYP constructs based on CO difference 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 nmolỈmg)1) CYP content Cells expressing + ⁄ 1A1 showed nearly equal CYP contents in the mitochondrial and microsomal fraction Cells expressing + 33 ⁄ 1A1 showed no detectable CYP in the microsomal fraction, but a high (3.5 nmolỈmg)1) level of CYP in mitochondria Cells expressing full-length CYP2E1 showed about 300 pmolỈmg)1 CYP in the microsomes and 55 pmolỈmg)1 in mitochondria The mitochondrial fraction from CYP2B1-expressing cells showed about 50 pmolỈmg)1 CYP As shown in Fig 8A, the microsomal fraction from full-length CYP1A1- and + ⁄ 1A1-expressing cells B CYP450 ND ND 1A1 6500 ND Vector 0.205 pmole/mg microsomal pmole/mg mitochondrial protein protein +331A1 mito 0.155 +51A1 0.005 400 440 460 480 500 3500 DHFR 1A1 420 200 ND ND ND +331A1 (m) 0.055 250 +331A1 0.105 ND ND 2E1 300 55 Wave length (nm) 2B1 4622 Not determined 50 Fig Mitochondrial CYP contents in yeast cells expressing CYP1A1, CYP2E1 and CYP2B1 proteins (A) The reduced CO spectra 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 microsomes 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 FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS Mitochondrial targeting of rat CYPs in yeast cells 1.8 1.6 1.4 1.2 0.8 0.6 0.4 0.2 Mito 1A1 C Fig Metabolic activities of the microsomal and mitochondrial CYPs in transformed yeast cells Microsomes and digitonin-treated mitoplasts from cells expressing various CYP variants were assayed for their enzyme activities as indicated 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 B EROD ACTIVITY Micro +5 pmoles of resorufin/min/mg nmoles/mg/min A nmoles/mg/min N B V Sepuri et al 2.5 ERND ACTIVITY Micro Mito 1.5 0.5 +33 1A1 +5 +33 33(M) BROD ACTIVITY 0.25 Micro Mito 0.2 0.15 0.1 0.05 CYP2B1: - - + + + + Anti-2B1: - - - - + + showed high activity of ethoxyresorufin O-dealkylation (EROD), which is a specific marker enzyme for ERassociated CYP1A1 +33 ⁄ 1A1-expressing cells, however, 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 + ⁄ 1A1 showed relatively low ERND activity Similarly, consistent with the low or nonsignificant mitochondrial localization of full-length CYP1A1, mitochondria from these cells also showed very low activity The mitochondrial isolates from + ⁄ 1A1- and + 33 ⁄ 1A1-expressing cells, however, showed high ERND activity (2.0–2.5 nmolỈmg)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 mitochondriatargeted 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 mitochondrial 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 preincubated with CYP2B1 antibody, indicating the specificity of the assay As shown in Fig 9A, both microsomal and mitochondrial 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 preincubated 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 endogenous FDX1 + FDXR in these mitochondrial preparations (Fig 9A) Additionally, the activity with the mitochondrial fraction was inhibited by 50% after addition 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 FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS 4623 Mitochondrial targeting of rat CYPs in yeast cells 4.5 3.5 2.5 1.5 0.5 Wt cells expressing CYP2E1 Micro Mito Cell Fractions NADPH FDR& FDX Ant-FDX SKF-525 Anti-2E1 Anti-IgG B FDXR depleted cells expressing CYP2E1 4.5 3.5 2.5 1.5 0.5 Cell Fractions NADPH FDR& FDX nmoles/HCHO/min/mg nmoles/HCHO/min/mg A N B V Sepuri et al Micro Mito Fig 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 Experimental procedures, using 50 lg of mitochondria or microsomal proteins NADPH (1 mM), FDX + FDXR (1 lg each), antibody to FDX (2 lg), antibody to CYP2E1 (1 lg), preimmune IgG (anti-IgG) and SKF-525 (0.1 mM) were added to the reaction before initiating the enzyme activity by adding the dimethylnitrosomine (4 mM) Depletion of FDXR was carried out as described in Experimental procedures Details of enzyme assays are given in Experimental procedures CYP2E1 in Yah1-depleted cells turned out to be lethal We therefore analyzed the Arh1-depleted cells expressing CYP2E1 The catalytic activity of the mitochondrial fraction was reduced by about 60% in FDXR-depleted cells (Fig 9B) However, the activity with the mitochondrial fraction was restored by the addition of purified bovine FDX + Fdr These results confirm that yeast mitochondrial FDX + FDXR is capable of supporting 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 mitochondrial 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-associated proteins lack the canonical mitochondria-targeting signals, and the precise mechanisms by which these proteins are translocated to the mitochondrial compartment 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 4624 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 noncanonical signal-containing proteins to the mitochondrial 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 capable of targeting proteins to both the ER and mitochondria [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 targeting 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 (+ ⁄ 1A1 or + 33 ⁄ 1A1) of CYP1A1 exposes the cryptic mitochondria-targeting sequence at positions 33–39 of the protein, thus targeting the + 331A1 protein FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS N B V Sepuri et al exclusively to the mitochondria In the present study, we observed no significant targeting of intact CYP1A1 to mitochondria, possibly due to the absence of cytosolic endoprotease specific for cleavage at the + and + 33 positions (Fig 2) Consistent with this possibility, both the + ⁄ 1A1 and + 33 ⁄ 1A1 proteins are efficiently targeted to the yeast mitochondrial compartment in transformed cells (Fig 2) Mitochondriatargeting sequences are usually rich in positively charged residues, either as part of an amphiphilic helical structure or as random structures The charged residues have been suggested to play an important role in binding to TOM20 and TOM22 outer membrane receptors of the import complex [41] The positive residues at the + 34 and + 39 positions of + 331A1 are indeed important for targeting of the protein to mitochondria, as substitutions at these positions with neutral or hydrophobic amino acids essentially abolished mitochondrial import (Fig 2D,E) Our results also showed that the + 51A1 construct was mostly targeted to mitochondria, with a minor fraction being found in association with the ER (Fig 2A) An N-terminally blocked CYP1A1 (DHFR-1A1 fusion) was not targeted to either the ER or the mitochondria, whereas the full-length was CYP1A1 targeted efficiently to the ER, suggesting that an open N-terminal signal domain is critical for protein targeting to the ER (Fig 2D) In contrast to the targeting of CYP1A1 requiring N-terminal truncation, we have shown that intact CYP2B1 and CYP2E1 proteins are targeted to mitochondria in inducer-treated rat livers [5], in transiently transfected COS cells, and also in an in vitro transport system with isolated rat liver mitochondria [6,10,12] 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 mitochondria-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 hepatoma cells is targeted to mitochondria as a 50 kDa soluble protein [21], suggesting proteolytic processing at an uncharacterized internal site These same investigators 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 controls 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 Mitochondrial targeting of rat CYPs in yeast cells intact protein, and that PKA-mediated phosphorylation is critical for mitochondrial targeting These findings are consistent with results from three different groups showing nearly identical gel migration of mitochondrial 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 difference in the targeting patterns of CYP2E1 remains unclear, it is likely that the hepatoma cells used by Neve & Ingelman-Sundberg [22] contain an unusual cytosolic endoprotease that clips the protein and activates an internal cryptic mitochondria-targeting signal It is known that randomly generated peptides, even from Escherichia coli, can potentially function as mitochondria-targeting signals when fused to the N-terminus of a reporter protein, DHFR [44] This does not mean that such internal signals are functional under physiologic settings while working with an intact protein A major difference that may account for the observed difference in the results is that we have used well-defined and intact mitochondrial isolates for analyzing CYP proteins, whereas Neve & Ingelman-Sundberg [21,22,22,23] have consistently used total cell homogenates with undefined mitochondrial integrity In this study, we have ensured mitochondrial purity and integrity by the use of several criteria: (a) mitochondria sedimented through a 0.65 m sucrose layer, which reduces microsomal contamination; (b) mitochondria intact as seen by scanning electron microscopy; and (c) mitochondrial markers for the outer membrane, intermembrane space, inner membrane and matrix compartments sequentially released at expected digitonin concentrations We have observed that mitochondria-associated CYP2E1 is resistant to limited protease treatment, similar to a bona fide matrix protein, Put2 (Fig 4A) However, both CYP2E1 and Put2 are readily degraded by trypsin following treatment with Triton X-100 (data not shown) Furthermore, mitochondrial CYP2E1 was released by digitonin only when the matrix protein Put2 was released (Fig 4B) These results provide rigorous proof for the intramitochondrial location of CYP2E1 Furthermore, as shown for rat liver mitochondrial and COS cell mitochondrial CYP2E1, the yeast mitochondrial CYP2E1 is readily extracted by alkaline Na2CO3, 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 FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS 4625 Mitochondrial targeting of rat CYPs in yeast cells 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 PKAmediated 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 the targeting of proteins lacking the canonical signals to mitochondria In mammalian cells, mitochondrial CYP activity is dependent on FDX and FDXR proteins Yeast homologs of FDXR, Arh1, and FDX, Yah1, are known to be involved in both heme A biosynthesis and iron–sulfur cluster assembly [49] It is not known whether these yeast homologs can serve as electron donors for mammalian CYPs Our results show that yeast homologs can serve as electron donors for mammalian CYPs (Fig 9), as mitochondria without externally added FDX + FDXR are able to support CYP-mediated ERND, BROD and NDMA-d metabolism CYP2E1 expressed in yeast strains with depleted FDXR resulted in decreased activity (Fig 9B) that was restored by adding bovine FDX + FDXR (Fig 9B) Additionally, the enzyme activity was inhibited by antibody to human FDX (Fig 9A) These results provide evidence that yeast FDX + FDXR fully supports that activity of mammalian CYPs As found before with rat liver mitochondrial enzymes, yeast mitochondrial CYP1A1 shows high ERND activity and mitochondrial CYP2E1 exhibits high NDMA-d activity The activities of both microsomal and mitochondrial enzymes were dependent on addition of NADPH and inhibited by SKF-525 In summary, our results show that the mitochondrial import of mammalian CYP family and family proteins is highly conserved in yeast, except that the cytosolic processing activity for CYP1A1 seems to be 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 mitochondria-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] 4626 N B V Sepuri et al 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 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 ⁄ c D strain MATa tpk1::URA3 tpk2::HIS3 leu2-3112 ura3-1 trp1-1 his311,15 ade2-1 can1-100, PKA a ⁄ b D strain MAa tpk1::URA3 tpk3::TRP1 leu2-3112 ura3-1 trp1-1 his3-11,15 ade2-1 can1100 and PKA b ⁄ c D strain 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 URA3::pGalArh1pHA and MATa ura3-52 lys2-80(amber) his3-D200 trp1-D63 leu2-D1 URA3::pGalYah1pHA were kind gifts from A Dancis (University of Pennsylvania) Plasmids were transformed into different yeast strains by using the standard LioAc method The yeast cells were grown to log phase at 30 °C in supplemental minimal medium containing 2% dextrose (SD) or 2% raffinose (SG), and appropriate amino acids was used to select the various plasmids To induce the Gal promoter (Gal-Yah1 or Gal-Arh1), cells were grown in 2% raffinose and 0.5% galactose Expression from the Gal promoter was turned off by shifting the cultures to identical medium without galactose [51] Plasmids and cloning strategy The full-length and truncated CYP cDNAs were amplified by PCR using Taq polymerase (Qiagen, Chatsworth, CA, USA) [8] An EcoRI site was used for the in-frame fusion of DHFR to the N-terminus of CYP1A1 by overlap PCR The double mutants (R34D and K39D) and single (R34D) mutants of + 33 ⁄ 1A1 were generated by using appropriately substituted forward primers in PCR reactions The S129A mutant CYP2E1 cDNA was generated as described previously [10] The cDNAs were cloned into the lm vector pTEF-URA3 or Leu2 or the centromeric pTEF-URA3 or Leu2 plasmid [52] The details of cDNAs, restriction sites used and plasmid designations are listed in Table Subcellular fractionation and isolation of mitochondria and mitoplasts Mitochondria were isolated from yeast strains containing high copy levels of various CYP cDNAs as described previously [20] Briefly, yeast strains expressing various CYPs were grown on selective synthetic medium in the presence FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS N B V Sepuri et al Mitochondrial targeting of rat CYPs in yeast cells Table Plasmids used in this study Plasmid, gene Marker Cloning Source ⁄ Reference pNS45, 1A1 pTEF 2l-Ura3 [5] pNS47, DHFR1A1 pTEF 2l-Ura3 pNS48, + 331A1 point mutation (R34D) pTEF 2l-Ura3 pNS49, + 331A1 pNS56, + 51A1 pNS58, + 331A1 (double mutant, R34D; K39I) pNS59, 2B1 pTEF2l-Ura3 pTEF2l-Ura3 pTEF2l-Ura3 5¢ BamH1 sense and 3¢ HindIII antisense primers were used to amplify the full length of 1A1 and cloned into the same sites of yeast vector 5¢ BamH1 sense and 3¢ HindIII antisense primers were used to amplify the DHFR ⁄ 1A1 and cloned into the same sites of yeast vector The point mutant generated by incorporating appropriate base substitutions in the forward primer and cloned into yeast vector as above Cloned as above Cloned as above The double mutant was generated as pNS48 pNS61, 2E1 pTEF2l-Ura3 pNS62, 2E1 (point mutation S128A) pTEF2l-Ura3 pNS69, su9-DHFR pTEF2l-Ura3 pNS120, 2E1 pTEF2l-Leu2 pTEF2l-Ura3 5¢ EcoRI sense and 3¢ XhoI antisense primers were used to clone into yeast vector as above 5¢ EcoRI sense and 3¢ XhoI antisense primers were used to clone into yeast vector as above 5¢ EcoRI sense and 3¢ XhoI antisense primers were used to clone the phosphomutant into yeast vector as above 5¢ BamH1 sense and 3¢ HindIII antisense primers were used to amplify the su9-DHFR and cloned into the same sites of yeast vector pNS61 was cut with Spe1 and Xho1 and cloned into same sites of yeast vector 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, mm EDTA, 20 mm Mops, pH 7.2) containing protease inhibitors and 0.4% BSA The homogenate was centrifuged twice for at 2500 g (Sorvall RC5B, SA600) to remove unbroken cells and nuclei; the supernatant was then centrifuged at 12 000 g for 10 (Sorvall RC5B, SA600) The pellet was washed twice with SEM buffer, 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, mm EDTA, 0.1 mm phenylmethanesulfonyl 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 routinely assessed by immunoblotting the subcellular fractions with antibodies specific for mitochondria (TIM23), microsomes (DMPS), and cytosol (3-phosphoglycerate kinase) To remove proteins that are peripherally associated with the mitochondrial fraction, isolated mitochondria were treated with trypsin in SEM buffer for 20 on ice Trypsin was then inhibited by adding a 10-fold excess of soybean trypsin inhibitor followed by washing the mitochondria [5] This study [1] [1] This study [3] [7] [7] This study This study with SEM buffer containing phenylmethanesulfonyl fluoride Digitonin fractionation Isolated mitochondria (100 lg) were resuspended in SEM buffer containing indicated concentrations of digitonin (0.05% to 0.4%, w ⁄ v) for 30 on ice with occasional mixing Digitonin-soluble and digitonin-insoluble fractions were separated by centrifugation at 12 000 g for 15 (Labnet, Hermlez-233M, 220.59 rotor), and the pellet was washed once again with SEM buffer The pellet and the supernatant fractions were mixed with SDS sample buffer and processed as described [53] Alkaline extraction of membrane proteins To determine the mode of association of CYPs with the membrane, we used an alkaline extraction method as described by Clark & Waterman [54] Mitochondrial and microsomal fractions were treated with 0.1 m Na2CO3 (pH 11.0) for approximately 20 on ice, and both the soluble and insoluble fractions were separated by centrifugation (Labnet, Hermlez-233M, 220.59 rotor); the samples were processed as described by Anandatheerthavarada et al [6] FEBS Journal 274 (2007) 4615–4630 ª 2007 University of Pennsylvania Journal compilation ª 2007 FEBS 4627 Mitochondrial targeting of rat CYPs in yeast cells Scanning electron microscopy Mitochondria were isolated as described above, and the final mitochondrial pellets were fixed in NaCl ⁄ Pi containing 4% formaldehyde and 2% glutaraldehyde Mitochondria were dehydrated through a graded ethanol series and embedded in hard grade LR White Resin (Sigma-Aldrich, St Louis, MO, USA) The sections were then examined and photographed using a JEOL (Hammarbaccan, Sollen Tuna, Sweden) 100CX electron microscope Catalytic activity The EROD and BROD activities were determined by measuring the formation of resorufin as described previously [55] The mitoplasts were isolated by treating the mitochondrial fraction with 0.0075% digitonin and isolation through a sucrose cushion The reaction mixture was composed of 20 mm Tris ⁄ HCl (pH 7.8), 20 mm MgCl2, 10 lm dicumarol, 3.2 mgỈmL)1 BSA, 200 lg of protein, and 20 lm resorufin derivatives as a substrate Reactions were initiated by the addition of mm NADPH, and incubation was continued for 30 at 37 °C in a shaking water bath The reactions were terminated by adding mL of ice-cold methanol, and insoluble particles were sedimented by centrifugation at 10 000 g for 10 at room temperature (Labnet, Hermlez233M, 220.59 rotor) Spectrophotometric determinations of the supernatant containing resorufin were made at the excitation 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 MgCl2, 500 lg of protein, and mm erythromycin, was preincubated for The reaction was initiated by the addition of mm NADPH, and continued for 30 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 zerotime 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 MgCl2, 215 mm KCl, mm NADPH, and mm NDMA in a 1.0 mL final volume The reaction mixture was incubated at 37 °C for 30 min, and the reaction was stopped by the addition of 0.1 mL of 25% zinc sulfate and 0.1 mL of saturated barium hydroxide After centrifugation at 2000 g for 10 (Labnet, Hermlez-233M, 220.59 rotor), 0.7 mL of the supernatant was mixed with an equal amount of Nash reagent The 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Bhagwat SV, Biswas G, Anandatheerthavarada HK, Addya S, Pandak W & Avadhani NG (199 9) Dual targeting property of the N-terminal signal sequence of P450 1A1 Targeting of heterologous proteins to... open N-terminal signal domain is critical for protein targeting to the ER (Fig 2D) In contrast to the targeting of CYP 1A1 requiring N-terminal truncation, we have shown that intact CYP2B1 and CYP2E1. .. that xenobiotic-inducible CYPs such as rat CYP 1A1 , CYP2E1 and CYP2B1, and mouse CYP 1A1 , contain chimeric noncanonical -targeting signals that are capable of targeting proteins to both the ER and