Bimodal Targeting of Cytochrome P450s to Endoplasmic Reticulum and Mitochondria: The Concept of Chimeric Signals

Summary

Targeting signals are critical for proteins to find their specific cellular destination. Signals for protein targeting to the endoplasmic reticulum (ER), mitochondria, peroxisome and nucleus are distinct and the mechanisms of protein translocation across these membrane compartments also vary markedly. Recently, however, a number of proteins have been shown to be present in multiple cellular sites such as mitochondria and ER, cytosol and mitochondria, plasma membrane and mitochondria, and peroxisome and mitochondria suggesting the occurrence of multimodal targeting signals in some cases. Cytochrome P450 monooxygenases (CYPs), which play crucial roles in pharmacokinetics and pharmacodynamics of drugs and toxins, are the prototype of bimodally targeted proteins. Several members of family 1, 2 and 3 CYPs have now been reported to be associated with mitochondria and plasma membrane in addition to the ER. This review highlights the mechanisms of bimodal targeting of CYP1A1, 2B1, 2E1 and 2D6 to mitochondria and ER. The bimodal targeting of these proteins is driven by their N-terminal signals which carry essential elements of both ER targeting and mitochondria targeting signals. These multimodal signals have been termed chimeric signals appropriately to describe their dual targeting property. The cryptic mitochondrial targeting signals of CYP2B1, 2D6, 2E1 require activation by PKA or PKC mediated phosphorylation at sites immediately flanking the targeting signal, and/or membrane anchoring regions. The cryptic mitochondria targeting signal of CYP1A1 requires activation by endoproteolytic cleavage by a cytosolic endoprotease, which exposes the mitochondrial signal. This review discusses both mechanisms of bimodal targeting and toxicological consequences of mitochondria targeted CYP proteins.

Introduction

Each subcellular compartment of eukaryotic cells is endowed with distinct structural/functional attributes; although, several overlapping functions are performed by different organelles. The signal sequence hypothesis for protein targeting, proposed by Blobel and Sabatini [1, 2] elegantly described how the signal sequence contained in each protein acts as an address code for its delivery to a specific sub-cellular compartment of the cell. The signal hypothesis, initially proposed for secreted proteins was composed of three important components: 1) Proteins destined to be secreted from the cell contain an amino-terminal signal sequence that is recognized by a soluble factor, now known to be SRP (signal recognition particle). 2) The SRP transports the nascent chain-ribosome complex to the endoplasmic reticulum (ER). Continued translation then threads the protein into the lumen of the ER. 3) The signal sequence tag may be removed from the secretory proteins after translocation. Studies by Blobel and others confirmed this scientific dogma, and the hypothesis was extended to include several additional postulates [3-5]. 1) Protein transport across the ER membrane proceeds through a protein conducting channel. Integral membrane proteins might also use a signal sequence to initiate translation and allow a partial translocation of a segment of the chain to the trans side of the membrane. 2) Transport of proteins across the membranes of other cellular organelles, like the inner and outer membranes of the mitochondria, is mediated by a signal sequence that is distinct from that of a secretory protein [2].

In support of the signal hypothesis, at least four major families of primary targeting signals have been characterized for the delivery of the proteins to the ER, mitochondria, peroxisomes and the nucleus. The ER targeting signals reside mostly at the N-termini of proteins, although internal signals have been reported in some cases [6]. The ER targeting signals generally consist of a hydrophobic stretch of amino acids, which favors an α-helical organization. These signals are recognized by soluble SRP complexes in the cytosol and the SRP bound proteins are escorted to the ER [7]. Once the protein is associated with the ER, additional secondary signals present within the protein, in conjunction with the various ER-Golgi sorting machineries, determine its destination---some proteins are retained in the ER and the others are targeted to the Golgi, lysosomes, plasma membrane or are secreted outside the cells. ER resident proteins are either localized to the ER lumen, or the ER membrane depending on the location of transmembrane domains and the stop-transfer signals. Two different peroxisome targeting signals, one located at the C-terminus (sequence SLK termed PTS1) and the other located at the N-terminus (R/KL/I/VX₅H/QL/A, termed PTS2) have been identified in yeast and mammalian cells [8,9]. The signal containing proteins bind to their respective cytosolic chaperones which escort the cargo to the docking complexes on the peroxisome membrane. This is the critical step for protein translocation into the peroxisomal matrix. Proteins translocated to the nuclear compartment contain a stretch of 7-12 positively charged amino acids with prototypical targeting signal of PKKKRKV, or a variation there of, called Nuclear Localization Signal (NLS) [10]. The proteins are translocated to the nucleoplasm through the nuclear membrane pores with the help of a set of proteins called importins. The protein translocation is an energy driven process which requires the hydrolysis of ATP or GTP. Fully folded proteins are translocated into the nucleus by this mechanism [11]. The nuclear protein translocation is a gated transport which is fundamentally different from the membrane translocation mechanism seen in the ER, mitochondria and peroxisome targeting. Some proteins lacking either peroxisomal or nuclear targeting signals can enter these organelles on a “piggy back” basis in association with other signal containing proteins [12]. The mitochondrial targeting signals are quite distinct, and are characterized by a stretch of hydrophobic and positively charged residues present either at the N-terminus or at the interior positions of the proteins [13, 14]. For a majority of proteins targeted to the mitochondrial matrix, TCA cycle proteins and other proteins associated with mitochondrial metabolism, the N-terminal signal is clipped after the protein enters the matrix compartment. Although the mitochondrial signal was initially predicted to be an N-terminal amphipathic helix, mitochondrial signals with β-sheet, and even unstructured secondary structure have been reported [6, 15]. For several proteins targeted to mitochondrial inner membrane and intermembrane space N-terminal or internal uncleaved signals have also been reported [16]. The currently held view is that the spacing and positions of the positively charged residues are critical for the signal function [17]. As opposed to the nuclear protein import, only unfolded nascent polypeptides are imported through the mitochondrial outer and inner membrane receptor complexes and also through ER membrane. [18]. Also, the entry of nascent chains into the matrix compartment requires adequately developed transmembrane potential and ATP as an energy source. Based on the known characteristics of mitochondrial import channels, it is unlikely that “piggy back import” or assisted import of proteins associated with the primary cargo occurs in the mitochondrial protein import pathway.

Since Blobel's signal hypothesis was proposed nearly four decades ago, several studies have shown that a number of proteins are localized in more than one subcellular location. For example, several signaling and other proteins that are normally found in the cytosol and nucleus are also localized in mitochondria [see accompanying review by Yogev et al., 19]. These include: the mitochondrial cysteine desulfurase, Nfs1; the main adenylate kinase, Aky2 [19] ; and the DNA repair enzyme, apurinic/apyrimidinic endonuclease I (Apn I) [20]. Several mammalian and unicellular eukaryotic proteins are also targeted bimodally to at least two distinct cellular compartments. These proteins include: the catalytic subunit of protein kinase A (PKA) [21], glutathione S- transferase (GST-A4-4) [see review by H. Raza, 22], NADH- cytochrome b5 reductase [23], cytochrome P450 enzymes [24], Alzheimer's amyloid precursor protein (APP) [25], α-synuclein [26], PKC isoforms, Akt, GSK3 [27], STAT 3 [28], acetyl CoA desaturase, Mia4, acyl CoA synthase, hydroxymethylglutaryl CoA lyase [29-31], proteins involved in oncogensis and apoptosis including p53, NFkB, Bcl XL, Bcl2 [32,33], nuclear receptors such as estrogen receptor, T3, GR, and RxR [34], and others. Accumulation of mammalian prion protein (PrP) in the cytosol due to temporary halt of its translocation to ER under stress has been suggested as a mechanism for the production of infectious prion particles (35). Similarly bimodal targeting of APP protein to mitochondria, leading to mitochondrial dysfunction, may be a contributing factor in Alzheimer's disease [25]. Recent proteomic studies suggest that more than 1000 proteins, many of which may lack canonical cleavable targeting signals may be translocated to mitochondria [36]. Recently the Pines group [37] suggested that nearly one third of the yeast mitochondrial proteins are also localized in other cellular compartments.

Utilizing protein purification and protein/peptide sequencing initially we observed that several of the ER associated CYPs, such as CYP1A1, 2B1, and 2E1 are also located inside the inner membrane compartment of mitochondria [38-46], most part exposed to the matrix. Furthermore, chronic treatment of rats with β-naphthoflavone (BNF) or Phenobarbital (PB), resulted in preferential clustering of +33/1A1 (N-terminal truncated CYP1A1) and intact CYP 2B1, respectively, in the mitochondrial compartment of the liver and brain. The mitochondrial -CYPs become the major part of the tissue pool, ranging up to ∼30% [45, 47, 48]. Since these initial observations, a number of investigators have observed CYP1A1, CYP2B1/2, CYP2D6, 3A1/2 and 2E1 forms in the mitochondrial compartments of xenobiotic-treated rat liver, lung and brain, or untreated monkey and human brains [44-57, See Table 1]. We proposed the concept of chimeric signal to account for the bimodal targeting of these echoproteins to ER and mitochondria. This review will focus primarily on the nature of chimeric signals of CYPs and the mechanism of their mitochondrial import. The review will also summarize pathophysiological significance of mitochondria-targeted CYPs.

Table 1. List of Microsomal CYPs Bimodally Targeted to Mitochondria.

CYPs Targeted	Molecular forms	References
CYP1A1	N-terminal truncated	38-50,
CYP1A2	unknown	44, 50
CYP1B1	unknown	87 **
CYP2B1	intact/phosphorylated	39, 40, 43, 50, 85
CYP2E1	intact/phosphorylated	40, 51, 52-56, 77, 85, 86
CYP2D6	intact/phosphorylated	47, 48, 87
CYP2C11	Unknown	50, 44
CYP2C6	Intact/phosphorylated	**
CYP3A2	unknown	44, 50, **
CYP4A1	unknown	44, 50

The table lists the full length and truncated mc-CYPs targeted to mitochondrial compartment. The references are listed in the List of References at the end of the article.

^**refers to unpublished data from the authors' laboratory. The precise signal domains for CYP1A1, 2B1, and 2D6 have been shown in Figure 1.

General functions of Cytochrome P450s

The cytochrome P-450s (CYPs) belong to a multi-gene family of heme proteins, which catalyzes the metabolism of a wide variety of endogenous as well as exogenous substrates including lipids, lipophilic drugs, and xenobiotic chemicals. They play a central role in modulating the pharmacokinetic and pharmacogenetic parameters of drugs and pharmaceutical products. These enzymes are also involved in the metabolism of toxic chemicals, as well as physiological substrates such as arachidonic acid, eicosanoids, cholesterol and steroids, bile-acids, vitamin D_3, and retinoic acid [58, 59]. CYPs belonging to different families show only a marginal to low sequence divergence, although they show a remarkable difference in substrate specificity and catalytic properties. Structurally, CYPs belonging to different families show a high conservation of general topography and structural fold [60]. The most highly conserved region among all the CYPs is the core of the protein around the heme moiety. This reflects a common mechanism of electron transfer and oxygen activation [61]. The two most variable regions are the substrate recognition site and the amino-terminal anchoring, and or, targeting signal domain [61].

CYPs catalyze more than 20 different types of reactions including carbon hydroxylation; heteroatom oxygenation; heteroatom release or dealkylation; and epoxidation. Some of the more unusual reactions catalyzed by CYPs are: reduction; desaturation; oxidative ester cleavage; ring expansions; and ring formation [62]. The catalytic function of CYPs requires NADPH as the source of electrons and appropriate electron transfer proteins which donate electrons from NADPH to CYP protein [61]. Recent studies suggest that CYPs are functionally associated with olfaction, cardiovascular function, neuronal function, digestion and others [63,64]. Furthermore, CYP expression is altered in pathophysiological states including tumorigenesis, cardiovascular injury, renal and pulmonary diseases, neuronal dysfunction and diabetes.

Mechanisms of targeting of canonical ER-specific and mitochondria-specific CYPs

Endoplasmic reticular (routinely called microsomal) CYPs are integral membrane proteins whose targeting is consistent with the postulates of Blobel's ER targeting signal hypothesis. The microsomal CYPs are anchored on the ER membrane through a single transmembrane domain located close to the N-terminus. This domain in many CYP proteins also contains a ER targeting domain and stop transfer signal. The Pro-rich regions around amino acid residues 35-40 in different microsomal CYPs enables proper folding of the cytosol exposed catalytic domain of the protein. CYPs are translated on membrane-free ribosomes in the cytosol and, as soon as the hydrophobic NH₂-terminus emerges from the ribosome, the translation is halted through the binding of the signal recognition particle (SRP) [65]. The hydrophobic signal sequence binds to the 54 kDa subunit of the SRP, which contains 7 protein subunits and a 7S RNA molecule [6, 7] and is then transported to the ER membrane. Translation resumes when the complex composed of the ribosome, nascent chain, and SRP is bound to the SRP receptor present in the ER membrane. SRP is then released and the ribosome docks onto the cytosolic side of the ER membrane. Nascent chains are inserted into the translocation channel, the sec61 complex, as they emerge from the ribosome. This allows completion of polypeptide translation and translocation to occur [66]. As soon as the hydrophobic residues have passed through the ER membrane, the translocation halts and the remainder of the protein is translated at the cytoplasmic side of the ER membrane [65, 67, 68]. Once translocation is stopped, the signal sequence moves laterally out of the sec61 pore into the lipid bilayer.

CYPs are retained in the ER membrane by either direct retention, in which the proteins are restricted to the ER, or retrieval, in which the proteins are transported out of the ER to an intermediate pre-Golgi fraction and returned to the ER by a receptor-mediated process [69, 70]. Direct retention of CYPs requires the signal sequence, which, in addition to its targeting function, also serves as a stop transfer signal to halt the translocation of the protein through the membrane. The transmembrane domains of many CYPs have been shown to contain stop-transfer domains [71]. The mechanism of retrieval remains unclear since CYPs do not have any identifiable retrieval signals.

Mitochondrial CYPs involved in the physiological pathways of cholesterol, sterol and vitamin D3 metabolism such as CYP27A, CYP24A, CYP11A and CYP11B, have canonical and cleavable mitochondrial targeting signals at their N-termini that are distinctly different than the signal sequences of microsomal CYPs. These CYPs are translated on membrane-free ribosomes in the cytosol as pre-proteins with a cleavable pre-sequence at their amino-terminus. The proteins are post-translationally targeted to the mitochondria. A recent study investigated the import of mitochondrial CYP27A1 in detail and found that it requires the cytosolic chaperone Hsp70, but not Hsp90, for initial targeting to the mitochondria, and that it requires all three of the receptor proteins in the TOM complex, TOM70, TOM20, and TOM22, for translocation across the mitochondrial outer membrane [70]. After translocation of the preprotein into the matrix, and N-terminal processing, the mature CYPs then associate with the inner membrane as a membrane extrinsic protein with the catalytic domain exposed to the matrix.

Mechanisms of bimodal targeting of CYPs to the ER and mitochondria

As briefly stated in the Introduction, numerous proteins are targeted to two or more separate subcellular destinations. Table 1 shows a list of CYP proteins bimodally targeted to the ER and mitochondria. The signal hypothesis elegantly predicted the targeting mechanism of proteins that are localized in the ER, mitochondria, or other organelles. However, it did not predict how the same protein may be targeted to more than one cellular destination. We proposed the chimeric signal hypothesis to account for the bimodal targeting properties of various CYP and other proteins [24, 40-43]. The chimeric signals of CYP1A1, 2B1, 2E1 and 2D6 which drive the protein targeting to ER and mitochondria are shown in Figure 1. By definition, the chimeric signals of CYPs consist of an ER targeting signal at the N-terminus, which is also part of the transmembrane anchor region, flanked by a cryptic mitochondria targeting signal located at amino acid residues 20-36 in different CYP proteins (Figure 1). [41-44,47, 48, 51,52]. The mitochondrial targeting signal regions (32 to 44 amino acid residues for CYP1A1 and 20 to 33 amino acid residues for CYP1B1, 2E1 and 2D6) characteristically contain 2-5 positively charged residues that are critical for mitochondrial import (Figure 1). The cryptic mitochondrial targeting signals of chimeric signals are difficult to identify by using computer algorithms. The cryptic mitochondria targeting signal requires activation by post-translational modification, which is either sequence-specific processing by a cytosolic endoprotease, as in the case of CYP1A1 (Figure 1A and 2A), or PKA-mediated phosphorylation, as in the case of CYP2B1, CYP2E1, and CYP2D6 (Figure 1B and Figure 2B) [43, 39, 52, 58]. Notably, none of the chimeric signal sequences of the CYP proteins imported into mitochondria are cleaved by the matrix metalloprotease. In the case of CYP1A1 the cleavage occurs in the cytosol before the protein enters mitochondria. A minor cleavage site is located between amino acid residues 4 and 5 and a major cleavage site is located between residues 32 and 33 (see Figure 1A).

Figure 1.

Chimeric signals of CYP echoproteins that are bimodally targeted to mitochondria and endoplasmic reticulum. A, The ER and mitochondria targeting domains, the endoprotease processing sites (between the 4^th and 5^th residies and between 32^nd and 33^rd residues) of CYP1A1, and the PKC phosphorylation site which alters its SRP binding affinity are shown. B, Alignment of signal sequences of CYP2B1, and 2D6 showing different signal domains and the cryptic mitochondrial targeting signals. Also shown are the conservation of PKA phosphorylation sites between residues 128 to 135 of CYP2B1, and 2D6. The Pro-rich region in all cases helps proper folding of the catalytic domain which mostly faces the cytosolic side of the ER.

Figure 2.

Models showing distinct modes of signal activation and mitochondrial targeting of CYPs belonging to different families. A; mechanism of targeting of CYP1A1. The cartoon shows that nascent chains which escape SRP binding are translated as membrane free proteins and prime candidates for mitochondrial translocation while the nascent chains that are bound to SRP are translocated to the ER. The model proposes that nascent chains which escape SRP binding are cleaved by a cytosolic Ser protease in sequence specific manner to activate the cryptic mitochondria signal of CYP1A1. These nascent chains that are translated as membrane free proteins are translocated to mitochondria with the help of HSP70 and HSP90 chaperones. B: mechanism of mitochondria targeting of CYP2B1 and other family 2 CYPs. Under low PKA conditions, when the nascent chains are not phosphorylated, SRP binds to the nascent chain and the passenger protein is guided to the ER membrane for translocation. Under high PKA conditions, phosphorylation reduces the affinity of nascent chains for SRP and the protein is translated as membrane free protein. The translated protein is then guided to mitochondria for importation by HSP70 and HSP90 chaperones.

In contrast to the canonical mitochondrial targeting signal, which is considered to be a structural element that binds to chaperones and receptors facilitating the targeting, the chimeric signal is more dynamic in nature-----it targets the protein to mitochondria or ER depending upon the physiologic demand of the cell. The cryptic mitochondria-targeting signals can either be activated by cytosolic proteases or protein kinases in the cytosol which in turn activate the cryptic mitochondrial targeting signal [71]. Protease protection and bicarbonate extraction experiments have shown that, once the chimeric signal-containing CYPs are inside the mitochondria, they associate with the inner membrane in a membrane-extrinsic orientation [40, 46, 72]. It is interesting that the orientation is the same irrespective of the presence (CYP2D6, CYP2E1 and CYP2B1) or absence (CYP1A1) of the N-terminal trans-membrane domain, and that this orientation is similar to that of the mitochondrial CYPs with canonical targeting signals.

Mechanism 1: Endoproteolytic processing of the passenger protein for activation of the cryptic signal

When CYP1A1 was first purified from mitochondria from β-napthoflavone (BNF)-induced rat liver, it was found to be present in two forms, +5/1A1 which lacks the N-terminal 4 amino acid residues and +33/1A1, which lacks the N-terminal 32 amino acid residues [41]. Additional in vitro studies demonstrated that +5/1A1 form was targeted to mitochondria at a relatively low level, while the +33/1A1 form was targeted at a high level. Furthermore the +33/1A1 was found in much higher abundance than the +5/1A1. The amino acid sequence at positions 33 to 40 exhibited properties expected of a mitochondrial targeting signal with respect to α-helicity and spatial distribution of positively charged amino acid residues (Figure 1). Substitutions of the positively charged residues at positions 34 and 39 essentially eliminated the mitochondrial targeting of +33/1A1. In addition, incubation of full-length CYP1A1 translation product with rat liver cytosol fraction converted CYP1A1 into two products that resembled +5/1A1 and +33/1A1 [41]. Together these results suggested that the mitochondrial targeting of CYP1A1 requires endoproteolytic cleavage by a cytosolic protease, which removes the 32 amino acids from the N-terminus and thereby activates the cryptic mitochondrial targeting signal located between residues 33-40 (Figure 2A) [41]. In further support of this mechanism, +5/1A1 and +33/1A1 were also purified from BNF-induced rat brain mitochondria, which indicates that the endoprotease processing sites are specific and that this is a general mechanism that occurs in multiple organs [45].

To further investigate the nature of the chimeric signal and the requirement for endoproteolytic cleavage, the N-terminal signal sequence (residues 1-44) of CYP1A1 as well as the processed sequence with the activated mitochondrial targeting signal (residues 33-44) were fused to both the cytosolic protein, DHFR, and the mature portion of rat CYP27A1, a mitochondrial CYP, and the targeting of the fusion proteins was investigated [74]. The N-terminally fused 1-44 signal sequence of CYP1A1 targeted the heterologous proteins, DHFR and mature portion of CYP27 to both ER and mitochondria, whereas the 33-44 sequence functioned strictly as a mitochondrial targeting signal. Site-specific mutations of the positively charged residues at the 34^th and 39^th positions eliminated the mitochondrial targeting without significantly affecting the ER targeting [42].

The endoprotease responsible for the activation of the N-terminal targeting signal of CYP1A1 was purified from the liver cytosol of BNF-treated rats and found to be a member of the Ser-protease family. This enzyme is a dimer of 90 kDa and 40 kDa subunits, designated p90 and p40, each containing Ser protease domains [74]. LC/MS/MS analysis demonstrated that p90 is the rat homolog of human Ser protease 9, while p40 is a Ser protease 9-like protein that shows homology to a splice variant of this protease. Both subunits are important for the processing activity of the endoprotease [74]. The processing of CYP1A1 is sequence specific -- both the core cleavage site (+32/+33) and the flanking amino acids (30-VRVTRT-35) are required for processing [74]. Interestingly, this protease was also found to process p53 (PAGSR), GR (ILLDFSK), RXR (ASFTK), and CYP1B1 (RLLRQR) which all carry Ser-protease consensus sites, shown in parentheses, and the processed form of each protein targets to mitochondria [74 and our unpublished data].

Mechanism 2: Role of protein phosphorylation in bimodal targeting of CYPs to mitochondria

PKA-mediated phosphorylation has been shown to activate the cryptic mitochondrial targeting signal in CYP2B1, CYP2E1, and CYP2D6 (Figure 1B) [43, 51, 52]. Initial studies employing protein fingerprint analysis and sequencing of peptides or MS/MS analysis of tryptic fragments showed that mitochondria imported CYP2B1 represent full length proteins. More recent studies also show that nearly intact CYP2D6 and protein is imported into mitochondria [47, 48]. In all cases, immuno-detection using anti Ser-phosphate antibody demonstrated that the mitochondrial proteins were phosphorylated to a higher level than the microsomal proteins [43, 47, 52]. LC/MS analysis identified Ser-129 as the site of phosphorylation of CYP2E1 [51]. CYP2E1 and 2B1 contain single PKA phosphorylation sites at Ser129 and Ser128, respectively. CYP2D6 contains a high consensus PKA target site at Ser 135, (Figure 1B) and potential sites of lower consensus at Ser148 and Ser 217 (not shown) [47, 48]. Mechanism of targeting of CYP2E1 has been discussed in details in the accompanying review by Knockaert et al.

Interestingly, phosphorylation by cytosolic PKA increased mitochondrial translocation under in vitro conditions and increased mitochondrial accumulation in whole cells transiently transfected with CYP cDNAs in all three cases (Figure 2B). PKA-mediated phosphorylation of CYP2B1 markedly reduced interaction of nascent chains with SRP and the phosphorylation site mutant (S128A). CYP2B1 was poorly imported into mitochondria both under in vitro (with isolated mitochondria) and in vivo (whole cell) conditions [43]. In the case of CYP2D6, PKA mediated phosphorylation increased mitochondrial targeting, although mutation at Ser135 only partly inhibited the import. It is likely that other less consensus PKA sites at Ser 148 and/or Ser 217 may also play a role [47,48]. Another notable difference with CYP2D6 was that it also contains a PKC consensus site at Tyr138 (RRFSVSTLR), whose role in the activation of the cryptic mitochondria targeting signal remains unclear. Calculation of the association constant for CYP2B1 binding to SRP demonstrated that the N-terminal chimeric signal of CYP2B1 has a significantly higher affinity for SRP than that of CYP2E1, and that phosphorylation reduces the interaction between CYP2B1 and SRP (see Figure 2B). The SRP binding affinity of CYP2D6, as tested by membrane flotation assay, was reduced by PKA mediated phosphorylation of nascent chains [48]. This suggests that, similar to that observed with CYP2B1, phosphorylation modulates SRP binding affinity and thus mitochondrial targeting of CYP2D6 (Figure 2B).

While the upstream effects of phosphorylation on the interaction with SRP and cytosolic Hsp70 (heat shock protein 70) are different for each of the proteins, the downstream results, at the level of the mitochondria, are similar in that they increase the interaction between the CYPs and the mitochondrial translocase proteins. Cross-linking studies showed that phosphorylated CYP2B1 interacted strongly with yeast TOM 40, TIM44, and matrix Hsp70 during in vitro mitochondrial import, but their respective phosphorylation site mutant, S128A/2B1, had no interaction with the mitochondrial translocase proteins [43]. Mitochondrial targeting studies with CYP1A1 and three family II CYPs have prompted a general hypothesis that SRP binding to nascent chain is a major regulatory step in the bimodal protein targeting process. The nascent chains that escape SRP binding because of either inherent lower affinity (CYP2E1), or lowering of affinity by PKC-mediated phosphorylation (CYP1A1) [42] or PKA-mediated phosphorylation (CYP2B1 and CYP2D6) are translated as soluble proteins and become prime candidates for mitochondrial translocation (see Figure 2B).

This hypothesis was tested by altering the hydrophobicity of the N-terminal SRP recognition domain which corresponds to the N-terminal 25-30 amino acid stretch of most microsomal -CYPs. In order to test whether modulation of SRP binding could alter the mitochondrial or microsomal targeting levels of CYP2D6 in the cell, we generated mutant constructs that were predicted to have either increased affinity (+SRP) or decreased affinity (-SRP) for the chaperone. SRP binds preferentially to sequences that are more hydrophobic and α-helical [75,76]. Therefore, we increased hydrophobicity of the ER targeting signal to increase the affinity for SRP, and decreased hydrophobicity of the signal to decrease the affinity for SRP, while maintaining the α-helicity of this region [42, 48, 77]. The effect of these mutations on the binding of SRP to CYP2D6 was tested using an ER membrane integration assay. Wild-type CYP2D6 integrated efficiently into the ER membrane, with approximately 60% of the total protein associated with the membrane. Mutation of the ER targeting signal to increase hydrophobicity in +SRP/2D6 greatly increased the efficiency of ER membrane integration, with approximately 95% of the total protein associated with the membrane. Likewise, the decreased hydrophobicity of the ER targeting signal in–SRP/2D6 mutant greatly decreased the integration of the protein into the membrane with just 25% of the total CYP2D6 protein associated with the membrane. This strategy was used to generate stable cell lines expressing mostly mitochondria targeted or mostly microsome targeted CYP2D6 [47, unpublished] and CYP2E1 [see accompanying review by M-A. Robin]. Using a similar rationale, Nebert's group [49] has transgenically knocked in mutations at the putative targeting domain and generated mice expressing mostly mt-CYP1A1 and mostly mc-CYP1A1 for use in toxicity studies. These results suggest that the chimeric signal is dynamic and possibly evolved from a common ancestor. The stable cell lines and transgenic animals developed using the signal remodeling strategy, present valuable models for investigating the role of mt-CYPs in drug metabolism and toxicity as described later in this review.

The factors that modulate the microsomal and mitochondrial targeting of CYPs can be important targets for controlling rates of drug metabolism and toxicity in animal and humans. In studies with chimeric signal-containing CYP proteins, we believe that affinity of the nascent chains for SRP binding represents a critical regulatory point which determines the level of mitochondrial targeting of these proteins. Support for this possibility comes from our studies showing that PKC mediated phosphorylation of CYP1A1 at T35 or PKA mediated phosphorylation of CYP2B1, and 2D6 at Ser 128, and S135, respectively, decrease the affinity of nascent chains for SRP binding, thereby promoting higher mitochondrial targeting [42, 43, 52, 77]. Recently it was shown that PKA phosphorylation of TOM receptor proteins markedly increased import efficiency suggesting that this can also be an important point of regulation. A series of publications from the Hegde laboratory [78-80] showed reduced ER translocation of PrP protein under ER stress is probably because of reduced efficiency of the Sec61 complex and other ancillary factors involved in ER translocation. It is likely that ER stress conditions also indirectly promote mitochondrial translocation of chimeric signal-containing CYPs by way of increasing their levels in the cytosol.

Interaction of chimeric-signals of CYPs with the TOM complex

A recent study characterized the mechanism of delivery of chimeric signal containing CYPs to the peripheral and channel-forming TOM proteins during mitochondrial targeting. Interestingly, the different activated chimeric signals interact with different combinations of peripheral TOM proteins and, in some cases, can even bypass the peripheral proteins altogether and interact directly with TOM40 [71]. These experiments demonstrated that import of CYP +33/1A1 and CYP2B1 signals do not require any of the peripheral TOM proteins tested, which include TOM20, TOM22, and TOM70; whereas, import of CYP +5/1A1 and CYP2E1 requires TOM70 but not TOM20 or TOM22 [71].

Both Hsp70 and Hsp90 have been shown to be involved in pre-protein delivery and ATP-dependent translocation in mammalian cells, whereas, in yeast, Hsp70 is sufficient for pre-protein import [78, 79]. It has therefore been suggested that the pathway requiring Hsp90 in mammalian cells may represent a later stage of evolution of the import pathway [80]. The requirement of CYPc27 for Hsp70 alone may therefore represent a transition point from lower eukaryotes to mammals [71]. In contrast, the pathway for mitochondrial import of chimerical-signal containing CYPs may represent a much more recent evolutionary event, which is consistent with the increased metabolic and physiologic roles of mitochondria in mammalian cell function [71].

Physiological significance of bimodal targeting and the functions of mitochondrial targeted CYPs

An interesting feature of bimodally targeted mitochondrial CYPs with respect to their metabolic activity is their preference for the electron transport proteins. When localized in mitochondria, the metabolic activities of these CYPs were preferentially activated by mitochondrial ferredoxin and ferredoxin reductase (also termed Adx and Adr, but when localized in the ER the proteins were activated preferentially by cytochrome P450 reductase [CPR] [38-52, 81]. These results suggest an evolutionary adaptation of mammalian CYPs to both eukaryotic like microsomal Cytochrome P450 reductase and bacterial like Adx and Adr systems. These results also raise two important points regarding CYP structure and function: (1) different electron donor proteins may differently modulate the activities of CYPs; (2) different modes of interfacing with the electron donor protein, possibly affect the routes of electron flow, which may differently modulate the catalytic activities of mammalian CYP enzymes [81]. These aspects were discussed in a recent review [24].

Currently the physiological significance of the bimodal targeting mechanism and the role of mitochondrial localized CYPs in drug metabolism are under investigation in many laboratories (see the review by Robin M-A). A recent study used transgenic mice with knocked-in mutations in the N-terminal signal region that cause either mainly mtCYP1A1 or mainly mcCYP1A1 expression and then compared their role in reducing benzo(a)pyrene toxicity [49]. It remains to be seen whether xenobiotic metabolism by chimeric signal-containing CYP 2B1, 2E1 or 2D6 in the mitochondria will result in any discernible toxicity in the intact animal.

The participation of CYPs in the generation of reactive oxygen species (ROS) has also been identified as a significant source of cellular toxicity. ROS can directly modify cellular macromolecules leading to a variety of toxic effects including lipid peroxidation, protein dysfunction, nucleic acid oxidation, and cell death [82]. The production of electrophilic metabolites by CYPs can contribute to oxidative stress by depletion of reduced glutathione, a major cellular antioxidant, and by participation in redox cycling [83, 84].

A large number of proteins associated with mammalian mitochondria are likely to contain non-canonical signals. In the case of CYPs and other proteins that are bimodally targeted to ER and mitochondria, plasma membrane and mitochondria, or cytosol and mitochondria, the term “chimeric signal” was coined to illustrate a complex amalgamated signal that can drive the targeting of proteins to two different sub cellular compartments [24, 41-45]. Thus the targeting mechanisms reviewed here for the constitutive and inducible CYPs are likely to be applicable to a large number of mitochondria imported proteins lacking canonical import signals, that are also localized in extramitochondrial compartments.

The chimeric signals described for the bimodal targeting of CYPs to mitochondria and ER are not static structural entities, but are highly dynamic. They are activated either by sequence specific cleavage at the N-terminus immediately past the ER targeting domain for class I CYPs, or phosphorylation of the nascent protein close to the chimeric signal domain. Thus the steady state equilibrium for the translocation of apoproteins to the ER or mitochondria is markedly altered by the signal activation mechanisms. The observation that the cytosolic Ser-protease and various protein kinases, including PKA are activated by xenobiotics or physiological factors [24], point to the physiological significance of the bimodal protein targeting, and its regulation. In keeping with this hypothesis, mitochondrial contents of different CYPs including CYP2D6, CYP2E1 and 3A4 vary markedly such that the mt-CYPs represent nearly 50% of the tissue pool in some cases. These results provide a compelling reason for understanding mechanisms of mitochondria targeting of these and also other inducible and constitutive CYPs.

On a broader basis, characterization of chimeric signals of different proteins and the hypothesis that modulation of SRP binding efficiency as a key regulatory step of bimodal protein targeting has provided a valuable tool for genetically modulating signal activity, and thus modulating the steady state levels of these proteins in the two membrane compartments. This strategy was used for generating stable cell lines and mouse models predominantly expressing mitochondrial or predominantly microsomal -CYPs [42, 47, 49, 77]. These cell and animal systems should help address questions on the functional importance and pathological consequence of CYP targeted to the mitochondrial compartment.