Bimodal targeting of cytochrome P450s to endoplasmic reticulum and mitochondria: the concept of chimeric signals

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 protein kinase A or protein kinase C 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.     

Bimodal targeting of microsomal CYP2E1 to mitochondria through activation of an N-terminal chimeric signal by cAMP-mediated phosphorylation

Cytochrome P450 2E1 (CYP2E1) plays an important role in alcohol-induced toxicity and oxidative stress. Recently, we showed that this predominantly microsomal protein is also localized in rat hepatic mitochondria. In this report, we show that the N-terminal 30 amino acids of CYP2E1 contain a chimeric signal for bimodal targeting of the apoprotein to endoplasmic reticulum (ER) and mitochondria. We demonstrate that the cryptic mitochondrial targeting signal at sequence 21-31 of the protein is activated by cAMP-dependent phosphorylation at Ser-129. S129A mutation resulted in lower affinity for binding to cytoplasmic Hsp70, mitochondrial translocases (TOM40 and TIM44) and reduced mitochondrial import. S129A mutation, however, did not affect the extent of binding to the signal recognition particle and association with ER membrane translocator protein Sec61. Addition of saturating levels of signal recognition particle caused only a partial inhibition of CYP2E1 translation under in vitro conditions, and saturating levels of ER resulted only in partial membrane integration. cAMP enhanced the mitochondrial CYP2E1 (referred to as P450MT5) level but did not affect its level in the ER. Our results provide new insights on the mechanism of cAMP-mediated activation of a cryptic mitochondrial targeting signal and regulation of P450MT5 targeting to mitochondria.     

An unusual TOM20/TOM22 bypass mechanism for the mitochondrial targeting of cytochrome P450 proteins containing N-terminal chimeric signals

Previously we showed that xenobiotic-inducible cytochrome P450 (CYP) proteins are bimodally targeted to the endoplasmic reticulum and mitochondria. In the present study, we investigated the mechanism of delivery of chimeric signal-containing CYP proteins to the peripheral and channel-forming mitochondrial outer membrane translocases (TOMs). CYP+33/1A1 and CYP2B1 did not require peripheral TOM70, TOM20, or TOM22 for translocation through the channel-forming TOM40 protein. In contrast, CYP+5/1A1 and CYP2E1 were able to bypass TOM20 and TOM22 but required TOM70. CYP27, which contains a canonical cleavable mitochondrial signal, required all of the peripheral TOMs for its mitochondrial translocation. We investigated the underlying mechanisms of bypass of peripheral TOMs by CYPs with chimeric signals. The results suggested that interaction of CYPs with Hsp70, a cytosolic chaperone involved in the mitochondrial import, alone was sufficient for the recognition of chimeric signals by peripheral TOMs. However, sequential interaction of chimeric signal-containing CYPs with Hsp70 and Hsp90 resulted in the bypass of peripheral TOMs, whereas CYP27 interacted only with Hsp70 and was not able to bypass peripheral TOMs. Our results also show that delivery of chimeric signal-containing client proteins by Hsp90 required the cytosol-exposed N-terminal 143 amino acids of TOM40. TOM40 devoid of this domain was unable to bind CYP proteins. These results suggest that, compared with the unimodal mitochondria-targeting signals, the chimeric mitochondria-targeting signals are highly evolved and dynamic in nature.     

Mitochondrial Targeting of Cytochrome P450 (CYP) 1B1 and Its Role in Polycyclic Aromatic Hydrocarbon-induced Mitochondrial Dysfunction

We report that polycyclic aromatic hydrocarbon (PAH)-inducible CYP1B1 is targeted to mitochondria by sequence-specific cleavage at the N terminus by a cytosolic Ser protease (polyserase 1) to activate the cryptic internal signal. Site-directed mutagenesis, COS-7 cell transfection, and in vitro import studies in isolated mitochondria showed that a positively charged domain at residues 41–48 of human CYP1B1 is part of the mitochondrial (mt) import signal. Ala scanning mutations showed that the Ser protease cleavage site resides between residues 37 and 41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that was attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene. In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (adrenodoxin) and ferredoxin reductase showed high aryl hydrocarbon hydroxylase activity. Administration of benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzodioxin induced similar mitochondrial functional abnormalities and oxidative stress in the lungs of wild-type mice and Cyp1a1/1a2-null mice, but the effects were markedly blunted in Cyp1b1-null mice. These results confirm a role for CYP1B1 in inducing PAH-mediated mitochondrial dysfunction. The role of mitochondrial CYP1B1 was assessed using A549 lung epithelial cells stably expressing shRNA against NADPH-cytochrome P450 oxidoreductase or mitochondrial adrenodoxin. Our results not only show conservation of the endoprotease cleavage mechanism for mitochondrial import of family 1 CYPs but also reveal a direct role for mitochondrial CYP1B1 in PAH-mediated oxidative and chemical damage to mitochondria.     

Mitochondrial trafficking of APP and alpha synuclein: Relevance to mitochondrial dysfunction in Alzheimer's and Parkinson's diseases

Mitochondrial dysfunction is an important intracellular lesion associated with a wide variety of diseases including neurodegenerative disorders. In addition to aging, oxidative stress and mitochondrial DNA mutations, recent studies have implicated a role for the mitochondrial accumulation of proteins such as plasma membrane associated amyloid precursor protein (APP) and cytosolic alpha synuclein in the pathogenesis of mitochondrial dysfunction in Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. Both of these proteins contain cryptic mitochondrial targeting signals, which drive their transport across mitochondria. In general, mitochondrial entry of nuclear coded proteins is assisted by import receptors situated in both outer and inner mitochondrial membranes. A growing number of evidence suggests that APP and alpha synclein interact with import receptors to gain entry into mitochondrial compartment. Additionally, carboxy terminal cleaved product of APP, ～ 4 kDa Abeta, is also transported into mitochondria with the help of mitochondrial outer membrane import receptors. This review focuses on the mitochondrial targeting and accumulation of these two structurally different proteins and the mode of mechanism by which they affect the physiological functions of mitochondria.     

Trypanosome Alternative Oxidase Possesses both an N-Terminal and Internal Mitochondrial Targeting Signal

Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of Trypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria in vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a T. brucei mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.     

The mitochondrial import receptor Tom70: identification of a 25 kDa core domain with a specific binding site for preproteins

The mitochondrial import receptor of 70 kDa, Tom70, preferentially recognizes precursors of membrane proteins with internal targeting signals. We report the identification of a stably folded 25 kDa core domain located in the middle portion of Tom70 that contains two of the seven tetratricopeptide repeat motifs of the receptor. The core domain binds non-cleavable and cleavable preproteins carrying internal targeting signals with a specificity indistinguishable from the full-length receptor. Competition studies indicate that both types of preproteins interact with overlapping binding sites of the core domain and that at least one additional interaction site is present in the full-length receptor. We suggest a model of Tom70 function in import of membrane proteins whereby a hydrophobic preprotein concomitantly interacts with several binding sites of the receptor.     

Identification and characterization of a mitochondrial targeting signal in rat cytochrome P450 2E1 (CYP2E1)

Cytochrome P450 2E1 (CYP2E1) lacking the hydrophobic NH(2)-terminal hydrophobic transmembrane domain is specifically targeted to mitochondria, where it is processed to a soluble and catalytically active form (Delta2E1) with a mass of about 40 kDa. Small amounts of Delta2E1 were also observed in mitochondria isolated from rat liver, indicating that this form of CYP2E1 is also present in vivo. In the present study the mitochondrial targeting signal was identified and characterized by the use of several NH(2)-terminally truncated and mutated forms of CYP2E1 that were expressed in the mouse H2.35 hepatoma cell line. Two potential mitochondrial targeting sequences were identified in the NH(2) terminus of CYP2E1. Deletion of the first potential mitochondrial targeting sequence located between amino acids 50 and 65, as in Delta(2-64)2E1, still resulted in mitochondrial targeting and processing, but when, in addition to the first, the second potential mitochondrial targeting sequence located between amino acids 74 and 95 was also deleted, as in Delta(2-95)2E1, the mitochondrial targeting was abolished. Mutation of the four positively charged Arg and Lys residues present in this sequence to neutral Ala residues resulted in the abrogation of mitochondrial targeting. Deletion of a hydrophobic stretch of amino acids between residues 76 and 83 also abolished mitochondrial targeting and import. Once imported in the mitochondria, these constructs were further processed to the mature protein Delta2E1. It is concluded that mitochondrial targeting of CYP2E1 is mediated through a sequence located between residues 74 and 95 and that positively charged residues as well as a hydrophobic stretch present in the beginning of this sequence are essential for this process.     

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

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 (+ 5 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.     

Functions of outer membrane receptors in mitochondrial protein import

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins and are imported into mitochondria. The targeting signals for mitochondria are encoded in the presequences or in the mature parts of the precursor proteins, and are decoded by the receptor sites in the translocator complex in the mitochondrial outer membrane. The recently determined NMR structure of the general import receptor Tom20 in a complex with a presequence peptide reveals that, although the amphiphilicity and positive charges of the presequence is essential for the import ability of the presequence, Tom20 recognizes only the amphiphilicity, but not the positive charges. This leads to a new model that different features associated with the mitochondrial targeting sequence of the precursor protein can be recognized by the mitochondrial protein import system in different steps during the import.     

A receptor for protein import into potato mitochondria

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.     

MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19.

Recognition of targeting signals is a crucial step in protein sorting within the cell. So far, only a few components capable of deciphering targeting signals have been identified, and insights into the chemical nature of the interaction between the signals and their receptors are scarce. Using highly purified mitochondrial outer membrane vesicles, we demonstrate that MOM22 and MOM19, components of the protein import complex of the outer membrane, bind preproteins at the mitochondrial surface in a reversible fashion. Interaction specifically and directly occurs with the N-terminal presequence and is abolished after inactivation of either MOM22 or MOM19. Binding is salt sensitive, suggesting that recognition involves electrostatic forces between the positive charges of the presequence and the acidic cytosolic domain of MOM22. MOM19 and MOM22 can be cross-linked with high efficiency. We propose that the two proteins form a complex which functions as the presequence receptor at the mitochondrial surface and facilitates the movement of preproteins into the translocation pore.     

Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bbeta2 regulatory subunit of protein phosphatase 2A

Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B', and B') subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bbeta regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, Bbeta2, targets PP2A to mitochondria to promote apoptosis in PC12 cells (Dagda, R. K., Zaucha, J. A., Wadzinski, B. E., and Strack, S. (2003) J. Biol. Chem. 278, 24976-24985). Here, we report that Bbeta2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of Bbeta2. When fused to green fluorescent protein, the N terminus of Bbeta2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length Bbeta2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal beta-propeller of Bbeta2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and beta-propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of Bbeta2, which also requires association with the rest of the PP2A holoenzyme.     

Isolation and identification of a novel mitochondrial metalloprotease (PreP) that degrades targeting presequences in plants

Most of the nuclear encoded mitochondrial precursor proteins contain an N-terminal extension called the presequence that carries targeting information and that is cleaved off after import into mitochondria. The presequences are amphiphilic, positively charged, membrane-interacting peptides with a propensity to form alpha-helices. Here we have investigated the proteolysis of the presequences that have been cleaved off inside mitochondria. A presequence derived from the overexpressed F(1)beta subunit of the ATP synthase and specific synthetic fluorescent peptides (Pep Tag Protease assay) have been shown to undergo rapid degradation catalyzed by a matrix located protease. We have developed a three-step chromatographic procedure including affinity and anion exchange chromatography for isolation of the protease from potato tuber mitochondria. Two-dimensional gel electrophoresis of the isolated proteolytically active fraction followed by electrospray ionization-mass spectrometry/mass spectrometry and data base searches allowed identification of the presequence peptide-degrading protease in Arabidopsis thaliana data base as a novel mitochondrial metalloendoprotease with a molecular mass of 105 kDa. The identified metalloprotease contains an inverted zinc-binding motif and belongs to the pitrilysin family.     

Identification of a cryptic N-terminal signal in Saccharomyces cerevisiae peroxisomal citrate synthase that functions in both peroxisomal and mitochondrial targeting

Saccharomyces cerevisiae has three distinct citrate synthases, two located in mitochondria (mature Cit1p and Cit3p) and one in peroxisomes (mature Cit2p). While the precursor of the major mitochondrial enzyme, Cit1p, has a signal for mitochondrial targeting at its N-terminus (MTS), Cit2p has one for peroxisomal targeting (PTS1) at its C-terminus. We have previously shown that the N-terminal segment of Cit2p is removed during import into peroxisomes [Lee, H.S. et al. (1994) Kor. J. Microbiol. 32, 558-564], which implied the presence of an additional N-terminal sorting signal. To analyze the function of the N-terminal region of Cit2p in protein trafficking, we constructed the N-terminal domain-swapped versions of Cit1p and Cit2p. Both fusions, Cit1::Cit2 and Cit2::Cit1, complemented the glutamate auxotrophy caused by the double-disruption of the CIT1 and CIT2 genes. In addition, part of the Cit2::Cit1 fusion protein, as well as Cit1::Cit2, was shown to be transported into both mitochondria and peroxisomes. The subcellular localization of the recombinant fusion proteins containing various N-terminal segments of Cit2p fused to a mutant version of green fluorescent protein (GFP2) was also examined. As a result, we found that the 20-amino acid N-terminal segment of Cit2p contains a cryptic cleavable targeting signal for both peroxisomes and mitochondria. In addition, we show that the peroxisomal import process mediated by the N-terminal segment of Cit2p was not affected by the disruption of either PEX5 (encoding PTS1 receptor) or PEX7 (encoding PTS2 receptor).     

Roles of Tom70 in Import of Presequence-containing Mitochondrial Proteins

Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.     

N terminus of calpain 1 is a mitochondrial targeting sequence

The ubiquitous m- and mu-calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, mu-calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of mu-calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form mu-calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic alpha-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of mu-calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.     

Multiple 40-kDa heat-shock protein chaperones function in Tom70-dependent mitochondrial import

Mitochondrial preproteins that are imported via the translocase of the mitochondrial outer membrane (Tom)70 receptor are complexed with cytosolic chaperones before targeting to the mitochondrial outer membrane. The adenine nucleotide transporter (ANT) follows this pathway, and its purified mature form is identical to the preprotein. Purified ANT was reconstituted with chaperones in reticulocyte lysate, and bound proteins were identified by mass spectrometry. In addition to 70-kDa heat-shock cognate protein (Hsc70) and 90-kDa heat-shock protein (Hsp90), a specific subset of cochaperones were found, but no mitochondria-specific targeting factors were found. Interestingly, three different Hsp40-related J-domain proteins were identified: DJA1, DJA2, and DJA4. The DJAs bound preproteins to different extents through their C-terminal regions. DJA dominant-negative mutants lacking the N-terminal J-domains impaired mitochondrial import. The mutants blocked the binding of Hsc70 to preprotein, but with varying efficiency. The DJAs also showed significant differences in activation of the Hsc70 ATPase and Hsc70-dependent protein refolding. In HeLa cells, the DJAs increased new protein folding and mitochondrial import, although to different extents. No single DJA was superior to the others in all aspects, but each had a profile of partial specialization. The Hsp90 cochaperones p23 and Aha1 also regulated Hsp90-preprotein interactions. We suggest that multiple cochaperones with similar yet partially specialized properties cooperate in optimal chaperone-preprotein complexes.