Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal

Abstract

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH 2 -terminal-truncated molecular species, ؉91A1 and ؉331A1. Mutations Pro-2 3 Leu and Tyr-5 3 Leu, which increase the signal recognition particle ( Proteins targeted to mitochondria contain either NH 2 -terminal or internal signals, which include amphipathic ␣-helices, ␤-sheet, and random structures with spaced positively charged residues (1-3). A majority of the NH 2 -terminally located mitochondrial-targeting signals are cleaved by the mitochondrial matrix metalloprotease following their import, although in a number of cases the signals remain uncleaved (1-4). Recent proteomic studies indicate that yeast mitochondria contain as many as 800 proteins while the rodent heart and liver mitochondria may contain well over 1500 proteins (5-8). It is estimated that Ͼ50% of mitochondria-associated proteins in both yeast and mammalian cells lack canonical MT 4 -targeting signals, and the precise mode of targeting of these proteins as well as mechanism of their translocation across the MT membranes remain unclear. The bimodal targeting of CYPs to ER and MT, Alzheimer amyloid precursor protein to plasma membrane and MT, and translocation of cytosolic Glutathione S-transferases to MT (9 -14) may represent one mode of targeting of noncanonical signal containing proteins to the MT compartment.Recent studies from our laboratory showed that different xenobiotic-inducible CYPs such as rat CYP1A1, CYP2E1, and CYP2B1 and others that are widely recognized as microsomal proteins (MC-CYPs) are also targeted to varying degrees to mitochondria (9 -11). These studies led to the concept of a new family of chimeric non-canonical-targeting signals, which function both as ER-targeting and MT-targeting signals, under different physiological conditions. We proposed that bimodal targeting of these CYP proteins is facilitated by the cryptic MTtargeting signal domain (residues 29 -40), located between the transmembrane helical domain (residues 1-30) and the Prorich domain (residues 39 -44) in various members of the CYP1, CYP2, and CYP3 families. We have also shown that the cryptic MT-targeting signals of different CYPs are activated by two distinct mechanisms: (a) endoproteolytic cleavage of the NH 2 -

Mitochondrial P450-dependent arachidonic acid metabolism by TCDD-induced hepatic CYP1A5; conversion of EETs to DHETs by mitochondrial soluble epoxide hydrolase

“…Several P450 enzymes regarded as being localized to the ER have also been found in liver, brain and lung mitochondria, including mouse and rat CYPs 1A1, 1A2, 2A5, 2B1 2E1, 3A4, 4 [3][4][5][6][7][8]. We show here that TCDD induced enzymatically active CYPs 1A4 and 1A5, the avian orthologs of mammalian CYP1A1 and 1A2, in CE liver mitochondria in substantial amounts within 24 hr of TCDD treatment.…”

“…Mitochondrial CYP1A enzymes in CE liver were similar to the rat and mouse orthologs with respect to (a) relative amounts and activity levels as compared to those in microsomes (mitochondrial CYP1A was present in CE livers at 20 to 40% of the microsomal levels and in rat and mouse livers at 20 to 30% of the microsomal levels [3,8]), (b) strict requirement for NADPH for catalytic activity as for their microsomal counterparts, and (c) ability to use either ferredoxin reductase or P450 reductase for electron transfer from NADPH but with greater effectiveness of ferredoxin reductase for the mitochondrial P450s [2,7].…”

“…Fig. 1 shows an alignment of the N-terminal amino acid sequences for chicken CYPs 1A4 and 1A5, mouse and rat CYP1A1 and mouse CYP1A2, the CYP1A P450s that have been reported to translocate to mitochondria [3][4][5][6][7][8], and human CYP1A1. CYPs 1A4 and 1A5 both contain the highly conserved positively charged arginine and lysine residues aligning with amino acids 34, 39 and 42 in rat and mouse CYP1A1 and identified as mitochondrial targeting signals [2,3].…”

“…CYPs 1A4 and 1A5 both contain the highly conserved positively charged arginine and lysine residues aligning with amino acids 34, 39 and 42 in rat and mouse CYP1A1 and identified as mitochondrial targeting signals [2,3]. The chick CYP1A enzymes also contain the trypsin-like cleavage site (underlined) also in rat and mouse CYP1A1 and mouse 1A2 found in P450s that translocate to mitochondria [3,6], as well as putative PKC phosphorylation sites (circled), also suggested to be involved in mitochondrial translocation [7]. Human CYP1A1 contains the conserved positive amino acids, cleavage site and a putative serine phosphorylation site.…”

“…Like most P450s involved in xenobiotic metabolism, CYP1A enzymes are primarily located in microsomes (endoplasmic reticulum or ER) in contrast to P450 enzymes catalyzing steroid and bile acid synthesis and metabolism of Vitamin D which are selectively targeted to mitochondria [2]. It has become clear, largely based on work of Avadhani et al, that several P450s localized in the ER, including rat and mouse CYP1A1 in both liver and extrahepatic organs, can undergo processing with targeting to mitochondria [3][4][5][6][7]. It has recently been shown that TCDDinduced CYP1A2 is also found in mouse liver mitochondria [8].…”