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.
Department of Animal Biology, Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Marie-Anne
MA
Robin
Hindupur K
HK
Anandatheerthavarada
Gopa
G
Biswas
Naresh Babu V
NB
Sepuri
Donna M
DM
Gordon
Debkumar
D
Pain
Narayan G
NG
Avadhani
D000595:Amino Acid Sequence; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism*; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008862:Microsomes, Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction
D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D019392:Cytochrome P-450 CYP2E1