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

Mitochondrial targeting of rat CYP2B1 and CYP2E1 and N-terminal truncated CYP1A1 proteins in Saccharomyces cerevisiae – role of protein kinase A in the mitochondrial targeting of CYP2E1

Tên đề tài: Mitochondrial targeting of rat CYP2B1 and CYP2E1 and N-terminal truncated CYP1A1 proteins in Saccharomyces cerevisiae – role of protein kinase A in the mitochondrial targeting of CYP2E1

Tác giả: Naresh B. V. Sepuri, Sanjay Yadav, Hindupur K. Anandatheerthavarada, Narayan G. Avadhani

Lĩnh vực: Animal Biology, School of Veterinary Medicine, University of Pennsylvania

Nội dung tài liệu: Nghiên cứu này sử dụng hệ thống nấm men để xác định tính bảo tồn của các cơ chế định vị và bản chất của các protein đích đến ti thể. Các protein cytochrome P450 (CYP) đầy đủ là CYP2B1 và CYP2E1, cũng như CYP1A1 bị cắt ngắn ở đầu N (phiên bản +5 và +33) đã được đưa vào tế bào nấm men. Kết quả cho thấy CYP2B1 và CYP2E1 đầy đủ được định vị đến cả màng microsome và ti thể, trong khi CYP1A1 bị cắt ngắn (+5 và +33) chủ yếu được định vị đến ti thể. Nghiên cứu cũng chỉ ra rằng PKA (protein kinase A) đóng vai trò quan trọng trong quá trình định vị ti thể của CYP2E1 thông qua quá trình phosphoryl hóa. Hoạt động xúc tác của các protein CYP được định vị đến ti thể được hỗ trợ đầy đủ bởi ferredoxin và ferredoxin reductase của nấm men.

Mục lục chi tiết:

• Keywords
• Keywords
• Correspondence
• Chimeric targeting signals; CYP2E1; evolutionary conservations; mitochondrial protein targeting; xenobiotic metabolism
• Received 30 April 2007, revised 6 July 2007, accepted 13 July 2007
• doi:10.1111/j.1742-4658.2007.05990.x
• 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
• 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.
• 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 6 July 2007, accepted 13 July 2007)
• doi:10.1111/j.1742-4658.2007.05990.x
• 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 β-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, Δ1-pyrroline-5-carboxylate dehydrogenase; TIM, translocase of inner membrane; TOM, translocase of outer membrane.
• FEBS Journal 274 (2007) 4615-4630 © 2007 University of Pennsylvania. Journal compilation 2007 FEBS
• 4615