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Journal of Biochemistry Advance Access originally published online on April 19, 2008
Journal of Biochemistry 2008 144(2):187-196; doi:10.1093/jb/mvn057
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© 2008 The Japanese Biochemical Society

Evidence for Mitochondrial Localization of P5, a Member of the Protein Disulphide Isomerase Family

Taiji Kimura1,*, Tomohisa Horibe1,{dagger}, Chikara Sakamoto2, Yu Shitara2, Fuyuki Fujiwara1, Tohru Komiya2, Akitsugu Yamamoto2, Toshiya Hayano3,5, Nobuhiro Takahashi4 and Masakazu Kikuchi5,{ddagger}

1Department of Bioscience & Technology, Faculty of Science & Engineering, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu 525-8577; 2Department of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, 526-0829; 3Department of Applied Biological Science, Tokyo University of Agriculture and Technology and Integrated Proteomics System Project, Pioneer Research on Genome the Frontier, Ministry of Education, Culture, Sports, Science & Technology of Japan, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509; 4United Graduate School of Agriculture, Tokyo University of Agriculture & Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509; and 5Department of Bioscience & Bioinformatics, College of Information Science & Engineering, 1-1-1 Noji-Higashi, Kusatsu 525-8577, Japan

{ddagger}To whom correspondence should be addressed. Tel: +81 77 561 5065, Fax: +81 77 561 5065, E-mail: kikuchi{at}is.ritsumei.ac.jp

Received April 9, 2008; Accepted April 12, 2008


  Abstract

This report demonstrates for the first time that P5, a member of the protein disulphide isomerase (PDI) family, is present in the mitochondria. Various organelles were screened for proteins bearing the CGHC motif using an affinity column conjugated with the phage antibody 5E, which cross-reacts with PDI family proteins. P5 was found in bovine liver mitochondrial extract and identified by Western blot analysis using anti-P5 antibody and by mass spectrometric analysis. Results of cell fractionation, proteinase sensitivity experiments and immuno-electron microscopy supported the mitochondrial localization of P5 and also indicated the presence of ERp57, another PDI family protein, in mitochondria. Our findings will be useful for the elucidation of the translocation mechanism of PDI family proteins and their roles in mitochondria.

Key Words: ERp57, protein disulphide isomerase, mitochondria, P5, single-chain antibody fragment

Abbreviations: AIF, apoptosis inducing factor; bPDI, bovine PDI; bP5, bovine P5; ER, endoplasmic reticulum; hPDI, human PDI; hPDIR, human protein disulphide isomerase-related protein; hP5, human P5; hsc70, heat shock cognate70; hsp60, heat shock protein 60; IMS, intermembrane space; LC, liquid chromatography; MALDI-TOF/MS, matrix assisted laser desorption ionization-time of flight mass spectrometry; MS/MS, tandem mass spectrometry; PDI, protein disulphide isomerase; scFv, single-chain Fv fragment; Tim, Translocase of the inner mitochondrial membrane; Tom, Translocase of the outer mitochondrial membrane; TX, thioredoxin; yPDI, yeast PDI

*Present address: The Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101 Japan

{dagger}Present address: Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Yoshida Konoecho, Sakyoku, Kyoto, 606-8501, Japan


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