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Molecular & Cellular Proteomics 8:1265-1277, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Differential Permeabilization Effects of Ca²⁺ and Valinomycin on the Inner and Outer Mitochondrial Membranes as Revealed by Proteomics Analysis of Proteins Released from Mitochondria^*,

Akiko Yamada, Takenori Yamamoto^,¶, Naoshi Yamazaki^||, Kikuji Yamashita, Masatoshi Kataoka^**, Toshihiko Nagata, Hiroshi Terada and Yasuo Shinohara^,||,**,

From the School of Dentistry, University of Tokushima, Kuramotocho-3, Tokushima 770-8504, Japan,
Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan,
||Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan,
**Health Technology Research Center, National Institute for Advanced Industrial Science and Technology (AIST), 2217-14 Hayashicho, Takamatsu 761-0395, Japan, and
Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamazaki 2641, Noda 278-8510, Japan

It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca²⁺. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224–5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca²⁺-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca²⁺-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca²⁺ and valinomycin on the inner and outer mitochondrial membranes are discussed.

To whom correspondence may be addressed: Inst. for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan. Fax:81-88-633-9150; E-mail: yshinoha{at}genome.tokushima-u.ac.jp

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