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

It is well established that cytochrome cis 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 cfrom 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 cmay 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.