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 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.Mitochondria are well known as the organelle for energy conversion in all eukaryotes. This energy conversion, i.e. ATP synthesis, is performed by using the electrochemical gradient of H⁺ across the inner mitochondrial membrane. To enable effective energy conversion, the mitochondrial inner membrane is highly resistant to the permeation of solutes and ions. However, under certain conditions, such as in the presence of Ca²⁺ and inorganic phosphate, the permeability of this inner membrane is known to be markedly increased. This phenomenon is referred to as the permeability transition (PT)¹ and is believed to result from the formation of a proteinaceous pore, referred to as the PT pore, which makes the inner membrane permeable to various solutes and ions smaller than 1.5 kDa (1–3). The physiological importance of the PT has long been uncertain; however, recent studies have revealed that the changes in the permeability of the inner mitochondrial membrane due to the induction of PT cause the release of cytochrome c into the cytosol and that the released cytochrome c then triggers subsequent steps of programmed cell death, which is known as apoptosis (4–6). Thus, the PT is considered to be one of the major regulatory steps of apoptosis. However, the questions as to how the PT is induced and how cytochrome c is released accompanied by the induction of PT have remained unanswered.To characterize the features of the mitochondrial PT and to understand the mechanism underlying the release of cytochrome c from mitochondria, investigators have studied the effects of various agents on this organelle. As a result, the PT and the release of cytochrome c were found to be induced not only by Ca²⁺ but also by other agents (7–9). We also found that copper-o-phenanthroline (10), metal ions (11), and cyanine dyes (12, 13) induced this PT and the release of cytochrome c from mitochondria. Furthermore we reported that valinomycin, known as a potassium-selective ionophore, also induces the release of cytochrome c from mitochondria but without the induction of PT (14). This finding indicated that cytochrome c could be released from mitochondria in two different manners: one with the induction of PT and the other without it. To understand how cytochrome c is released from mitochondria, it is very important to know what protein species are released from mitochondria concomitant with the release of cytochrome c. To address these questions, in the present study we used a mass spectrometry (LC-MS/MS system)-based proteome analysis approach, which allowed us to identify the protein species present in a limited amount of protein samples. Using proteomics techniques, we examined the protein species released from mitochondria treated with valinomycin or with Ca²⁺, and we discuss our findings on the status of inner and outer mitochondrial membranes treated with these agents.