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Evidence that coupling factor B is bound to the matrix side of the inner mitochondrial membrane
1Department of Cell Physiology, Boston Biomedical Research Institute, Mass. 02114.
Abstract:
Rotenone-sensitive NADH dehydrogenase activity and Lubrol stimulation of cytochrome oxidase activity were measured to assess the opposite membrane polarity of beef heart mitoplast and inside-out particle preparations. The ATP-Pi exchange activity of mitoplasts was not affected by their incubation at pH 8.9 in the presence of 5 mM EDTA (a treatment known to extract coupling factor B (F beta) from submitochondrial particles), nor was it stimulated by the addition of F beta to intact and alkaline treated mitoplast preparations. In contrast, the exchange activity of inside-out particles was decreased 18 fold by the alkaline/EDTA treatment and was almost completely restored by the addition of F beta to F beta-depleted particles. From these results it is concluded that in beef heart mitochondria, the coupling factor F beta is bound to the matrix-side of the inner mitochondrial membrane.
Insights
This study reveals that coupling factor B (Fβ) is located on the matrix side of the inner mitochondrial membrane in beef heart mitochondria. This finding is crucial for understanding mitochondrial energy production and Fβ
Area of Science:
- Mitochondrial biochemistry
- Bioenergetics
- Membrane protein localization
Background:
- Beef heart mitochondria possess inner membranes with distinct polarities crucial for ATP synthesis.
- Coupling factor B (Fβ) is a component of the mitochondrial respiratory chain, but its precise localization has been debated.
- Understanding Fβ localization is key to elucidating mechanisms of oxidative phosphorylation.
Purpose of the Study:
- To determine the sidedness of coupling factor B (Fβ) within the inner mitochondrial membrane.
- To investigate the functional role of Fβ in ATP-Pi exchange activity.
- To compare the behavior of mitoplasts and inside-out particles regarding Fβ extraction and restoration.
Main Methods:
- Assessed rotenone-sensitive NADH dehydrogenase and Lubrol-stimulated cytochrome oxidase activities to confirm membrane orientation.
- Utilized alkaline/EDTA treatment to extract coupling factor B (Fβ) from beef heart mitoplasts and inside-out particles.
- Measured ATP-Pi exchange activity before and after Fβ depletion and repletion.
Main Results:
- Alkaline/EDTA treatment did not affect mitoplast ATP-Pi exchange activity, even after Fβ addition.
- Inside-out particle ATP-Pi exchange activity decreased 18-fold after alkaline/EDTA treatment.
- Restoration of Fβ to depleted particles fully recovered ATP-Pi exchange activity in inside-out preparations.
Conclusions:
- Coupling factor B (Fβ) is predominantly bound to the matrix-side of the inner mitochondrial membrane in beef heart mitochondria.
- Fβ plays a critical role in the ATP-Pi exchange activity of inside-out submitochondrial particles.
- The matrix-side localization of Fβ is essential for its function in oxidative phosphorylation.