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Visualization of mitochondrial coupling factor F1(ATPase) by freeze-drying
Abstract:
It is known that the negatively stained preparations of inner mitochondrial membrane display characteristic approximately 9 nm F1 (ATPase) knobs projecting from the matrix surface. Freeze-etch studies have reported the absence of such knobs from the "etched" surface of the inner mitochondrial membranes. We have demonstrated their presence on the surface of SMP (submitochondrial particles) prepared by freeze-drying for transmission electron microscopy. This identification has been substantiated by comparison with freeze-dried TU particles (trypsin-urea treated SMP) that are devoid of F1 (ATPase). It has been suggested that a layer of water molecules is strongly adsorbed to the surface of SMP and does not sublime during normal freeze-"etching."
Insights
Submitochondrial particles (SMP) show F1 (ATPase) knobs on their surface, unlike freeze-etched membranes. Freeze-drying reveals these structures, confirming their presence on mitochondrial membranes.
Area of Science:
- Biochemistry
- Cell Biology
- Structural Biology
Background:
- Mitochondrial inner membranes contain F1 (ATPase) knobs.
- Previous freeze-etch studies suggested these knobs are absent from etched surfaces.
- Submitochondrial particles (SMP) are derived from the inner mitochondrial membrane.
Purpose of the Study:
- To investigate the presence of F1 (ATPase) knobs on submitochondrial particles (SMP).
- To reconcile conflicting observations from different electron microscopy techniques.
Main Methods:
- Transmission electron microscopy of freeze-dried SMP.
- Comparison with freeze-dried trypsin-urea treated (TU) particles, which lack F1 (ATPase).
- Analysis of negatively stained preparations.
Main Results:
- Freeze-dried SMP preparations clearly displayed the characteristic 9 nm F1 (ATPase) knobs.
- Freeze-dried TU particles, lacking F1 (ATPase), served as a control, showing no such knobs.
- These findings confirm the presence of F1 (ATPase) on the matrix side of the inner mitochondrial membrane.
Conclusions:
- The absence of F1 (ATPase) knobs in previous freeze-etch studies may be due to adsorbed water layers preventing sublimation.
- Freeze-drying is a suitable method for visualizing F1 (ATPase) on SMP.
- This study clarifies the structural organization of the mitochondrial ATP synthase complex.