Figure 5.
Biochemical Analysis of the Localization of Mitochondrial Actin in Different Mitochondrial Subcompartments.
(A) Immunoblot analysis indicates that mitochondrial actin resides in the inner membrane, matrix, contact sites, and, to a lesser extent, in the intermembrane space and in the outer membrane.
(B) The presence of actin in mitoplasts after most of the mitochondrial outer membrane was removed by different concentrations of digitonin. The mitoplast fraction after treatment with 0.25 to 0.5 mg digitonin per mg mitochondrial protein was purified as described in Methods, and immunoblot analysis was then performed. M indicates mitochondria.
(C) Mitoplast actin is slightly sensitive to high-salt treatment and highly sensitive to proteinase K treatment. Purified mitoplasts (marked as mp) isolated from digitonin-treated mitochondria were subjected to high salt (1.2 M), proteinase K (10 and 20 μg mL−1), or Lat B (20 μM) treatments. Immunoblot analysis was then performed after the mitoplasts were repurified on a Suc cushion. M indicates mitochondria.
Studies in (A) to (C) were repeated three to four times, and equivalent results were obtained each time.
(D) Immunogold labeling of an antiactin antibody indicates that actin resides in the mitochondrial matrix. Arrows in (a) and (b) indicate the gold particles representing actin inside mitochondrion ([b] is the enlargement area of [a] indicated by arrow); 5-nm gold labeling was applied in this experiment. Bars = 200 nm in (a) and 50 nm in (b).
(E) Immunogold-labeled negative staining ultrastructure analysis on contact sites purified by fractionation through two sequential Suc gradients (see Methods) revealed that gold labeling (18-nm gold) of an antiactin antibody appears coincident with ~5-nm filaments that pass across the mitochondrial membrane of the contact site. The inset box is the enlarged area indicated by the arrow. Bar = 200 nm.
Studies in (D) and (E) were repeated more than six and two times, respectively, and similar results were obtained each time.