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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01074

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Cross-linking ATP synthase complexes in vivo eliminates mitochondrial cristae

¹ Department of Biochemistry & Molecular Biology, and ARC Centre for Structural and Functional Microbial Genomics, Monash University, Clayton campus, Victoria 3800, Australia
² Monash Micro Imaging, School of Biomedical Sciences, Building 13C, Monash University, Clayton campus, Victoria 3800, Australia

View larger version (78K): [in a new window]   Fig. 1. Analysis of DsRed cross-linking of mtATPase complexes. Mitochondria were solubilised with dodecyl ß-maltoside (DM; 4 g/g protein; lanes 1, 2, 4 and 6) or digitonin (Dig; 4 g/g protein; lanes 3 and 5 or 10 g/g protein; lane 7) and samples of lysates subjected to CN-PAGE. Lysates were prepared from strains -27-DsRed (lanes 1 and 7), -27-DsRed-mtDsRed (lane 2), -27-GFP (lanes 3 and 4) and YRD15 (lanes 5 and 6). Gels were imaged for fluorescence due to DsRed (A; lanes 1, 2 and 7) or YEGFP3 (A; lanes 3-6), then subjected to in situ ATPase analysis (B). The positions of bands corresponding to mtATPase assemblies are indicated by lower case letters at the left of A (a, tetramer; b, trimer; c, dimer; and d, monomer). Bands containing mtATPase complexes were excised and subjected to second dimension SDS-PAGE, and gels silver stained (C). Subunit assignments were based on their mobility within the gel relative to size standards and by comparison with published mtATPase subunit profiles (Bateson et al., 1999). Lanes correspond to the numbered lanes from A, with lower case letters referring to mtATPase species a-d (above). The contrast in lane 7 for both A and C was altered independently of the rest of the gel in order to highlight bands of lower intensity. OSCP, oligomycin-sensitivity conferring protein.

View larger version (60K): [in a new window]   Fig. 2. Abnormal mitochondrial morphology of -27-DsRed cells. Yeast cells were grown in liquid SaccE medium at 28°C and subjected to fluorescence microscopy. Left panel, mitochondrial morphology revealed by fluorescence imaging; right panel, corresponding bright-field image. (A) YRD15-mtDsRed, (B) -27-GFP, (C and D) -27-DsRed. GFP (E) and DsRed (F) fluorescence in -27-DsRed-mtGFP cells. Bars, 2 µm.

View larger version (58K): [in a new window]   Fig. 3. Dividing -27-DsRed cells display multiple buds. -27-DsRed cells were examined using bright-field (A) and fluorescence microscopy (B). (C) Overlay of A and B. One dividing cell can be seen with five buds (upper), and a second can be seen with three (lower) (A). The lower mother cell burst during examination, releasing fluorescent mitochondria across the field of view (B). Bar, 2 µm. (D) The number of buds attached to dividing cells for YRD-15, -27-GFP, -27-DsRed and -27-DsRed-mtDsRed cultures.

View larger version (110K): [in a new window]   Fig. 4. -27-DsRed cells have abnormal mitochondrial ultrastructure. TEM was carried out on cell sections as described in Materials and Methods. Normal mitochondria are indicated by white arrows; abnormal mitochondria are indicated by black arrows. Images are presented in reverse contrast, with membranes being black. Bars, 0.5 µm. (A) YRD15 (B) -27-GFP, (C-F) -27-DsRed, (G,H) A20N.

View larger version (135K): [in a new window]   Fig. 5. Immunogold localisation of mtATPase subunit . Immunogold electron microscopy was performed as in Materials and Methods using a monoclonal antibody directed against the subunit of mtATPase. Arrowheads point to gold particles that identify the inner membrane. Bars, 0.5 µm. (A) YRD15 (B) -27-GFP, (C-E) -27-DsRed.

View larger version (43K): [in a new window]   Fig. 6. Mitochondrial membranes from -27-DsRed cells contain normal levels of subunits e and g. Mitochondrial membrane lysates (50 µg of protein) were subjected to SDS-PAGE and western analysis. Blots were probed with antibodies raised against subunits e and g.

View larger version (74K): [in a new window]   Fig. 7. Partial restoration of normal mitochondrial morphology in -27-DsRed-mtDsRed cells. TEM was performed on -27-DsRed-mtDsRed cell sections. White arrows, normal mitochondria; black arrows, abnormal mitochondria. Images are presented in reverse contrast, with membranes being black. Bars, 0.5 µm.

View larger version (67K): [in a new window]   Fig. 8. b-24-DsRed cells contain mtATPase oligomers and show abnormal mitochondrial ultrastructure. b-24-DsRed mitochondria were solubilised with dodecyl ß-maltoside (4 g/g protein) and subjected to CN-PAGE. The gel was imaged for DsRed fluorescence (A). Lower-case letters indicate the positions of mtATPase tetramer (a), trimer (b), dimer (c) and monomer (d). TEM was performed on b-24-DsRed sections (B) as described in Materials and Methods. White arrows, normal mitochondria; black arrows, abnormal mitochondria. Images are presented in reverse contrast, with membranes being black. Bars, 0.5 µm.

View larger version (37K): [in a new window]   Fig. 9. Tetrameric mtATPase assemblies in the membrane. The oligomerisation of identical mtATPase complexes has been proposed to form a rigid arc that leads to a protrusion of the inner mitochondrial membrane (A) (adapted from Allen, 1995). Here, for simplicity the contributing complexes are shown lacking the peripheral stator stalk and FO subunits aside from the subunit 9 (c) ring. Interactions between the DsRed moiety of mtATPase tetramers in -27-DsRed cells may prevent the component monomers from arcing away from each other and out from the membrane, thus promoting sheets of inner mitochondrial membrane lacking curvature (B).