Cardiolipin stabilized supercomplexes of Saccharomyces cerevisiae respiratory chain complexes III and IV (ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase, respectively), but was not essential for their formation in the inner mitochondrial membrane because they were found also in a cardiolipindeficient strain. Reconstitution with cardiolipin largely restored wild-type stability. The putative interface of complexes III and IV comprises transmembrane helices of cytochromes b and c 1 and tightly bound cardiolipin. Subunits Rip1p, Qcr6p, Qcr9p, Qcr10p, Cox8p, Cox12p, and Cox13p and cytochrome c were not essential for the assembly of supercomplexes; and in the absence of Qcr6p, the formation of supercomplexes was even promoted. An additional marked effect of cardiolipin concerns cytochrome c oxidase. We show that a cardiolipindeficient strain harbored almost inactive resting cytochrome c oxidase in the membrane. Transition to the fully active pulsed state occurred on a minute time scale.

Using the technique of blue native gel electrophoresis, the oligomeric state of the yeast mitochondrial F 1 F 0 -ATP synthase was analysed. Solubilization of mitochondrial membranes with low detergent to protein ratios led to the identification of the dimeric state of the ATP synthase. Analysis of the subunit composition of the dimer, in comparison with the monomer, revealed the presence of three additional small proteins. These dimer-specific subunits of the ATP synthase were identified as the recently described subunit e/Tim11 (Su e/Tim11), the putative subunit g homolog (Su g) and a new component termed subunit k (Su k). Although, as shown here, these three proteins are not required for the formation of enzymatically active ATP synthase, Su e/Tim11 and Su g are essential for the formation of the dimeric state. Su e/Tim11 appears to play a central role in this dimerization process. The dimer-specific subunits are associated with the membrane bound F 0 -sector. The F 0 -sector may thereby be involved in the dimerization of two monomeric F 1 F 0 -ATP synthase complexes. We speculate that the F 1 F 0 -ATP synthase of yeast, like the other complexes of oxidative phosphorylation, form supracomplexes to optimize transduction of energy and to enhance the stability of the complex in the membrane.

A mechanism decreasing oxidative metabolism during normal cell division and growth is expected to direct substrates toward biosyntheses rather than toward complete oxidation to CO(2). Hence, any event decreasing oxidative phosphorylations (OXPHOS) could provide a proliferating advantage to a transformed or tumor cell in an oxidative tissue. To test this hypothesis, we studied mitochondrial enzymes, DNA and OXPHOS protein content in three types of renal tumors from 25 patients. Renal cell carcinomas (RCCs) of clear cell type (CCRCCs) originate from the proximal tubule and are most aggressive. Chromophilic RCCs, from similar proximal origin, are less aggressive. The benign renal oncocytomas originate from collecting duct cells. Mitochondrial enzyme and DNA contents in all tumor types or grades differed significantly from normal tissue. Mitochondrial impairment increased from the less aggressive to the most aggressive RCCs, and correlated with a considerably decreased content of OXPHOS complexes (complexes II, III, and IV of the respiratory chain, and ATPase/ATP synthase) rather than to the mitochondrial content (citrate synthase and mitochondrial (mt)DNA). In benign oncocytoma, some mitochondrial parameters (mtDNA, citrate synthase, and complex IV) were increased 4- to 7-fold, and some were slightly increased by a factor of 2 (complex V) or close to normal (complexes II and III). A low content of complex V protein was found in all CCRCC and chromophilic tumors studied. However F(1)-ATPase activity was not consistently decreased and its impairment was associated with increased aggressiveness in CCRCCs. Immunodetection of free F(1)-sector of complex V demonstrated a disturbed assembly/stability of complex V in several CCRCC and chromophilic tumors. All results are in agreement with the hypothesis that a decreased OXPHOS capacity favors faster growth or increased invasiveness.

Vacuolar proton-translocating ATPase (holoATPase and free membrane sector) was isolated from bovine chromaffin granules by blue native polyacrylamide gel electrophoresis. A 5-fold excess of membrane sector over holoenzyme was determined in isolated chromaffin granule membranes. M9.2, a novel extremely hydrophobic 9.2-kDa protein comprising 80 amino acids, was detected in the membrane sector. It shows sequence and structural similarity to Vma21p, a yeast protein required for assembly of vacuolar ATPase. A second membrane sector-associated protein (M8-9) was identified and characterized by aminoterminal protein sequencing.Proton-translocating adenosine triphosphatases have fundamental roles in energy conservation, secondary active transport, the acidification of intracellular compartments, and cellular pH homeostasis. They fall into three broad classes, called F, P, and V (1), of which the vacuolar type (V-ATPases) 1 is both the most recently recognized and the least well characterized. ATPases of this class occur in endomembranes bounding the acidic compartments of animal, plant, and fungal cells (2) and also in the plasma membranes of some specialized cell types. They have been purified from several mammalian sources, including adrenal secretory vesicles (3, 4), brain clathrincoated vesicles, (5, 6), and kidney medulla microsomes (7), as well as from the vacuoles of fungi and higher plants. Most V-ATPases contain some 6 -10 different subunits (2), but subunit composition depends on the source of the enzyme, and tissue-specific isoforms exist (8). The V-type ATPases are structurally similar to those of the F-type, having a transmembrane proton-conducting sector and an extramembrane catalytic sector. By analogy with the two sectors of F-ATPases (9 -12), these are termed V 0 and V 1 , respectively. For a recent review, see Ref. 13.In this work, the recently developed technique of blue native polyacrylamide gel electrophoresis (BN-PAGE; Refs. 14 -17) was employed to purify vacuolar ATPase holoenzyme (V 1 V 0 ) and free membrane sector (V 0 ) simultaneously from adrenal secretory vesicle membranes. Combined with high resolution Tricine-SDS-PAGE in the second dimension, the subunit composition, particularly with respect to small polypeptides, was determined. Two novel proteins, 8 -9 and 9.2 kDa in size, were found in the membrane sector. Here we report the detailed analysis of the larger of these two polypeptides. EXPERIMENTAL PROCEDURESMaterials-Restriction enzymes and T4-DNA ligase were obtained from New England Biolabs. Taq DNA polymerase was from Stratagene, and TA Cloning Kit ® was from Invitrogen. Sequenase version 2.0 sequencing kit, [␣-35 S]dATP and Hybond N ϩ membranes were obtained from Amersham Pharmacia Biotech. ABI Prism™ dye terminator cycle sequencing kit was purchased from Perkin Elmer. The cDNA library from bovine adrenal medulla was a kind gift from Leonora Ciufo (University of Edinburgh, Edinburgh, Scotland, United Kingdom). Human EST clone ID 143553 (GenBank™accession number R75754) was obtained fr...