The assembly of cytochrome-c oxidase was studied in human cells cultured in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using twodimensional PAGE (blue native PAGE and tricine/SDS/PAGE) and subsequent western blots were developed with monoclonal antibodies against specific subunits of cytochrome-c oxidase. Proteins were also visualized using metabolic labeling followed by two-dimensional electrophoresis and fluorography. These techniques allowed identification of two assembly intermediates of cytochrome-c oxidase. Assembly of the 13 subunits of cytochrome-c oxidase starts with the association of subunit I with subunit IV. Then a larger subcomplex is formed, lacking only subunits VIa and either VIIa or VIIb.Keywords : cytochrome-c oxidase; assembly; respiratory chain ; cultured human cell.Cytochrome-c oxidase (COX) is the terminal enzyme of the in a stable intermediate complex made up solely of nuclear-encoded subunits [10,11]. respiratory chain located in the inner mitochondrial membrane. The enzyme complex catalyses the oxidation of cytochrome c Basic questions regarding the assembly of COX remain to be resolved. What is the order of assembly of the subunits? How by molecular oxygen; the energy released in this reaction is used to translocate protons across the inner mitochondrial membrane. is assembly regulated and what are the rate-limiting steps? Answers to these questions are not only important to understand The proton gradient resulting from the activity of the different respiratory-chain complexes is used to drive ATP synthesis. the function and regulation of the enzyme, but they will also provide information on causes of COX deficiency in patients. Mammalian COX contains 13 subunits, three of which (COX I, II and III) are encoded by the mitochondrial genome, while the Most investigations of the assembly pathway of COX employed metabolic labeling followed by immunoprecipitation [8Ϫ remaining ten subunits (COX IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc and VIII) are nuclear-encoded [1Ϫ5].10, 12]. This approach may give erroneous results because there are differences in epitope availability in partially assembled The recent resolution of bovine heart COX by X-ray analysis [6, 7] not only revealed the exact topology of its subunits, but complexes or because there is selective loss of subunits during immunoprecipitation. We have used two-dimensional electroalso assists studies aimed at clarifying the reaction mechanism of the enzyme. Moreover, the quarternary structure of the com-phoresis in combination with translational inhibitors and metabolic labeling to analyze COX assembly. Two assembly intermeplex puts certain structural constraints on models regarding the assembly pathway of COX. To resolve the assembly process of diates could be detected. the enzyme, however, additional experimental approaches are required. Previous studies of the assembly pathway of COX have shown that pools of unassembled subunits exist and that EXPERIMENTAL PR...

UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl) UvA-DARE (Digital Academic Repository)X-linked cardioskeletal myopathy and neutropenia (Barth syndrome): respiratory-chain abnormalities in cultured fibroblasts Barth, P.G.; van den Bogert, C.; Bolhuis, P.A.; Scholte, H.R.; van Gennip, A.H.; Schutgens, R.B.H.; Ketel, A.G.

To study the assembly of mitochondrial F1F0 ATP synthase, cultured human cells were labeled with [35S]methionine in pulse-chase experiments. Next, two-dimensional electrophoresis and fluorography were used to analyze the assembly pattern. Two assembly intermediates could be demonstrated. First the F1 part appeared to be assembled, and next an intermediate product that contained F1 and subunit c. This product probably also contained subunits b, F6 and OSCP, but not the mitochondrially encoded subunits a and A6L. Both intermediate complexes accumulated when mitochondrial protein synthesis was inhibited, suggesting that mitochondrially encoded subunits are indispensable for the formation of a fully assembled ATP synthase complex, but not for the formation of the intermediate complexes. The results and methods described in this study offer an approach to study the effects of mutations in subunits of mitochondrial ATP synthase on the assembly of this complex. This might be of value for a better understanding of deficiencies of ATP synthase activity in mitochrondrial diseases.