Assembly of mitochondrial ATP synthase in cultured human cells: implications for mitochondrial diseases

Abstract

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.

Assembly of cytochrome‐c oxidase in cultured human cells

“…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.…”

“…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.…”

“…Western blotting was performed as described [11], and blots methionine-free medium, [ 35 S]methionine (specific activity were developed with monoclonal antibodies [16] against COX I Ͼ 960 Ci/mmol) was added to 20 µCi/ml, at a cell density of 10 6 (1D6-E1-A8), COX II (12C4-F12) or COX IV (10G8-D12-cells/ml. After 1 h, cells were washed twice with NaCl/P i and C12).…”

“…After transfer to nitrocellulose membrane, the proteins were probed with a variety of COX antibodies. Four [11]. We used a similar approach in the present study to obtain more information on COX subcomplexes.…”

Reduced Respiratory Control with ADP and Changed Pattern of Respiratory Chain Enzymes as a Result of Selective Deficiency of the Mitochondrial ATP Synthase

“…Structural protein defects of the subunit a, which is encoded by the mitochondrial gene ATP6, have been shown to result in incomplete assembly of the complex V holoenzyme. This is known from studies in tissues and cultured cells with the mutation T8993G (10,30), associated with NARP disease but also from rho 0 cells, which lack mitochondrial DNA (31) as well as cells with inhibited mitochondrial protein synthesis (32), where increased content of unassembled or released F 1 of 390 kD and accumulation of atypical ATPase subcomplex of 460 kD was found (10,30). In the case of our patient, as detected by BN-PAGE and Western blots of solubilized OX-PHOS complexes, possibly some amount of F 1 -like intermediate could be present.…”