Expression of mtDNA and nDNA encoded respiratory chain proteins in chemically and genetically-derived Rho0 human fibroblasts: a comparison of subunit proteins in normal fibroblasts treated with ethidium bromide and fibroblasts from a patient with mtDNA depletion syndrome

Abstract

Although much progress has been made in identifying genetic defects associated with mitochondrial diseases, the protein expression patterns of most disorders are poorly understood. Here we use immunochemical techniques to describe subunit expression patterns of respiratory chain enzyme complexes II (succinate dehydrogenase: SD) and IV (cytochrome c oxidase: COX) in cultured cells lacking mtDNA (Rho0 cells) derived either chemically by exposure of normal cells to ethidium bromide, or genetically in cells derived from a patient with mtDNA depletion syndrome. Both control cells and early passage patient-derived cells express a normal complement of SD and COX subunit proteins. Ethidium bromide treatment of normal cells and in vitro cell proliferation of patient-derived cells caused both populations to acquire identical Rho0 phenotypes. As expected, they lack mtDNA-encoded subunits COX-I and COX-II. In contrast, nDNA-encoded subunits are affected differentially, with some (COX-VIc) lacking and others (COX-IV, COX-Va, SD 30 and SD 70) maintained at somewhat reduced levels. We suggest that the differential stability of nDNA-encoded subunits in the absence of intact enzyme complexes is due to the ability of some, but not all, subunits to associate as partial complexes in the absence of mtDNA-encoded subunits.

A Functionally Active Human F1F0 ATPase Can be Purified by Immunocapture from Heart Tissue and Fibroblast Cell Lines

“…Western blots of mitochondria from MRC5 and GM00028 were probed with an IF 1 -specific monoclonal antibody, together with a mixture of mAbs specific for subunits of the 5 OXPHOS complexes. As previously reported (25,38) this very sensitive method can reveal specific defects in the enzymes of the OXPHOS system. The results in Fig.…”

“…All cell cultures were maintained and mitochondria isolated by cell homogenization and differential centrifugation as previously described (25). The mitochondria were suspended in final wash buffer with inhibitors (see above), and protein concentrations were determined by measuring the A 280 of 10 l of mitochondria diluted in 1 ml of 0.6% SDS and heated to 94 -100°C for 4 min.…”

Unique domain anchoring of Src to synaptic NMDA receptors via the mitochondrial protein NADH dehydrogenase subunit 2

“…We found that ND2 was present in the PSD fraction, and we estimated the amount of ND2 in this fraction was Ϸ15% of that in total brain homogenate. In contrast to ND2, neither the oxidoreductase protein ND4, another mitochondrially encoded component of complex I (25)(26)(27), nor Cyto1, an inner mitochondrial membrane protein component of complex IV (30), was detectable in the PSD fraction. Conversely, Cyto1 and ND4, as well as ND2, were readily detected in proteins from brain mitochondria (Fig.…”

Immunological Phenotyping of Fibroblast Cultures from Patients with a Mitochondrial Respiratory Chain Deficit

“…Mouse monoclonal antibodies to the flavoprotein (clone 2E3-GC12-FB2-AE2) and iron-sulfur protein (clone 21A11-AE7) of complex II, the core 2 protein of complex III (clone 13G12-AF12-BB11), subunits I (clone 1D6-E1-A8), II (clone 12C4-F12), III (clone DA5), IV (clone 10G8-C12-D12), Va (clone 6E9-B12-D12), Vb (clone 16H12-H9), and VIc (clone 3G5-F7-G3) of complex IV, and subunit ␣ of complex V (clone 7H10-BD4) have been described elsewhere (Marusich et al, 1997;Rahman et al, 1999;Taanman and Capaldi, 1993;Taanman et al, 1996). In addition, a mouse monoclonal antibody directed against the 39-kd subunit of complex I (clone: 20C11-B11-B11) was used.…”