Inhibition of the synthesis of a cytochrome‐c‐oxidase subunit isoform by antisense RNA

Abstract

To investigate the role of subunit VIIe, an oxygen-regulated subunit isoform of Dictostelium discoideum cytochrome-c oxidase, the full-length cDNA was inserted into an expression vector under the control of an actin promoter in the sense and antisense orientation. The DNA constructs were used for stable transformation of the slime mold amoebae. In most of the 28 antisense clones tested, the concentration of cytochrome-c oxidase was lowered compared to the wild type, while no significant changes were found in the sense mutants. Antisense RNA was abundantly expressed, leading to a drastic reduction of the steady-state level of the endogenous subunit VIIe mRNA, which was decreased up to 20-30% the level observed in parent cells. In these transformants, the amount of the target polypeptide and cytochrome c oxidase was 40-50% and 60-70% of control, respectively. A similar decrease was found in the level of the remaining nuclear and mitochondrial subunits. Unexpectedly, these changes affected neither basal nor uncoupled cell respiration suggesting an increase of the enzyme specific activity. Hypoxia completely relieved the cytochrome-c-oxidase deficit. These results indicate that subunit VII is needed for an efficient assembly of the protein complex and provide evidence for its involvement in the modulation of the enzyme activity.Cytochrome-c oxidase couples electron transfer from cytochrome c to molecular oxygen with proton translocation across the plasma membrane of many aerobic bacteria and in the mitochondrial inner membrane. The process involves four redox centers (hemes a and a, and two copper atoms) located in the two largest enzyme subunits [l-41. This catalytic core, with a third large polypeptide, has been highly conserved during evolution. In eukaryotes, it is encoded by mitochondrial genes and assembled with 4 -10 additional subunits which are products of nuclear genes. The function of the nuclear subunits is unclear, although the presence of tissue-specific isoforms in mammals may suggest a regulatory role [S, 61. Recent evidence indicates that these polypeptides can influence the enzyme conformation and activity either directly or by interaction with allosteric effectors [7-91. In this connection, the low degree of similarity of homologous nuclear subunits is a puzzling aspect that does not support the existence of evolutionary conserved functions [lo].Amoeba-like Dictyostelium cells are an interesting model because they can, under certain conditions, aggregate and differentiate in a process that is reminiscent of some aspects of embryogenesis in higher eukaryotes [ll]. Dictyosteelium cytochrome oxidase contains only four nuclear subunits and

MITOCHIP assessment of differential gene expression in the skeletal muscle of Ant1 knockout mice: Coordinate regulation of OXPHOS, antioxidant, and apoptotic genes

“…Of the nuclear OXPHOS subunit mRNAs that are up-regulated, 3 belonged to complex I (Ndufs4, Ndufb5 and Ndufc2) while one belonged to complex IV (Cox7a2). Interestingly, Ndufs4 has been shown to be essential for the assembly of Complex I in humans [25], while Cox7a2 has been shown to be essential for assembly of Complex IV in yeast [26] and Dictyostelium [27]. The induction of mtDNA and nDNA OXPHOS proteins is consistent with the hyper-proliferation of mitochondrial and the increased complex II and IV staining in the Ant1−/− skeletal muscle [11,15,22].…”

“…However, this is futile due to the absence of Ant1, resulting in uncontrolled proliferation of the mitochondria [11]. All the mtDNA OXPHOS genes as well as four nuclear-encoded OXPHOS genes are significantly up-regulated in the Ant1 deficient muscle, with two of the nuclear encoded proteins, Ndufs4 and Cox7a2, being involved in the assembly of complex I and complex IV, respectively [25][26][27]. The fact that major up-regulated nuclear OXPHOS genes are involved in complex assembly suggests that most nuclear-encoded components of the ETC are not normally limiting in muscle and that the number of functional ETC complexes may be regulated at the level of complex assembly.…”