Expression and fate of the nuclearly encoded subunits of cytochrome-c oxidase in cultured human cells depleted of mitochondrial gene products.
Synthesis, import, assembly and turnover of the nuclearly encoded subunits of cytochrome-c oxidase were investigated in cultured human cells depleted of mitochondrial gene products by continuous inhibition of mitochondrial protein synthesis (OP- cells). Immunoprecipitation after pulse labeling demonstrated that the synthesis of the nuclear subunits was not preferentially inhibited, implying that there is no tight regulation in the synthesis of mitochondrial and nuclear subunits of mitochondrial enzyme complexes. Quantitative analysis of the mitochondrial membrane potential in OP- cells indicated that its magnitude was about 30% of that in control cells. This explains the normal import of the nuclearly encoded subunits of cytochrome-c oxidase and other nuclearly encoded mitochondrial proteins into the mitochondria that was found in OP- cells. The turnover rate of nuclear subunits of cytochrome-c oxidase, determined in pulse-chase experiments, showed a specific increase in OP- cells. Moreover, immunoblotting demonstrated that the steady-state levels of nuclear subunits of cytochrome-c oxidase were severely reduced in these cells, in contrast to those of the F1 part of complex V. Native electrophoresis of mitochondrial enzyme complexes showed that assembly of the nuclear subunits of cytochrome-c oxidase did not occur in OP- cells, whereas the (nuclear) subunits of F1 were assembled. The increased turnover of the nuclear subunits of cytochrome-c oxidase in OP- cells is, therefore, most likely due to an increased susceptibility of unassembled subunits to intra-mitochondrial degradation.
Nijtmans
L G
LG
Department of Neurology, University of Amsterdam, Academic Medical Center, The Netherlands.
Spelbrink
J N
JN
Van Galen
M J
MJ
Zwaan
M
M
Klement
P
P
Van den Bogert
C
C
D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000201:enzymology*; D002478:Cells, Cultured; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*
D003576:Electron Transport Complex IV