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Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei.
- Source :
-
Molecular microbiology [Mol Microbiol] 2005 Oct; Vol. 58 (1), pp. 116-30. - Publication Year :
- 2005
-
Abstract
- The function, stability and mutual interactions of selected nuclear-encoded subunits of respiratory complexes III and IV were studied in the Trypanosoma brucei procyclics using RNA interference (RNAi). The growth rates and oxygen consumption of clonal cell lines of knock-downs for apocytochrome c1 (apoc1) and the Rieske Fe-S protein (Rieske) of complex III, and cytochrome c oxidase subunit 6 (cox6) of complex IV were markedly decreased after RNAi induction. Western analysis of mitochondrial lysates using specific antibodies confirmed complete elimination of the targeted proteins 4-6 days after induction. The Rieske protein was reduced in the apoc1 knock-down and vice versa, indicating a mutual interdependence of these components of complex III. However, another subunit of complex IV remained at the wild-type level in the cox6 knock-down. As revealed by two-dimensional blue native/SDS-PAGE electrophoresis, silencing of a single subunit resulted in the disruption of the respective complex, while the other complex remained unaffected. Membrane potential was reproducibly decreased in the knock-downs and the activities of complex III and/or IV, but not complex I, were drastically reduced, as measured by activity assays and histochemical staining. Using specific inhibitors, we have shown that in procyclics with depleted subunits of the respiratory complexes the flow of electrons was partially re-directed to the alternative oxidase. The apparent absence in T. brucei procyclics of a supercomplex composed of complexes I and III may represent an ancestral state of the respiratory chain.
- Subjects :
- Animals
Blotting, Western
Cell Nucleus genetics
Cytochromes c analysis
Down-Regulation
Electron Transport
Electron Transport Complex I genetics
Electron Transport Complex III analysis
Electron Transport Complex III genetics
Electron Transport Complex IV genetics
Electrophoresis, Gel, Two-Dimensional
Gene Silencing
Iron-Sulfur Proteins analysis
Membrane Potentials
Oxygen Consumption
Protein Subunits genetics
Protozoan Proteins genetics
RNA Interference
Trypanosoma brucei brucei genetics
Trypanosoma brucei brucei growth & development
Electron Transport Complex I metabolism
Electron Transport Complex III metabolism
Electron Transport Complex IV metabolism
Protein Subunits metabolism
Protozoan Proteins metabolism
Trypanosoma brucei brucei enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0950-382X
- Volume :
- 58
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Molecular microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 16164553
- Full Text :
- https://doi.org/10.1111/j.1365-2958.2005.04813.x