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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
Author(s) -
Horváth Anton,
Horáková Eva,
Dunajčíková Petra,
Verner Zdeněk,
Pravdová Eliška,
Šlapetová Iveta,
Cuninková L’udmila,
Lukeš Julius
Publication year - 2005
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2005.04813.x
Subject(s) - biology , trypanosoma brucei , protein subunit , alternative oxidase , coenzyme q – cytochrome c reductase , respiratory chain , rna interference , biochemistry , oxidase test , electron transport complex iv , microbiology and biotechnology , mitochondrial respiratory chain , mitochondrion , cytochrome c oxidase , cytochrome c , rna , enzyme , gene
Summary 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 c 1 (apo c 1 ) 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 apo c 1 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.