The effect of down-regulation of mitochondrial RNA-binding proteins MRP1 and MRP2 on respiratory complexes in procyclic Trypanosoma brucei.

MRP1 and MRP2 are multifunctional mitochondrial RNA-binding proteins with a regulatory role in RNA editing and putative role(s) in RNA processing in Trypanosoma brucei. Silencing of MRP1 and/or MRP2 by RNA interference affected the assembly and functionality of respiratory complexes. The absence of several subunits of complexes I, III and IV resulted in their disintegration and subsequent decrease of specific activities and also caused a significant decrease of membrane potential. The overall respiration in the interfered cells decreased by only about 20%, since the trypanosome alternative oxidase effectively replaced the missing cytochromes and became the principal terminal oxidase.

Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei.

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.