ATP synthase is responsible for maintaining mitochondrial membrane potential in bloodstream form Trypanosoma brucei.
The mitochondrion of Trypanosoma brucei bloodstream form maintains a membrane potential, although it lacks cytochromes and several Krebs cycle enzymes. At this stage, the ATP synthase is present at reduced, although significant, levels. To test whether the ATP synthase at this stage is important for maintaining the mitochondrial membrane potential, we used RNA interference (RNAi) to knock down the levels of the ATP synthase by targeting the F1-ATPase alpha and beta subunits. RNAi-induced cells grew significantly slower than uninduced cells but were not morphologically altered. RNAi of the beta subunit decreased the mRNA and protein levels for the beta subunit, as well as the mRNA and protein levels of the alpha subunit. Similarly, RNAi of alpha subunit decreased the alpha subunit transcript and protein levels, as well as the beta-subunit transcript and protein levels. In contrast, alpha and beta RNAi knockdown resulted in a 60% increase in the F0 complex subunit 9 protein levels without a significant change in the steady-state transcript levels of this subunit. The F0-32-kDa subunit protein expression, however, remained stable throughout induction of RNAi for alpha or beta subunits. Oligomycin-sensitive ATP hydrolytic and synthetic activities were decreased by 43 and 44%, respectively. Significantly, the mitochondrial membrane potential of alpha and beta RNAi cells was decreased compared to wild-type cells, as detected by MitoTracker Red CMXRos fluorescence microscopy and flow cytometry. These results support the role of the ATP synthase in the maintenance of the mitochondrial membrane potential in bloodstream form T. brucei.
Brown
Silvia V
SV
Department of Microbiology and Immunology, 253 Biomedical Research Building, University at Buffalo, Buffalo, New York 14214, USA.
Hosking
Paul
P
Li
Jinlei
J
Williams
Noreen
N
D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D049109:Cell Proliferation; D005434:Flow Cytometry; D006868:Hydrolysis / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008928:Mitochondria / Q000502:physiology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*
D009840:Oligomycins; D012333:RNA, Messenger; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D025261:Mitochondrial Proton-Translocating ATPases; D013752:Tetracycline