title: Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes.
abstract: Kinetoplastid parasites cause lethal diseases in humans and animals. The kinetoplast itself contains the mitochondrial genome, comprising a huge, complex DNA network that is also an important drug target. Isometamidium, for example, is a key veterinary drug that accumulates in the kinetoplast in African trypanosomes. Kinetoplast independence and isometamidium resistance are observed where certain mutations in the F1-γ-subunit of the two-sector F1Fo-ATP synthase allow for Fo-independent generation of a mitochondrial membrane potential. To further explore kinetoplast biology and drug resistance, we screened a genome-scale RNA interference library in African trypanosomes for isometamidium resistance mechanisms. Our screen identified 14 V-ATPase subunits and all 4 adaptin-3 subunits, implicating acidic compartment defects in resistance; V-ATPase acidifies lysosomes and related organelles, whereas adaptin-3 is responsible for trafficking among these organelles. Independent strains with depleted V-ATPase or adaptin-3 subunits were isometamidium resistant, and chemical inhibition of the V-ATPase phenocopied this effect. While drug accumulation in the kinetoplast continued after V-ATPase subunit depletion, acriflavine-induced kinetoplast loss was specifically tolerated in these cells and in cells depleted for adaptin-3 or endoplasmic reticulum membrane complex subunits, also identified in our screen. Consistent with kinetoplast dispensability, V-ATPase defective cells were oligomycin resistant, suggesting ATP synthase uncoupling and bypass of the normal Fo-A6-subunit requirement; this subunit is the only kinetoplast-encoded product ultimately required for viability in bloodstream-form trypanosomes. Thus, we describe 30 genes and 3 protein complexes associated with kinetoplast-dependent growth. Mutations affecting these genes could explain natural cases of dyskinetoplasty and multidrug resistance. Our results also reveal potentially conserved communication between the compartmentalized two-sector rotary ATPases.
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom;
authors.forename: Nicola
authors.initials: N
authors.lastname: Baker
authors.affiliation: Wellcome Trust Centre of Molecular Parasitology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom.
authors.forename: Graham
authors.initials: G
authors.lastname: Hamilton
authors.affiliation: Wellcome Trust Centre of Molecular Parasitology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Wilkes
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom;
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hutchinson
authors.affiliation: Wellcome Trust Centre of Molecular Parasitology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Barrett
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom; d.horn@dundee.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Horn
mesh_terms: D000143:Acids / Q000378:metabolism; D000818:Animals; D002451:Cell Compartmentation / Q000187:drug effects; D018105:DNA, Kinetoplast / Q000378:metabolism*; D004351:Drug Resistance / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D010617:Phenanthridines / Q000737:chemistry / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D021122:Protein Subunits / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D034622:RNA Interference / Q000187:drug effects; D015203:Reproducibility of Results; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D000143:Acids; D018105:DNA, Kinetoplast; D010617:Phenanthridines; D021122:Protein Subunits; C000702:isometamidium chloride; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases
keywords: brucei; mitochondrion; nagana; petite; samorin