The F 1 -ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function

The mitochondrion of the parasitic protozoon Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by partial import of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single copy genes, suggesting the use of the same enzyme in the cytosol and in the mitochondrion. However, the T. brucei genome encodes two distinct genes for eukaryotic aspartyl-tRNA synthetase (AspRS), although the cell has a single tRNAAsp isoacceptor only. Phylogenetic analysis showed that the two T. brucei AspRSs evolved from a duplication early in kinetoplastid evolution and also revealed that eight other major duplications of AspRS occurred in the eukaryotic domain. RNA interference analysis established that both Tb-AspRS1 and Tb-AspRS2 are essential for growth and required for cytosolic and mitochondrial Asp-tRNAAsp formation, respectively. In vitro charging assays demonstrated that the mitochondrial Tb-AspRS2 aminoacylates both cytosolic and mitochondrial tRNAAsp, whereas the cytosolic Tb-AspRS1 selectively recognizes cytosolic but not mitochondrial tRNAAsp. This indicates that cytosolic and mitochondrial tRNAAsp, although derived from the same nuclear gene, are physically different, most likely due to a mitochondria-specific nucleotide modification. Mitochondrial Tb-AspRS2 defines a novel group of eukaryotic AspRSs with an expanded substrate specificity that are restricted to trypanosomatids and therefore may be exploited as a novel drug target.

Section: Discussionmentioning

confidence: 99%

Dual Targeting of a tRNAAsp Requires Two Different Aspartyl-tRNA Synthetases in Trypanosoma brucei

Charrière

O’Donoghue

Helgadöttir

et al. 2009

“…This atrophied organelle utilizes respiratory components that do not generate a proton gradient across the inner membrane. Instead, the BSF mitochondrion relies on reverse action of the ATP synthase to maintain the mitochondrial membrane potential (5,6). BSFs excrete pyruvate as the end product of glycolysis and therefore have a diminished need for mitochondrial metabolism (4).…”

mentioning

confidence: 99%

Mitochondrial Fatty Acid Synthesis in Trypanosoma brucei

Stephens¹,

Lee²,

Paul³

et al. 2007

111

Whereas other organisms utilize type I or type II synthases to make fatty acids, trypanosomatid parasites such as Trypanosoma brucei are unique in their use of a microsomal elongase pathway (ELO) for de novo fatty acid synthesis (FAS). Because of the unusual lipid metabolism of the trypanosome, it was important to study a second FAS pathway predicted by the genome to be a type II synthase. We localized this pathway to the mitochondrion, and RNA interference (RNAi) or genomic deletion of acyl carrier protein (ACP) and ␤-ketoacyl-ACP synthase indicated that this pathway is likely essential for bloodstream and procyclic life cycle stages of the parasite. In vitro assays show that the largest major fatty acid product of the pathway is C16, whereas the ELO pathway, utilizing ELOs 1, 2, and 3, synthesizes up to C18. To demonstrate mitochondrial FAS in vivo, we radiolabeled fatty acids in cultured procyclic parasites with

“…Surprisingly, mgi mutations were mapped to the ␣-, ␤-, or ␥-subunits of the ATP synthase. Similar, if not identical, mutations were identified using a similar selection scheme in yeast Saccharomyces cerevisae (13) and in a related approach using the blood form of Trypanosoma brucei (14). While the relationship between the effect of the mutations and the resulting phenotypes is still unclear, there is good biochemical and genetic evidence that when the corresponding mutations are present in the yeast ATP synthase, the enzyme is uncoupled (15).…”

mentioning

confidence: 89%

Crystal Structures of Mutant Forms of the Yeast F1 ATPase Reveal Two Modes of Uncoupling

Arsenieva

Symerský

Wang

et al. 2010

The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F 1 ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between ␥Leu83 and ␤ DP residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the ␥-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process.The mitochondrial ATP synthase is a molecular motor that couples the transport of protons down a potential gradient with the phosphorylation of ADP. This process can be reversed and the hydrolysis of ATP results in the pumping of protons out of the mitochondrial matrix into the cytoplasm generating a proton gradient. In the synthesis mode, the protomotive force is established by oxidation of NADH or succinate by the electron transport chain. Whereas much is known on the structure/ function of the ATP synthase and the mechanism of ATP hydrolysis, less is known on the molecular details on the coupling of proton transport and ATP synthesis. The structure/ function relationship of the ATP synthase is key to understanding the coupling mechanism; that is, identification of which intra-and intermolecular interactions within the ATP synthase critical for coupling.The ATP synthase is composed of two distinct components, the F 1 and the F o portion and as such is referred to as the F 1 F o ATP synthase. The F 1 portion contains the catalytic sites and is composed of ␣ 3 ␤ 3 ␥␦⑀ with an overall molecular mass of about 350 kDa (1). The active site is composed of the ␣␤ pair, and thus there are three catalytic sites per complex. The ␥␦⑀-subunits comprise the central stalk with the ␥-subunit situated in the middle of the ␣ 3 ␤ 3 subcomplex. The central stalk acts as a rotor and drives conformational changes in the active sites in a sequential manner, thereby effecting ATP synthesis. The ␦-and ⑀-subunits are critical for coupling (2, 3), but their roles are unclear, they may participate directly in the coupling process or needed merely for structural purposes.The F o portion of the ATP synthase acts as a proton turbine whose rotation is physically coupled to the central stalk and drives its rotation. F o is minimally composed of subunits abc 10 with the c 10 arranged as a cylinder within the membrane (4). Subunit a is thought to directly participate in the proton flow while subunit b is minimally involved in the structure of the peripheral stalk. The peripheral stalk is composed of subunits b, d, h, and subunit 5 (5-8) and acts as a stator connecting F 1 with ...