Futile import of tRNAs and proteins into the mitochondrion of Trypanosoma brucei evansi

Abstract

Trypanosoma brucei brucei has two distinct developmental stages, the procyclic stage in the insect and the bloodstream stage in the mammalian host. The significance of each developmental stage is punctuated by specific changes in metabolism. In the insect, T. b. brucei is strictly dependent on mitochondrial function and thus respiration to generate the bulk of its ATP, whereas in the mammalian host it relies heavily on glycolysis. These observations have raised questions about the importance of mitochondrial function in the bloodstream. Peculiarly, akinetoplastic strains of Trypanosoma brucei evansi that lack mitochondrial DNA do exist in the wild and are developmentally locked in the glycolysis-dependent bloodstream stage. Using RNAi we show that two mitochondrion-imported proteins, mitochondrial RNA polymerase and guide RNA associated protein 1, are still imported into the nucleic acids-lacking organelle of T. b. evansi, making the need for these proteins futile. We also show that, like in the T. b. brucei procyclic stage, the mitochondria of both bloodstream stage of T. b. brucei and T. b. evansi import various tRNAs, including those that undergo thiolation. However, we were unable to detect mitochondrial thiolation in the akinetoplastic organelle. Taken together, these data suggest a lack of connection between nuclear and mitochondrial communication in strains of T. b. evansi that lost mitochondrial genome and that do not required an insect vector for survival.

Trypanosome Letm1 Protein Is Essential for Mitochondrial Potassium Homeostasis

“…Digitonin Fractionation, Western Blot Analysis, and Triton X-114 Separation of Membrane Proteins-Digitonin fractionation of cells into cytosolic and mitochondrial compartments and Western blots were performed as described previously (36). Antibodies against the T. brucei mitochondrial heat shock protein, cytochrome c, TrCOIV, and the ␤-subunit of F 1 -ATPase (31, 45) were used at 1:1000 dilutions, whereas enolase was used at 1:10,000 dilutions.…”

“…Letm1 Is Dispensable for Mitochondrial Translation-Because T. brucei evansi has lost its mt genome, components responsible for gene expression in the organelle have been rendered redundant (31,36). Thus, the essential nature of Letm1 in these 0 trypanosomes argues ostensibly against a primary function in mt translation via its interaction with ribosomes.…”

“…Membrane potential is maintained by the remaining F O F 1 -ATP synthase, which hydrolyzes ATP to pump H ϩ out of the matrix (31,32). However, the BS mitochondrion is not dormant because organellar gene expression is still needed for cell viability (33)(34)(35)(36), and a handful of essential mt biochemical pathways have been revealed (31,32,(37)(38)(39).…”

Sex is a ubiquitous, ancient, and inherent attribute of eukaryotic life

“…This is exemplified by T. b. evansi surviving loss of mitochondrial (kinetoplast) DNA by skipping the tsetse fly vector, in which it is needed, altogether (compare its loss of sex; see above). Based on almost zero mutation rates of hundreds of unused genes encoding mitochondrial proteins (65) with proven lack of essentiality (81), it has been proposed that after relatively short periods of time, these lineages vanish, being replaced by new strains that, via disruption of kinetoplast DNA (and hence of sex), entered the path to their slow, yet inevitable demise (65,82).…”

Dynamics of Mitochondrial RNA-Binding Protein Complex in Trypanosoma brucei and Its Petite Mutant under Optimized Immobilization Conditions

“…Thus, these trypanosomes can be considered an analog to rho 0 petite mutant Saccharomyces cerevisiae, which also lacks mitochondrial DNA (9,21,22). Yet, AK T. b. evansi still imports the protein machinery required for RNA processing despite the lack of substrate nucleic acids, as well as all tRNAs (21,(23)(24)(25). Among the imported macromolecular complexes assembled from the imported proteins are the MRP1/2 heterotetramer and a catalytically active RNA-editing core complex (RECC) that coordinates the enzymatic steps required for U insertion/deletion (21,25).…”