Dual Functions of α-Ketoglutarate Dehydrogenase E2 in the Krebs Cycle and Mitochondrial DNA Inheritance in Trypanosoma brucei

Abstract

The dihydrolipoyl succinyltransferase (E2) of the multisubunit ␣-ketoglutarate dehydrogenase complex (␣-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional ␣-KD, bloodstream form (BF) trypanosomes express ␣-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, ␣-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for ␣-KDE2 in kDNA maintenance was revealed in ␣-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that ␣-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.

Tubulin-binding cofactor C domain-containing protein TBCCD1 orchestrates cytoskeletal filament formation

“…3G); the size and fluorescence intensity of the large kinetoplasts indicated kDNA replication still occurred. Similar increases in kinetoplast size, corresponding to continued kDNA replication were observed in RNAi mutants deficient in mitochondrial TAC components (Zhao et al, 2008;Sykes and Hajduk, 2013). Our observations indicate loss of centriolar TbTBCCD1 causes asymmetric mis-segregation of kDNA.…”

“…Detailed analyses of TAC-kinetoplast association are currently limited to mitochondrial proteins (Ochsenreiter et al, 2008;Zhao et al, 2008;Sykes and Hajduk, 2013). We therefore looked further, by using TEM, at the kDNA mis-segregation phenotype arising following RNAi against TbTBCCD1 (a nonmitochondrial, TAC-associated protein).…”

The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes

“…␣-KD is a vital component of energy metabolism in most aerobic prokaryotes and eukaryotes. ␣-KDE1, ␣-KDE2, and ␣-KDE3 mRNAs are constitutively expressed in both BF and PF T. brucei trypanosomes, yet a functional Krebs cycle and ␣-KD activity are present only in PF trypanosomes (12). We showed that ␣-KDE2 is a bifunctional protein that localized to the mitochondrion of BF T. brucei but was not involved in energy production.…”

In vivo analysis of trypanosome mitochondrial RNA function by artificial site-specific RNA endonuclease-mediated knockdown

“…To determine whether the A6-ASRE protein was synthesized and imported into the mitochondrion, total cell lysates were prepared from T. brucei transfectants and fractionated by differential centrifugation (Ochsenreiter and Hajduk 2006;Sykes and Hajduk 2013). Total cell (T), cytosolic (C), and mitochondrial (M) protein fractions were prepared either before (uninduced) or 24 h post induction with tetracycline (induced) (Fig.…”

“…Purified mitochondria were further purified into membrane and matrix fractions as previously described (Sykes and Hajduk 2013). In brief, purified mitochondria were incubated in 0.5% Triton X-100, 20 mM HEPES-NaOH (pH 7.6) with protease inhibitor cocktail (Roche) on ice for 45 min.…”