Abstract
Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasite Trypanosoma brucei was identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Deltaasct/Deltaasct knock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of beta-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of (13)C-enriched and (14)C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate in T. brucei.
Highlights
Acetyl:succinate coenzyme A (CoA)-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria
Identification of an Acetyl:succinate CoA-transferase (ASCT) Candidate Gene—The transfer of the coenzyme A (CoA) group is catalyzed by a large family of prokaryotic and eukaryotic CoA transferases [47] that share conserved protein domains (CoA_trans in protein families data base, accession PF01144) and have a broad range of substrate specificities
2) A quantitative correlation was documented between ASCT activity and the amount of protein encoded by the candidate ASCT gene
Summary
The digestion was stopped by adding EDTA on ice. ASCT Activity Assay—Homogenates of procyclic T. brucei were prepared from 5 ϫ 107 cells in 20 mM HEPES buffer, pH 7.4, containing 1% Triton X-100 using a Teflon glass homogenizer. Nuclear Magnetic Resonance (NMR) Experiments—4 ϫ 109 T. brucei procyclic cells grown in the SDM79 medium (up to 1 ϫ 107 cells1⁄7mlϪ1) were incubated in 10 ml of incubation buffer (PBS buffer supplemented with 24 mM NaHCO3, pH 7.3) containing 110 mole D-[1-13C]glucose (11 mM) for 90 –180 min at 27 °C as described before [11, 39]. After removal of carbon dioxide, the acidified supernatant was separated from the cells by centrifugation (4 °C, 10 min at 500 ϫ g) and neutralized by the addition of 40 l of 6 M NaOH. Using defatted and dialyzed bovine serum albumin (Roche Applied Science) as the standard [46]
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