Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism.
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.
Laboratoire de Genomique Fonctionnelle des Trypanosomatides, UMR-5162 CNRS, Université Victor Segalen Bordeaux II, 33076 Bordeaux Cedex, France.
Loïc
L
Rivière
Susanne W H
SW
van Weelden
Patricia
P
Glass
Patricia
P
Vegh
Virginie
V
Coustou
Marc
M
Biran
Jaap J
JJ
van Hellemond
Frédéric
F
Bringaud
Aloysius G M
AG
Tielens
Michael
M
Boshart
D000818:Animals; D015256:Coenzyme A-Transferases / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D019906:Nuclear Magnetic Resonance, Biomolecular; D034622:RNA Interference; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D015256:Coenzyme A-Transferases; C049804:acetate-succinate CoA-transferase; D005947:Glucose