Acetyl:Succinate CoA-transferase in Procyclic Trypanosoma brucei

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 Δasct/Δasct 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 β-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of 13C-enriched and 14C-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. 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 Δasct/Δasct 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 β-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of 13C-enriched and 14C-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. Acetyl:succinate CoA-transferase (ASCT) 1The abbreviations used are: ASCT, acetyl:succinate CoA-transferase; BSD, blasticidin; BLE, phleomycin; PBS, phosphate-buffered saline; RNAi, RNA interference; wt, wild type. is an enzyme that transfers the CoA moiety of acetyl-CoA to succinate, yielding acetate and succinyl-CoA. ASCT was earlier characterized as an acetate-producing activity in Trypanosomatidae (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar). Trypanosomatidae are the earliest branching eukaryotes that retained mitochondria. They belong to the order Kinetoplastida, characterized by the kinetoplast, a highly concatenated form of the genome of the single mitochondrion. These unicellular eukaryotes contain unique organelles, such as glycosomes, which harbor part of the glycolytic pathway (2Opperdoes F.R. J. Bioenerg. Biomembr. 1994; 26: 145-146Crossref PubMed Scopus (11) Google Scholar, 3Hannaert V. Bringaud F. Opperdoes F.R. Michels P.A. Kinetoplastid. Biol. Dis. 2003; 2: 11Crossref PubMed Scopus (139) Google Scholar). The energy metabolism of Trypanosomatidae is characterized by excretion of mainly partially oxidized end products like pyruvate, succinate, and acetate. All of the Trypanosomatidae investigated produce acetate to a certain extent during their life cycle (4Cazzulo J.J. FASEB J. 1992; 6: 3153-3161Crossref PubMed Scopus (175) Google Scholar, 5Blum J.J. Parasitol. Today. 1994; 9: 118-122Abstract Full Text PDF Scopus (57) Google Scholar, 6Opperdoes F.R. Marr J.M. London A. Biochemistry and Molecular Biology of Parasites. Academic Press, Inc., London, UK1995: 19-31Crossref Google Scholar, 7Van Hellemond J.J. Van Der Meer P. Tielens A.G.M. Parasitology. 1997; 114: 351-360Crossref Scopus (36) Google Scholar), but the amount of acetate produced differs widely depending on the species and the stage of development. Trypanosoma brucei, one of the causative agents of African trypanosomiasis, alternates during its life cycle between the bloodstream of its mammalian host and the blood-feeding tsetse insect vector Glossina spp. In the mammalian bloodstream long slender-form parasites proliferate. At the peak of parasitemia, nonproliferative short stumpy cells develop that are prepared to differentiate to procyclic insect stage parasites. The long slender stage depends entirely on glycolysis for energy generation and excretes pyruvate as the main end product of carbohydrate metabolism (8Fairlamb A.H. Opperdoes F.R. Morgan M.J. Carbohydrate Metabolism in Cultured Cells. Plenum Publishing Corp., New York1986: 183-224Crossref Google Scholar, 9Opperdoes F.R. Annu. Rev. Microbiol. 1987; 41: 127-151Crossref PubMed Scopus (429) Google Scholar, 10Clayton C. Michels P.A. Parasitol. Today. 1996; 12: 465-471Abstract Full Text PDF PubMed Scopus (106) Google Scholar). The procyclic stage, on the other hand, can also use amino acids for its energy generation, and its glucose metabolism is completely reorganized, with succinate and acetate as main end products. Recently it was shown that inside the glycosome succinate is produced by a soluble fumarate reductase (11Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In the procyclic stage the mitochondrion is directly involved in the degradation of substrates, in contrast to the situation in the long slender bloodstream stage. Pyruvate enters the mitochondrion and is converted by pyruvate dehydrogenase into acetyl-CoA. This acetyl-CoA is not degraded to carbon dioxide via the Krebs cycle but is converted into acetate by ASCT (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar, 12Van Weelden S.W. Fast B. Vogt A. Van Der Meer P. Saas J. Van Hellemond J.J. Tielens A.G. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The ASCT reaction concomitantly produces succinyl-CoA, which is recycled to succinate by succinyl-CoA synthetase, an enzyme that also forms part of the Krebs cycle and produces ATP. ASCT activity is also known to occur in the anaerobically functioning mitochondria of metazoa that produce acetate, such as parasitic helminths like Fasciola hepatica and Ascarissuum (13Saz H.J. deBruyn B. de Mata Z. J. Parasitol. 1996; 82: 694-696Crossref PubMed Scopus (15) Google Scholar, 14McLaughlin G.L. Saz H.J. deBruyn B.S. Comp. B. PubMed Scopus Google Scholar). ASCT is also a enzyme in the metabolism of a of such as such as and such as A. M. Parasitol. PubMed Scopus Google Scholar, Microbiol. PubMed Scopus Google Scholar, A.H. Biol. PubMed Scopus Google Scholar). In ASCT is inside their hydrogenosomes, are to and in the M. A.G. Proc. Biol. Sci. PubMed Scopus Google Scholar, T. J. 1996; PubMed Scopus Google Scholar). of and the of at the of that mitochondrial showed that and mitochondria are Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, A. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, S. Proc. Biol. Sci. 1996; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). They from the of the of and it is that by to M. 1998; PubMed Scopus Google Scholar). The between mitochondria and the of however, the main in energy generation of mitochondria and is the of pyruvate and the of not The cycle is the only pathway to mitochondria and (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar). that of from a to the of the organelles, the of hydrogenosomes, and functioning mitochondria. the of not a single ASCT gene is known identified the gene encoding ASCT of T. brucei. and studies used to the between ASCT gene and mitochondrial ASCT enzyme of 13C-enriched and 14C-labeled glucose to wild and transgenic of T. brucei. The and in the gene of T. brucei ASCT and also that this enzyme is indeed involved in acetate ASCT is not an gene as ASCT is not the only enzyme acetate in T. brucei. from The for and was from M. A. The of that part of is from the genome it is with of of of the of was and in the by a M. A. 1994; PubMed Scopus Google from of C. the from by a from S. C. Parasitol. PubMed Scopus Google to the and the with produce RNA the of the T. brucei ASCT to was from and into S. Parasitol. PubMed Scopus Google to a a of the T. brucei ASCT in the expression vector by and S. C. Parasitol. PubMed Scopus Google and was as for of a protein gene expression F. S. Biteau N. Baltz Baltz T. Parasitol. PubMed Scopus Google Scholar). in T. brucei, the was from a product from T. brucei with the and and from a from with and The into a M. Van J. A. Van N. P. 1998; 95: Full Text Full Text PDF PubMed Scopus Google Scholar). was as Weelden S.W. Fast B. Vogt A. Van Der Meer P. Saas J. Van Hellemond J.J. Tielens A.G. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (99) Google with an of the by and procyclic T. brucei cells at in and Weelden S.W. Fast B. Vogt A. Van Der Meer P. Saas J. Van Hellemond J.J. Tielens A.G. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, M. Google at between and The procyclic used in this was from a tsetse with bloodstream M. Van N. J. PubMed Scopus Google Scholar). The procyclic was from the bloodstream by in Boshart M. Parasitol. 1996; 82: PubMed Scopus Google Scholar). The and the procyclic cells the RNA gene and the of a RNA are in Bringaud F. S. Biteau N. Baltz Baltz T. Parasitol. PubMed Scopus Google and in S. C. Parasitol. PubMed Scopus Google Scholar), and of as P. Boshart M. Biol. Google with the for and was used at and cells procyclic cells directly in with generation of the cells and in and in of to the amino acids of ASCT by a was in the the expression vector of at with the cells by and by to the in by at of of on and with The was on protein acids to an in vector with for was by in and was on the and the protein lysates of procyclic form T. brucei by and on Press, Scholar). The with PBS, for at and in PBS, with T. brucei brucei ASCT brucei protein for at the was and with the cells with as F. Baltz Baltz T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). with and the F. S. Baltz C. Baltz T. Parasitol. PubMed Scopus Google was by and with a and by a and and in as in Saas J. A. Fast B. Boshart M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was from N. J. Biol. Chem. PubMed Scopus Google Scholar). The and in of J. A. Fast B. Boshart M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and of on at the reaction was by of in on the and in and of The and with and of for at The was by on ASCT of procyclic T. brucei prepared from cells in a was by for at The activity of ASCT was by a as (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar). In the succinate, and The was by the of the at for and by the of products by and by cells by at for The cells and in was to the and on for protein in to the and for at The was by for The was and the with and in the the and the for ASCT lysates with only used as the brucei procyclic cells in the to in of with for at as (11Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, V. S. Biran M. P. V. P. Michels P.A. Canioni P. Baltz T. Bringaud F. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). The and not during the in the was at the and the end of the with the The was and in of and of was as an as (11Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, V. S. Biran M. P. V. P. Michels P.A. Canioni P. Baltz T. Bringaud F. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). The was for a The of acetate and succinate and was from J. Scholar, Proc. Natl. Acad. Sci. U. S. A. 82: PubMed Scopus Google as V. S. Biran M. P. V. P. Michels P.A. Canioni P. Baltz T. Bringaud F. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). The and of the of succinate, acetate, pyruvate, the on the Biran and P. The amount of 13C-enriched was on the of the the amount of from the to the with the T. brucei cells at for at in of the for with a of and The the of of by the of of to the to was the and the on the with for at In this carbon dioxide was acetate in the The carbon dioxide was in a of with of and of to the by PubMed Scopus Google Scholar, Tielens A.G. Parasitology. PubMed Scopus Google Scholar). of carbon the was from the cells by at and by the of Analysis of the end products by on a in form A.G. Meer P. Parasitol. PubMed Scopus Google Scholar). The was with of of and of The and with of in a All for end products identified by their a F. Scholar). was with a and as the A. PubMed Scopus Google Scholar). of an ASCT of the is by a of and CoA J. PubMed Scopus Google that protein in protein and a of a of mammalian is with acetate, the of for gene in the T. brucei genome a single was and the to amino its with transferase from single and not of this gene to of the T. brucei genome The gene a protein amino with CoA transferase in the of CoA 2002; 41: PubMed Scopus Google Scholar). All amino acids in of the in the of transferase 2002; 41: PubMed Scopus Google are in the new CoA transferase identified in T. brucei. also identified in T. amino and amino of ASCT expression of the ASCT gene with ASCT activity, procyclic T. brucei for of the of the was between of a and was into the host for the and for of RNA At with the ASCT activity was as (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google in lysates of and The activity to the produced expression in and used for of the protein by by with The of an correlation between the amount of the protein and ASCT activity the of the protein was at the of of from The a long 2) also with and from a to produce expression of an of the that into an RNA protein and ASCT activity of that the procyclic host and are in the shown in and that the gene of ASCT was from the growth The population of and increased from to and from to upon of ASCT in procyclic T. brucei and from cells with the cells with at and and for The ASCT activity in was as the of the activity in the The at the of the lysates used for the enzyme with the the for the and are as of and of T. brucei the of the ASCT procyclic with gene of ASCT with and and BSD, and and of the in the was by of the in in out of only out of confirmed as homozygous gene and by and This of and the in of the ASCT gene This was also by a severe growth of the knock out The population increased from to and a of was by of in The absence of ASCT protein on and a ASCT activity in knock out confirmed of The enzyme activity by the ASCT in knock out was to an acetate-producing activity that is of succinate from ASCT not of the Δasct/Δasct and of ASCT, into the which into the of This in a of ASCT activity the correlation between ASCT expression and enzyme protein of the activity the between ASCT protein and enzyme activity upon The a population of but only of the growth was in the wild in a growth the of the growth of is to to the to of the the protein in depletion of ASCT activity from lysates of wild procyclic cells and of the transgenic ASCT activity in the which is to of the to the of activity to mitochondria of procyclic T. brucei (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar). the of the ASCT gene product with and that a identified as the mitochondrion by the in cells but not in Δasct/Δasct cells in procyclic cells also by of and ASCT protein was by in the soluble and and mitochondrial as The of ASCT was to that of indicating a mitochondrial This was confirmed by of with and ASCT in the mitochondrial but was completely in the soluble In with this a short amino mitochondrial was by via and by to transferase The colocalization of ASCT activity (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google and the gene product in the mitochondrion for and of the ASCT of ASCT by by soluble and mitochondrial to and with and with of P. by in the quantitative of the from A. The of a protein into the was from the of and the of of soluble and mitochondrial with Analysis of Metabolism of the T. brucei ASCT used a carbon to and the end products excreted by the wild and The parasites in as the only carbon to of was by The was by The wild and procyclic cells mainly 13C-enriched succinate, acetate, and and of the 13C-enriched excreted with of and and (11Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, V. S. Biran M. P. V. P. Michels P.A. Canioni P. Baltz T. Bringaud F. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). the of acetate excretion in the and was in the and of 13C-enriched excreted the of succinate excretion increased by in the the amount of 13C-enriched end products excreted by the increased by the of glucose was The glucose with the This was during the for as as in in The of excretion was in the as with cells of the 13C-enriched excreted of the ASCT gene in a of acetate production, other for of acetate excretion in the absence of ASCT The but not the wild also excreted of 13C-enriched pyruvate and β-hydroxybutyrate end products of metabolism by procyclic T. end products in of 13C-enriched excreted of of are the are shown of the is of the is of the is between and of the is of the is of the is of the is between and of the is of the is of the is of the is of the is of the is of the is of the is of the is of the is between and of the is of the is of the is of the is of the is between and of the is of the is of the is of the is of the is of the is of the is of the is of the is between and of the is end products in of 13C-enriched excreted of glucose during the in this of glucose by the for in at of glucose of of the is of the is of the is of the is The of are the are shown of the is of the is between and of the is of glucose during the in this of glucose by the for in at in a new The Δasct/Δasct showed of glucose and end product excretion to and excretion of pyruvate and β-hydroxybutyrate was for of ASCT protein and activity the amount of excreted acetate increased from in the wild to of 13C-enriched excreted protein in the the of Δasct/Δasct cells was by ASCT the between the and ASCT in and cells with the an of the of glucose and end product a of the of acetate and succinate production, and excretion of β-hydroxybutyrate The and an This was not as a certain of the expression in T. brucei, which of is S. C. Parasitol. PubMed Scopus Google Scholar, F. S. Biteau N. Baltz Baltz T. Parasitol. PubMed Scopus Google Scholar, V. S. Biran M. P. V. P. Michels P.A. Canioni P. Baltz T. Bringaud F. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). the amount of ASCT protein in and of the protein in the wild not In to the which only on cells at in a also investigated the end product in wild and procyclic cells The end products excreted during growth in glucose acetate and succinate as Weelden S.W. Fast B. Vogt A. Van Der Meer P. Saas J. Van Hellemond J.J. Tielens A.G. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The of that in of the of acetate and only acetate and succinate are in The absence of ASCT not in a in the of acetate and succinate a but not in acetate is by the of the wild and Δasct/Δasct cells This is in with the with 1) and also with the of end products excreted by Δasct/Δasct and and The degradation from the of excreted was not increased in cells with the wild that the of and are and that showed the of Δasct/Δasct activity and end products of glucose in procyclic T. end of of from the amount of end of product of protein was the of the amount of protein from to end of the of glucose of of activity was shown to a acetate from from wt, Δasct/Δasct of from wt, from wt, from wt, from wt, from wt, from the amount of end product of protein was the of the amount of protein from to end of the of Δasct/Δasct of This activity was shown to a acetate from from wt, Δasct/Δasct of in a new Analysis of the that ASCT activity 3) showed a in acetate production, succinate was This is in with the studies and for an of ASCT in acetate ASCT enzyme activity in T. brucei is by a identified member of the CoA transferase gene family. This is the of a gene encoding of the 1) The genome of T. brucei only one member of the highly and CoA transferase gene J. PubMed Scopus Google Scholar). 2) quantitative correlation was between ASCT activity and the amount of protein by the ASCT with was 3) of the ASCT gene in protein product and activity the 4) of the gene product in T. brucei in increased ASCT activity and increased acetate 5) the gene product and ASCT activity are in procyclic form T. brucei but not in the bloodstream stage of the parasite not of the ASCT gene product was by and to mitochondrial (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google of the ASCT ASCT activity by with a the ASCT gene expression of ASCT protein in and in protein with In procyclic T. brucei, acetyl-CoA produced from glucose is converted into acetate and is not the Krebs cycle Weelden S.W. Fast B. Vogt A. Van Der Meer P. Saas J. Van Hellemond J.J. Tielens A.G. Boshart M. J. Biol. Chem. 2003; 278: 12854-12863Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Van Hellemond (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google characterized a mitochondrial ASCT activity in involved in acetate from acetyl-CoA. investigated the of ASCT for acetate in wild and by and quantitative of the excreted end products of glucose the of acetate excretion was only slightly in the of ASCT activity, an involved in acetate from In a acetate from acetyl-CoA was (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar). This activity increased in the Δasct/Δasct as an during of the knock out acetate excretion is only slightly the show a of ASCT 1) The Δasct/Δasct pyruvate and which are not in the of the wild type. In β-hydroxybutyrate is the end product of a pathway used to of acetyl-CoA. of the are in the T. brucei genome not a pathway is to in this In pyruvate is the of the ASCT acetyl-CoA. the excretion of β-hydroxybutyrate and pyruvate the of acetyl-CoA accumulation in cells, which are in acetyl-CoA into acetate to the absence of is that the acetate not to the produced acetyl-CoA. 2) The of glucose increased as a of ASCT gene of ASCT gene expression depending on the and the produced by ASCT can converted into succinate by the succinyl-CoA with a of by (1Van Hellemond J.J. Opperdoes F.R. Tielens A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3036-3041Crossref PubMed Scopus (114) Google Scholar). The of glucose in the absence of ASCT, an of energy from 3) The and show of their growth which that ASCT activity is for functioning of procyclic T. brucei. The was and the growth was partially by ASCT the of ASCT deficiency, which to to accumulation of and the that of ASCT in a of acetate excretion in that ASCT is a for acetate in the ASCT is not an gene and an acetate-producing the importance of the ASCT for functioning is 1) The of upon of the was and 2) that in of the vector was not in the ASCT a event in T. brucei, is the Δasct/Δasct cells are short not the for an Δasct/Δasct are to the wild growth The of this long is not In the and the studies show that the identified gene for the ASCT enzyme in T. brucei. via ASCT in parasites as as but is in their The enzyme to a for This not to African trypanosomiasis, ASCT is not in the bloodstream stage of T. brucei the mammalian ASCT however, in of other The gene of T. brucei ASCT of ASCT in such as N. and T. which contain the of studies on ASCT, one of the shared by and mitochondria of on the between mitochondria and the of M. A. for genome to the and for from the at The for for the of and and for vector and Boshart by of and is for of product