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Acetate Produced in the Mitochondrion Is the Essential Precursor for Lipid Biosynthesis in Procyclic Trypanosomes

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Acetate Produced in the Mitochondrion Is the Essential Precursor for Lipid Biosynthesis in Procyclic Trypanosomes Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes Loı¨cRivie`rea, Patrick Moreaub, Stefan Allmannc, Matthias Hahnc, Marc Birand, Nicolas Plazollesa, Jean-Michel Franconid, Michael Boshartc, and Fre´de´ ric Bringauda,d,1 aLaboratoire de Microbiologie Cellulaire et Mole´culaire et Pathoge´nicite´, Unite´Mixte de Recherche 5234 Centre National de la Recherche Scientifique, bLaboratoire de Biogene`se Membranaire, Unite´Mixte de Recherche 5200 Centre National de la Recherche Scientifique, and dRe´sonance Magne´tique des Syste`mes Biologiques, Unite´Mixte de Recherche 5536, Centre National de la Recherche Scientifique, Universite´Victor Segalen Bordeaux 2, 146 rue Le´o Saignat, 33076 Bordeaux cedex, France; and cBiozentrum, Department Biologie I, Genetik, Ludwig-Maximilians-Universita¨t Mu¨ nchen, D-82152 Planegg-Martinsried, Germany Edited by M. Daniel Lane, Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 11, 2009 (received for review March 27, 2009) Acetyl-CoA produced in mitochondria from carbohydrate or amino synthesis of fatty acids (10) are noteworthy. Indeed, whereas acid catabolism needs to reach the cytosol to initiate de novo most cells use either a type I or type II synthase for de novo fatty synthesis of fatty acids. All eukaryotes analyzed so far use a acid synthesis, T. brucei is the only known organism using a third citrate/malate shuttle to transfer acetyl group equivalents from the mechanism for this process. This parasite uses microsomal mitochondrial matrix to the cytosol. Here we investigate how this elongases to synthesize short-, medium-, and long-chain fatty acetyl group transfer occurs in the procyclic life cycle stage of acids from butyryl-CoA, by using malonyl-CoA as a C2 donor, Trypanosoma brucei, a protozoan parasite responsible of human whereas other organisms only can extend premade medium and sleeping sickness and economically important livestock diseases. long FAs by elongases to make long and very long fatty acyl Deletion of the potential citrate lyase gene, a critical cytosolic chains (11). In addition, we show here that the procyclic try- enzyme of the citrate/malate shuttle, has no effect on de novo panosomes also use a pathway to produce cytosolic acetyl-CoA to feed de novo biosynthesis of fatty acids. biosynthesis of fatty acids from 14C-labeled glucose, indicating that In nonphotosynthetic eukaryotes, nearly all of the acetyl-CoA another route is used for acetyl group transfer. Because acetate is used in fatty acid synthesis is formed in mitochondria from produced from acetyl-CoA in the mitochondrion of this parasite, pyruvate oxidation and from the catabolism of the carbon we considered genes encoding cytosolic enzymes producing skeletons of amino acids. It is widely accepted that all eukaryotes acetyl-CoA from acetate. We identified an acetyl-CoA synthetase analyzed so far use the citrate/malate shuttle to transfer acetyl gene encoding a cytosolic enzyme (AceCS), which is essential for group equivalents from the mitochondrial matrix to the cytosol cell viability. Repression of AceCS by inducible RNAi results in a across the acetyl-CoA-impermeable mitochondrial inner mem- 20-fold reduction of 14C-incorporation from radiolabeled glucose brane [see (12)]. In this shuttle, intramitochondrial acetyl-CoA or acetate into de novo synthesized fatty acids. Thus, we demon- first reacts with oxaloacetate to form citrate, in the citric acid strate that the essential cytosolic enzyme AceCS of T. brucei is cycle (TCA) reaction catalyzed by citrate synthase (CS). Citrate responsible for activation of acetate into acetyl-CoA to feed de passes through the mitochondrial inner membrane on a citrate novo biosynthesis of lipids. To date, Trypanosoma is the only transporter or a citrate/malate exchanger, and is converted back known eukaryotic organism that uses acetate instead of citrate to to acetyl-CoA and oxaloacetate by the cytosolic citrate lyase transfer acetyl groups over the mitochondrial membrane for cyto- (CL) (see Fig. 1). In trypanosomes, existence of a citrate/malate solic lipid synthesis. shuttle was assumed to meet the needs of cytosolic fatty acid biosynthesis, although this view is not supported by any exper- acetyl-CoA synthetase ͉ citrate/malate shuttle ͉ de novo lipid imental data (11, 13). Here we demonstrate that this assumption biosynthesis ͉ mitochondrial acetate is incorrect for the procyclic stage of T. brucei. Instead, this parasite expresses a cytosolic ‘‘AMP forming’’ acetyl-CoA syn- thetase (AceCS), which is essential for incorporation of radio- Trypanosoma brucei is an unicellular eukaryote, belonging to the labeled glucose (or acetate) into fatty acids and essential for protozoan order Kinetoplastida, that causes sleeping sickness in growth. Thus, acetyl-CoA is converted into acetate in the humans and economically important livestock diseases. This mitochondrion (7), which cross the mitochondrial inner mem- parasite undergoes a complex life cycle during transmission from brane to be converted into acetyl-CoA by the cytosolic AceCS. the bloodstream of a mammalian host (bloodstream stages of the We have demonstrated that acetate can be used to exchange parasite) to the alimentary tract (procyclic stage) and salivary acetyl group equivalents between the mitochondrial and cyto- glands (epimastigote and metacyclic stages) of a bloodfeeding solic compartments. insect vector, the tse-tse fly. In addition to their relevance for health and development in subsaharan Africa, trypanosomes are Results famous for a variety of very unusual genetic and biochemical A Citrate/Malate Shuttle Is Not Required for Lipid Biosynthesis. It was features that stimulate broad scientific and evolutionary interest. assumed by analogy (8) that acetyl-CoA is exchanged in try- These include exotic mechanisms of gene expression like poly- cistronic transcription of genes (1), maturation of premessenger Author contributions: L.R., P.M., M. Boshart, and F.B. designed research; L.R., P.M., S.A., RNA by trans-splicing and extensive editing of mitochondrial M.H., M. Biran, N.P., and F.B. performed research; J.-M.F. contributed new reagents/analytic RNAs (2, 3), and sophisticated mechanisms of immune evasion tools; L.R., P.M., S.A., M.H., M. Biran, M. Boshart, and F.B. analyzed data; and L.R., P.M., M. like antigenic variation and antibody endocytosis (4, 5). In the Boshart, and F.B. wrote the paper. context of this report, metabolic peculiarities like the compart- The authors declare no conflict of interest. mentalization of glycolysis in glycosomes, which are peroxisome- This article is a PNAS Direct Submission. like organelles (6), the presence of a single, developmentally 1To whom correspondence should be addressed. E-mail: [email protected]. regulated mitochondrion per cell with unusual enzyme activities This article contains supporting information online at www.pnas.org/cgi/content/full/ (7, 8), energy metabolism (9) and unusual pathways for de novo 0903355106/DCSupplemental. 12694–12699 ͉ PNAS ͉ August 4, 2009 ͉ vol. 106 ͉ no. 31 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903355106 Downloaded by guest on September 28, 2021 Glucose Glycosomes Mitochondrion Succinate Succinate -1 cells) Mal TCA KG 8 G3P DHAP 2 Cit 1 4 CPM • (10 PEP Pyr AcCoA Acetate 5 6 1 23 4 1 23 4 1 23 4 1 23 4 1 23 4 7 PC PI PE TAG FFA Fatty acids AcCoA Acetate Fig. 2. D-[U-14C]-glucose incorporation into lipids of the EATRO1125.T7T 3 Cit and ⌬cl/⌬cl cell lines. This figure shows the relative amounts of 14C-labeled phospholipids (PC, PI, and PE) and 14C-labeled neutral lipids (TAG and FFA) Lipids extracted from the EATRO1125.T7T (black columns labeled 1) and ⌬cl/⌬cl knock-out (gray columns labeled 2, 3, and 4 for ⌬cl/⌬cl-C9, ⌬cl/⌬cl-E9, and Fig. 1. Schematic representation of lipid biosynthesis from D-glucose me- ⌬cl/⌬cl-B6, respectively) cell lines incubated in the presence of D-[U-14C]- tabolism in the procyclic form of T. brucei. Black arrows represent enzymatic glucose. Error bars indicate m Ϯ SD of 3 independent experiments. steps of D-glucose metabolism, and excreted end products are boxed. Dashed arrows indicate steps, which are supposed to occur at a background level or not at all. The enzymes targeted by RNAi are indicated by a boxed number. De which is a major source of acetyl-CoA in standard growth novo lipid biosynthesis is schematically represented by double line arrows. The glycosomal compartments, the mitochondrial compartment and the tricar- conditions (15)). Under these conditions, acetyl-CoA and glyc- 14 boxylic acid cycle (TCA) are indicated. Abbreviations: AcCoA, acetyl-CoA; Cit, erol-3-phosphate (G3P) produced from D-[U- C]-glucose ca- citrate; DHAP, dihydroxyacetone phosphate; G3P, glycerol 3-phosphate; KG, tabolism (Fig. 1) are major precursors for label incorporation 2-ketoglutarate; MAL, malate; PEP, phosphoenolpyruvate; Pyr, pyruvate. In- into the fatty acid and glycerol parts, respectively, of the glyc- dicated steps are: 1, citrate synthase (CS); 2, citrate transporter or citrate/ erolipids. Indeed, L-proline is a poor acetyl-CoA producer and malate exchanger; 3, citrate lyase (CL); 4, acetate:succinate CoA-transferase does not produce G3P in the presence of D-glucose (16). The (ASCT); 5, unknown enzyme; 6, passive diffusion or acetate carrier; 7, ‘‘AMP- major fatty-acid containing lipids labeled de novo were the forming’’ acetyl-CoA synthetase (AceCS). phospholipids (PC, phosphatidylcholine; PE, phosphatidyleth- anolamine;
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