Mitochondrial calcium transport in trypanosomes

Abstract

The biochemical peculiarities of trypanosomes were fundamental for the recent molecular identification of the long-sought channel involved in mitochondrial Ca(2+) uptake, the mitochondrial Ca(2+) uniporter or MCU. This discovery led to the finding of numerous regulators of the channel, which form a high molecular weight complex with MCU. Some of these regulators have been bioinformatically identified in trypanosomes, which are the first eukaryotic organisms described for which MCU is essential. In trypanosomes MCU is important for buffering cytosolic Ca(2+) changes and for activation of the bioenergetics of the cells. Future work on this pathway in trypanosomes promises further insight into the biology of these fascinating eukaryotes, as well as the potential for novel target discovery.

Keywords: Acidocalcisome; Calcium; Inositol 1,4,5-trisphosphate receptor; Mitochondrial calcium uniporter.

Fig. 1

Domain organization of MCU and MCUb proteins highlighting two highly conserved transmembrane domains and one putative pore region, from Trypanosoma brucei (TbMCU, Tb427tmp.47.0014; TbMCUb, Tb427.10.300), Trypanosoma cruzi (TcMCU, TcCLB.503893.120; TcMCUb, TcCLB.504069.4), Leishmania major (LmMCU, LmjF.27.0780; LmMCUb, LmjF.21.1690), and Homo sapiens (HsMCU, NP_612366.1; HsMCUb, NP_060388.2). Two critical conserved substitutions from MCU to MCUb are boxed and indicated with arrows. MTS, mitochondrial targeting sequence; coil, coiled-coil domain; TM, transmembrane domain; DIME, functional “DIME” motif, the putative Ca²⁺ selectivity filter.

Fig. 2

Schematic representation of the mitochondrial calcium uniporter complex (MCUC) organization of mammals (A) and trypanosomes (B), was based on our interpretation of published data, and modified from Pendin et al. (52) and Sancak et al. (57). IMS: intermembrane space. Black balls represent Ca²⁺.

Fig. 3

Domain organization of MICU1 and MICU2 proteins highlighting two conserved EF hands, from Trypanosoma brucei (TbMICU1, Tb427.08.1850; TbMICU2, Tb427.07.2960), Trypanosoma cruzi (TcMICU1, TcCLB.511391.210; TcMICU2, TcCLB.510525.130), Leishmania major (LmMICU1, LmjF.07.0110), and Homo sapiens (HsMICU1, NP_006068.2; HsMICU2, NP_689939.1). MTS, mitochondrial targeting sequence; EF, EF-hand domain for Ca²⁺ binding

Fig. 4

Representative tracings of Ca²⁺ uptake by digitonin-permeabilized wild-type procyclic (A) and bloodstream (B) forms of T. brucei. (A). The reaction buffer contained 2 mM succinate, and 1 μM Calcium Green 5-N. After several pulses of Ca²⁺ (5 μM final concentration), 5 × 10⁷ procyclic forms were added to the reaction medium (2.45 ml) and the reaction was started adding 50 μM digitonin. Blue arrow indicates addition of digitonin to permeabilize the cells. No Ca²⁺ uptake was detected in the absence of succinate (not shown). (B) The reaction containing 2 × 10⁸ blodstream forms with 1 μM Calcium Green 5-N was started adding 40 μM digitonin in the presence of 1 mM ATP and 500 μM sodium orthovanadate. Ruthenium red (RR, 40 μM, red tracing), FCCP (5 μM) or oligomycin (Oligo, 2.5 μg/ml) were added where indicated. No Ca²⁺ uptake was detected in the absence of ATP (not shown). A decrease in fluorescence indicates decreasing medium Ca²⁺ or increasing mitochondrial Ca²⁺. Other conditions as in reference .

Fig. 5

Scheme of the energy metabolism in procyclic trypomastigotes. Arrows indicate steps of glucose, proline, and threonine metabolism and glycerolipid biosynthesis; dashed arrows indicate steps for which no evidence of flux is available. Abbreviations: A, ATPase; AcCoA, acetyl-CoA; Cit, citrate, CoASH, coenzyme A; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; G3P, glycerol 3-phosphate; KG, 2-ketoglutarate; Mal, malate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Resp. Ch., respiratory chain; TCA, tricarboxylic acid cycle. Enzymes are: 1, pyruvate dehydrogenase; 2, acetyl-CoA thioesterase; 3, acetate:succinate CoA-transferase; 4, succinyl-CoA synthetase; 5, F₀F₁-ATP synthase; 6, acetyl-CoA synthetase; 7, threonine dehydrogenase; 8, proline dehydrogenase. Activities potentially stimulated by Ca²⁺ are indicated. Reprinted with permission from ref. .

Fig. 6

Scheme of the energy metabolism in bloodstream trypomastigotes. Abbreviations and symbols as in Fig. 5, except for: A, ATPase; FAS II, type II fatty acid biosynthesis pathway; GPDH, glycerol 3-phosphate dehydrogenase; UQ, ubiquinone; TAO, trypanosome alternative oxidase. Pyruvate is mostly excreted out of the cells. Activity potentially stimulated by Ca²⁺ is indicated. Reprinted with permission from ref. .