Mitochondrial calcium transport in trypanosomes

Cell Biology International

Bertolini

et al. 2018

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The presence of a conserved mechanism for mitochondrial calcium uptake in trypanosomatids was crucial for the molecular identification of the mitochondrial calcium uniporter (MCU), a long-sought channel present in most eukaryotic organisms. Since then, research efforts to elucidate the role of MCU and its regulatory elements in different biological models have multiplied. MCU is the pore-forming subunit of a multimeric complex (the MCU complex or MCUC) and its predicted structure in trypanosomes is simpler than in mammalian cells, lacking two of its subunits and probably possessing other unidentified components. MCU protein has been characterized in Trypanosoma brucei and Trypanosoma cruzi, the causative agents of African and American trypanosomiasis, respectively. Contrary to its mammalian homolog, TbMCU was found to be essential for cell growth and survival, while its paralog MCUb is an essential protein in T. cruzi. These findings could be further exploited for chemotherapeutic purposes. The emergence of new molecular tools for the genetic manipulation of trypanosomatids has been determinant for the functional characterization of the MCUC components in these organisms. However, further research has to be done to determine the role of each component in intracellular calcium signaling and cell bioenergetics. In this mini-review we summarize the original results on mitochondrial calcium uptake in trypanosomes, how did they contribute to the molecular identification of the MCU, and the functional characterization of the MCUC subunits that has so far been studied in these peculiar eukaryotes.

Section: The Mcu Complex In Trypanosomessupporting

confidence: 81%

Section: Introductionmentioning

confidence: 99%

The mitochondrial calcium uniporter complex in trypanosomes

Cell Biology International

Bertolini

et al. 2018

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“…We next investigated the localization of two proteins for which either no previous localization studies have been reported (TcMCU) (20) or for which its localization has been disputed (TcIP 3 R) (12). TcMCU is the T. cruzi orthologue of the recently discovered MCU from vertebrate cells (8,9) and of TbMCU (10).…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor

Journal of Biological Chemistry

Storey

et al. 2016

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Edited by Dennis VoelkerMethods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIP 3 R (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and acidocalcisomes, respectively. As expected, TcMCU was colocalized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIP 3 R, endogenously tagged TcIP 3 R showed co-localization with antibodies against VP1 to acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi.The application of the CRISPR/Cas9 technology to the study of protist parasites has dramatically increased the tools available for their genetic manipulation (1). Trypanosoma cruzi, the etiologic agent of Chagas disease, which is a significant cause of morbidity and mortality from the South of the United States to the South of Argentina and Chile, has been particularly refractory to genetic manipulation. However, the recent use of the CRISPR/Cas9 technology to knock down or knock out genes (2, 3) has revolutionized their study.The localization of proteins is important to determine their cellular function, and previous studies in T. cruzi have used either antibodies or gene tagging methods with vectors that overexpressed the proteins (4). Although specific antibodies are useful to detect the endogenous proteins, it is not always possible to obtain them because either the proteins have low antigenicity or the antibodies cross-react with other proteins. Plasmids that enable the tagging of genes at their endogenous loci are not available for T. cruzi, and a major drawback of the overexpression of tagged proteins is that the proteins of interest sometimes are retained in the endoplasmic reticulum (ER) 4 or localize to other compartments.Here we have adapted the CRISPR/Cas9 system to tag genes of T. cruzi at their endogenous loci and tested this system with two genes encoding proteins of well recognized localization (flagellar calcium binding protein or TcFCaBP and the acidocalcisome vacuolar proton pyrophosphatase or TcVP1) and two encoding proteins of undefined or disputed localizati...

“…The finding of MCU activity in trypanosomatids, such as Trypanosoma cruzi (13,14), the agent of Chagas disease, and T. brucei (15), which belongs to the group of parasites that cause sleeping sickness, was important for the discovery of a gene encoding a modulator of the uniporter (mitochondrial calcium uptake 1, or MICU1) (16) and the pore subunit of the channel or MCU (17)(18)(19). After the molecular identification of MCU, other components of the MCU complex were described (20 -23), but only orthologs of MCU, MCUb, MICU1, and MICU2 are present in trypanosomes, whereas MCU regulator (MCUR1) and essential MCU regulator (EMRE) are absent (24,25). Mitochondrial Ca 2ϩ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca 2ϩ increases, for the bioenergetics of the cells, and for viability and infectivity (26,27).…”

mentioning

confidence: 99%

Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of Trypanosoma cruzi

Journal of Biological Chemistry

Bertolini

et al. 2018

Self Cite

In vertebrate cells, mitochondrial Ca 2؉ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca 2؉-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1␣ subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation/dephosphorylation cycle for the E1␣ subunit of PDH from nonvertebrate organisms has been described, the Ca 2؉-mediated PDP activation has not been studied. In this work, we investigated the Ca 2؉ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and T. brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP's role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca 2؉-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP/ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting cultured host cells. We conclude that TcPDP is a Ca 2؉-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity, and energy metabolism in T. cruzi. Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.