myo-Inositol Uptake Is Essential for Bulk Inositol Phospholipid but Not Glycosylphosphatidylinositol Synthesis in Trypanosoma brucei

González-Salgado, Amaia; Steinmann, Michael E.; Greganova, Eva; Rauch, Monika; Mäser, Pascal; Sigel, Erwin; Bütikofer, Peter

doi:10.1074/jbc.m112.344812

Cited by 34 publications

(67 citation statements)

References 63 publications

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“…Membrane transporters are of particular importance for these pathogens to acquire essential nutrients from their respective hosts and offer attractive targets for rational drug design and/or the delivery of cytotoxic substrate analogues. The reported dependence of T. brucei procyclic forms in culture on the presence of exogenous myo-inositol (13,14) validates HMIT as a potential drug target.…”

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confidence: 79%

“…Overexpression of C-terminal 3ϫ hemagglutinin (HA)-tagged TbHMIT was performed using inducible vector pALC14 as described previously (14), resulting in plasmid pAG3020-BSF2. Before transfection of T. brucei bloodstream forms, plasmid DNA was linearized with NotI and isolated with phenol and chloroform.…”

Section: Methodsmentioning

confidence: 99%

“…For immunolocalization of HA-tagged TbHMIT, trypanosomes were cultured in the presence of tetracycline for 24 h to induce protein expression and processed as described previously (14). HA-tagged proteins were detected using monoclonal mouse anti-HA antibody (Covance, Munich, Germany) at a dilution of 1:250 in PBS for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Direct evidence for the presence of PI synthase in the Golgi complex was obtained from immunolocalization studies in Trypanosoma brucei bloodstream forms (13), showing that the enzyme has a dual localization in the ER and Golgi complex. In support of the idea of the Golgi complex being the site of bulk PI synthesis in trypanosomes (13), a subsequent study revealed that T. brucei procyclic forms express a plasma membrane-and Golgi complex-localized proton-linked myo-inositol transporter, TbHMIT (14). Downregulation of TbHMIT in-hibited bulk PI formation but had no effect on GPI synthesis, demonstrating that PI synthesis in T. brucei is compartmentalized, with the Golgi complex representing the site of synthesis of bulk membrane PI utilizing exogenous myo-inositol (14) and the ER being the site of PI synthesis for GPI production utilizing de novosynthesized myo-inositol (15).…”

mentioning

confidence: 88%

“…In support of the idea of the Golgi complex being the site of bulk PI synthesis in trypanosomes (13), a subsequent study revealed that T. brucei procyclic forms express a plasma membrane-and Golgi complex-localized proton-linked myo-inositol transporter, TbHMIT (14). Downregulation of TbHMIT in-hibited bulk PI formation but had no effect on GPI synthesis, demonstrating that PI synthesis in T. brucei is compartmentalized, with the Golgi complex representing the site of synthesis of bulk membrane PI utilizing exogenous myo-inositol (14) and the ER being the site of PI synthesis for GPI production utilizing de novosynthesized myo-inositol (15). Transporter-mediated myo-inositol uptake has also been characterized in other protozoan parasites, including Leishmania donovani (16)(17)(18)(19) and Trypanosoma cruzi (20,21).…”

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confidence: 88%

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Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo -Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

et al. 2015

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c myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na ؉ -or H ؉ -linked myo-inositol transporters. While Na ؉ -coupled myo-inositol transporters are found exclusively in the plasma membrane, H ؉ -linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complexlocalized T. brucei H ؉ -linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol. In all eukaryotes, myo-inositol is the precursor for all inositolcontaining phospholipids, including phosphatidylinositol (PI), phosphatidylinositol (poly)phosphates, inositol phosphorylceramide (IPC), and glycosylphosphatidylinositol (GPI). In mammalian cells, it is taken up from the environment via sodium/myoinositol cotransporters (SMITs) or proton-linked myo-inositol transport (T. brucei H ϩ -linked myo-inositol transporter [TbHMIT]). Human SMIT1 and SMIT2 belong to the sodium/ glucose transporter family, SGLT/SLC5, whose members in general mediate uptake of sugars and osmolytes in the gastrointestinal tract and the kidney (1). They are localized in the plasma membrane and, besides myo-inositol, also transport xylose and glucose (2, 3). In contrast, the human HMIT belongs to the sugar/polyol transport facilitator family, GLUT/SLC2A (4). While most members of this family are also located in the plasma membrane and regulate sugar homeostasis within the body, members of the family of subclass III transporters, to which HMIT belongs, are typically localized intracellularly (5, 6). Interestingly, GLUT12/ SLC2A12 and HMIT/SLC2A13 have been found to colocalize with Golgi complex markers (7,8). Although the substrate specificities of the subclass III GLUT/SLC2A transporters have been studied in model systems, their physiological roles have not been firmly established (5, 6). Notably, HMIT completely lacks sugar transport activity but instead transports myo-inositol with a K m of approximately 100 M in a Xenopus oocyte expression system (8).As an alternative to uptake, myo-inositol can...

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confidence: 79%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 88%

mentioning

confidence: 88%

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Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo -Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

et al. 2015

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The Importance of Targeting Lipid Metabolism in Parasites for Drug Discovery

Young¹,

Roberts²,

Smith³

2016

Comprehensive Analysis of Parasite Biology: From Metabolism to Drug Discovery

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H+-dependent inorganic phosphate uptake in Trypanosoma brucei is influenced by myo-inositol transporter

Russo‐Abrahão

Koeller

Steinmann

et al. 2017

J Bioenerg Biomembr

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Trypanosoma brucei is an extracellular protozoan parasite that causes human African trypanosomiasis or "sleeping sickness". During the different phases of its life cycle, T. brucei depends on exogenous inorganic phosphate (P), but little is known about the transport of P in this organism. In the present study, we showed that the transport of P across the plasma membrane follows Michaelis-Menten kinetics and is modulated by pH variation, with higher activity at acidic pH. Bloodstream forms presented lower P transport in comparison to procyclic forms, that displayed an apparent K = 0.093 ± 0.008 mM. Additionally, FCCP (H-ionophore), valinomycin (K-ionophore) and SCH28080 (H, K-ATPase inhibitor) inhibited the P transport. Gene Tb11.02.3020, previously described to encode the parasite H:myo-inositol transporter (TbHMIT), was hypothesized to be potentially involved in the H:P cotransport because of its similarity with the Pho84 transporter described in S. cerevisiae and other trypanosomatids. Indeed, the RNAi mediated knockdown remarkably reduced TbHMIT gene expression, compromised cell growth and decreased P transport by half. In addition, P transport was inhibited when parasites were incubated in the presence of concentrations of myo-inositol that are above 300 μM. However, when expressed in Xenopus laevis oocytes, two-electrode voltage clamp experiments provided direct electrophysiological evidence that the protein encoded by TbHMIT is definitely a myo-inositol transporter that may be only marginally affected by the presence of P. These results confirmed the presence of a P carrier in T. brucei, similar to the H-dependent inorganic phosphate system described in S. cerevisiae and other trypanosomatids. This transport system contributes to the acquisition of P and may be involved in the growth and survival of procyclic forms. In summary, this work presents the first description of a P transport system in T. brucei.

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myo-Inositol Uptake Is Essential for Bulk Inositol Phospholipid but Not Glycosylphosphatidylinositol Synthesis in Trypanosoma brucei

Cited by 34 publications

References 63 publications

Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo -Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo -Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

The Importance of Targeting Lipid Metabolism in Parasites for Drug Discovery

H+-dependent inorganic phosphate uptake in Trypanosoma brucei is influenced by myo-inositol transporter

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