Stress-regulated Arabidopsis GAT2 is a low affinity -aminobutyric acid (GABA) transporter.

The four carbon non-proteinogenic amino acid γ-aminobutyric acid (GABA) accumulates to high levels in plants in response to various abiotic and biotic stress stimuli, and plays a role in C:N balance, signaling and as a transport regulator. Expression in Xenopus oocytes and voltage-clamping allowed characterizing Arabidopsis GAT2 (At5g41800) as low affinity GABA transporter with a K0.5GABA~8 mM. L-alanine and butylamine represented additional substrates. GABA-induced currents were strongly dependent on the membrane potential, reaching highest affinity and highest transport rates at strongly negative membrane potentials. Mutation of Ser17, previously reported to be phosphorylated in planta, did not result in altered affinity. In short term stress experiment, AtGAT2 mRNA levels were upregulated at low water potential and under osmotic stress (polyethylene glycol, mannitol). Furthermore, AtGAT2 promoter activity was detected in vascular tissues, in maturating pollen, and the phloem unloading region of young seeds. Even though this suggested a role of AtGAT2 in long distance transport and loading of sink organs, under the conditions tested neither AtGAT2 overexpressing plants nor atgat2 or atgat1 T-DNA insertion lines, or atgat1 atgat2 double knockout mutants differed from wild type plants in growth on GABA, in amino acid levels or resistance to salt and osmotic stress.

Nutrient availability regulates proline/alanine transporters in Trypanosoma brucei.

Trypanosoma brucei is a species of unicellular parasite that can cause severe diseases in livestock and humans, including African trypanosomiasis and Chagas disease. Adaptation to diverse environments and changes in nutritional conditions is essential for T. brucei to establish an infection when changing hosts or during invasion of different host tissues. One such adaptation is the ability of T. brucei to rapidly switch its energy metabolism from glucose metabolism in the mammalian blood to proline catabolism in the insect stages and vice versa. However, the mechanisms that support the parasite's response to nutrient availability remain unclear. Using RNAseq and qRT-PCR, we investigated the response of T. brucei to amino acid or glucose starvation and found increased mRNA levels of several amino acid transporters, including all genes of the amino acid transporter AAT7-B subgroup. Functional characterization revealed that AAT7-B members are plasma membrane-localized in T. brucei and when expressed in Saccharomyces cerevisiae supported the uptake of proline, alanine, and cysteine, while other amino acids were poorly recognized. All AAT7-B members showed a preference for proline, which is transported with high or low affinity. RNAi-mediated AAT7-B downregulation resulted in a reduction of intracellular proline concentrations and growth arrest under low proline availability in cultured procyclic form parasites. Taken together, these results suggest a role of AAT7-B transporters in the response of T. brucei to proline starvation and proline catabolism.

Branched late-steps of the cytosolic iron-sulphur cluster assembly machinery of Trypanosoma brucei.

Fe-S clusters are ubiquitous cofactors of proteins involved in a variety of essential cellular processes. The biogenesis of Fe-S clusters in the cytosol and their insertion into proteins is accomplished through the cytosolic iron-sulphur protein assembly (CIA) machinery. The early- and middle-acting modules of the CIA pathway concerned with the assembly and trafficking of Fe-S clusters have been previously characterised in the parasitic protist Trypanosoma brucei. In this study, we applied proteomic and genetic approaches to gain insights into the network of protein-protein interactions of the late-acting CIA targeting complex in T. brucei. All components of the canonical CIA machinery are present in T. brucei including, as in humans, two distinct CIA2 homologues TbCIA2A and TbCIA2B. These two proteins are found interacting with TbCIA1, yet the interaction is mutually exclusive, as determined by mass spectrometry. Ablation of most of the components of the CIA targeting complex by RNAi led to impaired cell growth in vitro, with the exception of TbCIA2A in procyclic form (PCF) trypanosomes. Depletion of the CIA-targeting complex was accompanied by reduced levels of protein-bound cytosolic iron and decreased activity of an Fe-S dependent enzyme in PCF trypanosomes. We demonstrate that the C-terminal domain of TbMMS19 acts as a docking site for TbCIA2B and TbCIA1, forming a trimeric complex that also interacts with target Fe-S apo-proteins and the middle-acting CIA component TbNAR1.

The intermembrane space protein Erv1 of Trypanosoma brucei is essential for mitochondrial Fe-S cluster assembly and operates alone.

Sulfhydryl oxidase Erv1 is a ubiquitous and conserved protein of the mitochondrial intermembrane space that plays a role in the transport of small sulfur-containing proteins. In higher eukaryotes, Erv1 interacts with the mitochondrial import protein Mia40. However, Trypanosoma brucei lacks an obvious Mia40 homologue in its genome. Here we show by tandem affinity purification and mass spectrometry that in this excavate protist, Erv1 functions without a Mia40 homologue and most likely any other interaction partner. Down-regulation of TbErv1 caused a reduction of the mitochondrial membrane potential already within 24h to less than 50% when compared with control cells. The depletion of TbErv1 was accompanied by accumulation of trCOIV precursor, with a concomitant reduction of aconitase activity both in the cytosol and mitochondrion. Overall, TbErv1 seems to have a role in the mitochondrial translocation and Fe-S cluster assembly in the organelle.

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 µM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-ß-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 µM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

Minimal cytosolic iron-sulfur cluster assembly machinery of Giardia intestinalis is partially associated with mitosomes.

Iron-sulfur (Fe-S) clusters are essential cofactors that enable proteins to transport electrons, sense signals, or catalyze chemical reactions. The maturation of dozens of Fe-S proteins in various compartments of every eukaryotic cell is driven by several assembly pathways. The ubiquitous cytosolic Fe-S cluster assembly (CIA) pathway, typically composed of eight highly conserved proteins, depends on mitochondrial Fe-S cluster assembly (ISC) machinery. Giardia intestinalis contains one of the smallest eukaryotic genomes and the mitosome, an extremely reduced mitochondrion. Because the only pathway known to be retained within this organelle is the synthesis of Fe-S clusters mediated by ISC machinery, a likely function of the mitosome is to cooperate with the CIA pathway. We investigated the cellular localization of CIA components in G. intestinalis and the origin and distribution of CIA-related components and Tah18-like proteins in other Metamonada. We show that orthologs of Tah18 and Dre2 are missing in these eukaryotes. In Giardia, all CIA components are exclusively cytosolic, with the important exception of Cia2 and two Nbp35 paralogs, which are present in the mitosomes. We propose that the dual localization of Cia2 and Nbp35 proteins in Giardia might represent a novel connection between the ISC and the CIA pathways.

Cytosolic iron-sulphur protein assembly is functionally conserved and essential in procyclic and bloodstream Trypanosoma brucei.

Cytosolic and nuclear iron-sulphur (Fe/S) proteins include essential components involved in protein translation, DNA synthesis and DNA repair. In yeast and human cells, assembly of their Fe/S cofactor is accomplished by the CIA (cytosolic iron-sulphur protein assembly) machinery comprised of some 10 proteins. To investigate the extent of conservation of the CIA pathway, we examined its importance in the early-branching eukaryote Trypanosoma brucei that encodes all known CIA factors. Upon RNAi-mediated ablation of individual, early-acting CIA proteins, no major defects were observed in both procyclic and bloodstream stages. In contrast, parallel depletion of two CIA components was lethal, and severely diminished cytosolic aconitase activity lending support for a direct role of the CIA proteins in cytosolic Fe/S protein biogenesis. In support of this conclusion, the T. brucei CIA proteins complemented the growth defects of their respective yeast CIA depletion mutants. Finally, the T. brucei CIA factor Tah18 was characterized as a flavoprotein, while its binding partner Dre2 functions as a Fe/S protein. Together, our results demonstrate the essential and conserved function of the CIA pathway in cytosolic Fe/S protein assembly in both developmental stages of this representative of supergroup Excavata.