RESUMEN
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.
RESUMEN
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.
