The branched-chain amino acid transporter AAT10.2 of Trypanosoma brucei is regulated by substrate availability

Amino acid transporters (AATs) are essential for the uptake of exogenous amino acids, and their regulation allows the protozoan parasite Trypanosoma brucei to rapidly adapt to changing environmental conditions when alternating between the mammalian and the insect host, or when invading different host tissues. With a phosphoproteome analysis of procyclic form T. brucei we identified many AATs of the amino acid/auxin permease (AAAP) family with phosphorylated residues exclusively in their hydrophilic N-terminal domain. AAT10.2, which contained multiple phosphorylated residues, was further characterized and shown to be localized at the plasma membrane and the flagellar pocket. RNAi in procyclic form T. brucei revealed that AAT10.2 is essential and the main transporter for branched-chain amino acids (BCAA) under the conditions tested. High affinity and selective transport of BCAA was verified by expressing AAT10.2 in Saccharomyces cerevisiae. In T. brucei, AAT10.2 protein abundance was regulated by BCAA levels. However, mutation of the identified phosphorylation sites or truncation of the first 35 amino acids did not affect affinity, localization, or the BCAA-dependent regulation of AAT10.2. The non-phosphorylatable 3xTy-STA-AAT10.2∗ variant, in which N-terminal serine and threonine residues were mutated to alanine, showed increased protein levels compared to native Ty-tagged AAT10.2∗ or the phosphomimicking variant (3xTy-STD-AAT10.2∗) with aspartate replacing serine and threonine in the N-terminal region. Truncation of the N-terminal domain reduced the overall AAT10.2 abundance and BCAA levels in short-term experiments, and decreased T. brucei growth when BCAA availability was limited. These findings establish AAT10.2 as an essential, substrate-regulated BCAA transporter in procyclic form T. brucei.