Glycosomal ABC transporters of Trypanosoma brucei: characterisation of their expression, topology and substrate specificity.

Metabolism in trypanosomatids is compartmentalised with major pathways, notably glycolysis, present in peroxisome-like organelles called glycosomes. To date, little information is available about the transport of metabolites through the glycosomal membrane. Previously, three ATP-binding cassette (ABC) transporters, called GAT1-3 for Glycosomal ABC Transporters 1 to 3, have been identified in the glycosomal membrane of Trypanosoma brucei. Here we report that GAT1 and GAT3 are expressed both in bloodstream and procyclic form trypanosomes, whereas GAT2 is mainly or exclusively expressed in bloodstream-form cells. Protease protection experiments showed that the nucleotide-binding domain of GAT1 and GAT3 is exposed to the cytosol, indicating that these transporters mediate the ATP-dependent uptake of solutes from the cytosol into the glycosomal lumen. Depletion of GAT1 and GAT3 by RNA interference in procyclic cells grown in glucose-containing medium did not affect growth. Surprisingly, GAT1 depletion enhanced the expression of the very different GAT3 protein. Expression knockdown of GAT1, but not GAT3, in procyclic cells cultured in glucose-free medium was lethal. Depletion of GAT1 in glucose-grown procyclic cells caused a modification of the total cellular fatty-acid composition. No or only minor changes were observed in the levels of most fatty acids, including oleate (C18:1), nevertheless the linoleate (C18:2) abundance was significantly increased upon GAT1 silencing. Furthermore, glycosomes purified from procyclic wild-type cells incorporate oleoyl-CoA in a concentration- and ATP-dependent manner, whilst this incorporation was severely reduced in glycosomes from cells in which GAT1 levels had been decreased. Together, these results strongly suggest that GAT1 serves to transport primarily oleoyl-CoA, but possibly also other fatty acids, from the cytosol into the glycosomal lumen and that its depletion results in a cellular linoleate accumulation, probably due to the presence of an active oleate desaturase. The role of intraglycosomal oleoyl-CoA and its essentiality when the trypanosomes are grown in the absence of glucose, are discussed.