Abstract
FTY720 is a sphingoid base analog that acts as an anticancer agent in animal models. Its effect on tumor cells stems largely from its ability to trigger endocytosis of several nutrient transporters. The observation that FTY720 similarly stimulates downregulation of amino acid permeases in yeast suggests that the cellular mechanisms it targets, which are still poorly characterized, are evolutionarily conserved. We here report that adding FTY720 to yeast cells results in rapid inhibition of the intrinsic activity of multiple permeases. This effect is associated with inhibition of the TORC1 kinase complex, which in turn promotes ubiquitin-dependent permease endocytosis. Further analysis of the Gap1 permease showed that FTY720 elicits its ubiquitylation via the same factors that promote this modification when TORC1 is inhibited by rapamycin. We also show that FTY720 promotes endocytosis of the LAT1/SLC7A5 amino acid transporter in HeLa cells, this being preceded by loss of its transport activity and by mTORC1 inhibition. Our data suggest that in yeast, TORC1 deactivation resulting from FTY720-mediated inhibition of membrane transport elicits permease endocytosis. The same process seems to occur in human cells even though our data and previous reports suggest that FTY720 promotes transporter endocytosis via an additional mechanism insensitive to rapamycin.
Highlights
2-Amino-2-[2-(4-octylphenyl)]-1,3-propanediol hydrochloride, known as FTY720 or fingolimod, is a synthetic derivative of myriocin, a natural antibiotic isolated from the pathogenic fungus Isaria sinclairii[1]
We provide evidence that the intrinsic activity of multiple nutrient permeases is reduced upon FTY720 addition, this being associated with rapid inhibition of TORC1, which in turn promotes Ub-dependent permease endocytosis
We show that FTY720 triggers LAT1 endocytosis in HeLa human cells, and that this effect is preceded by a reduction of LAT1 activity and inhibition of mTORC1
Summary
2-Amino-2-[2-(4-octylphenyl)]-1,3-propanediol hydrochloride, known as FTY720 or fingolimod, is a synthetic derivative of myriocin, a natural antibiotic isolated from the pathogenic fungus Isaria sinclairii[1]. The drug notably promotes downregulation of Cat-1 (cationic amino acid transporter 1), Glut[1] (glucose transporter 1), and 4F2hc This last, named CD98 or SLC3A29, is a transmembrane protein which associates with various transporters via a disulfide bridge and is required for their proper cell-surface secretion. Recent work has revealed that FTY720 contributes to tumor cell death via yet another mechanism: inhibition of PI(3)P 5-kinase, the enzyme producing PI(3,5)P2, through mislocalization[16]. This inhibition causes accumulation of enlarged endosomes (vacuoles) containing intraluminal vesicles, along with inhibition of autophagosome formation and www.nature.com/scientificreports/. We discuss models according to which transporter inactivation coupled to TORC1 inhibition could contribute importantly to transporter endocytosis in FTY720-treated yeast and human cells
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