Myriocin is a potent inhibitor of sphingolipid biosynthesis that increases lifespan in a variety of model organisms, but how slowing sphingolipid biosynthesis promotes longevity remains unknown. Previously, we reported that myriocin treatment of yeast cells triggers an acute decline in the abundance of most amino acids, resulting in a state of amino acid restriction. Myriocin also triggers the endocytic downregulation of the methionine transporter Mup1, although its effect on other nutrient transporters is unknown. Here, we characterized a panel of different PM proteins – including amino acid transporters, hexose transporters, proton pumps, and signaling receptors – during a myriocin treatment time course. In contrast to Mup1, all other PM proteins examined were either unaffected or accumulated at the PM in response to myriocin treatment. Notably, myriocin treatment inhibits bulk endocytosis after 4 hours of treatment, which may account for the accumulation of various PM proteins in response to myriocin. Our data indicates that the mechanism of myriocin-induced Mup1 endocytosis is distinct from methionine-induced Mup1 endocytosis in that it is dependent on the Rsp5 adaptor Art2, C-terminal lysine residues, and the formation of K63-linked ubiquitin polymers. These findings shed new light on how cells adapt to sphingolipid depletion and reveal a novel mechanism for endocytic clearance of Mup1.

Accumulating evidence in several model organisms indicates that reduced sphingolipid biosynthesis promotes longevity, although underlying mechanisms remain unclear. In yeast, sphingolipid depletion induces a state resembling amino acid restriction, which we hypothesized may be due to altered stability of amino acid transporters at the plasma membrane. To test this, we measured surface abundance for a diverse panel of membrane proteins in the presence of myriocin, a sphingolipid biosynthesis inhibitor. Unexpectedly, we found that surface levels of most proteins examined were either unaffected or increased during myriocin treatment, consistent with an observed decrease in bulk endocytosis. In contrast, sphingolipid depletion triggered selective endocytosis of the methionine transporter Mup1. Unlike methionine-induced Mup1 endocytosis, myriocin triggers Mup1 endocytosis that requires the Rsp5 adaptor Art2, C-terminal lysine residues, and the formation of K63-linked ubiquitin polymers. These findings reveal cellular adaptation to sphingolipid depletion by ubiquitin-mediated remodeling of nutrient transporter composition at the cell surface.