Abstract
Sphingoid long chain bases have many effects on cells including inhibition or stimulation of growth. The physiological significance of these effects is unknown in most cases. To begin to understand how these compounds inhibit growth, we have studied Saccharomyces cerevisiae cells. Growth of tryptophan (Trp-) auxotrophs was more strongly inhibited by phytosphingosine (PHS) than was growth of Trp+ strains, suggesting that PHS diminishes tryptophan uptake and starves cells for this amino acid. This hypothesis is supported by data showing that growth inhibition is relieved by increasing concentrations of tryptophan in the culture medium and by multiple copies of the TAT2 gene, encoding a high affinity tryptophan transporter. Measurement of tryptophan uptake shows that it is inhibited by PHS. Finally, PHS treatment induces the general control response, indicating starvation for amino acids. Multiple copies of TAT2 do not protect cells against two other cationic lipids, stearylamine, and sphingosine, indicating that the effect of PHS on tryptophan utilization is specific. Other data demonstrate that PHS reduces uptake of leucine, histidine, and proline by specific transporters. Our data suggest that PHS targets proteins in the amino acid transporter family but not other distantly related membrane transporters, including those necessary for uptake of adenine and uracil.
Highlights
The long chain base component of sphingolipids inhibits cell growth under many conditions and is cytotoxic to some cell types, but the mechanisms underlying these phenomenona are not well characterized
Since pRS314 carries a portion of the GAL3 gene that is linked to the centromeric plasmid pRS314 (TRP1), we verified that cells transformed with a plasmid carrying GAL3 remained sensitive to PHS
We conclude from these data that tryptophan auxotrophs (TrpϪ) of S. cerevisiae are more sensitive to growth inhibition by PHS than are tryptophan prototrophs (Trpϩ)
Summary
Trast, the activity of some, but not all, specific amino acid permeases is constitutive and not repressed by preferred nitrogen sources (19). We demonstrate here that PHS blocks transport of tryptophan, leucine, histidine, and proline by their specific amino acid transporters. Inhibition of amino acid transport explains why PHS blocks growth of auxotrophic S. cerevisiae strains and why prototrophic strains are resistant to the drug
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