One of the most recent functions assigned to the TOR signaling pathway in yeast is the coordination of the transcription of genes involved in nutrient utilization. Here we show that transcription of ENA1, a gene encoding a lithium and sodium ion transporter essential for salt tolerance in yeast, is controlled by the TOR signaling pathway. First, ENA1 expression is strongly induced under TOR-inactivating conditions. Second, the absence of the TOR-controlled GATA transcription factors GLN3 and GAT1 results in reduced basal and salt-induced expression of ENA1. Third, a gln3 gat1 mutant displays a pronounced sensitivity to high concentrations of lithium and sodium. Fourth, TOR1, similar to ENA1, is required for growth under saline stress conditions. In summary, our results suggest that TOR plays a role in the general response to saline stress by regulating the transcription of ENA1 via GLN3 and GAT1.

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Ergosterol is the yeast functional equivalent of cholesterol in mammalian cells. Deletion of the ERG6 gene, which encodes an enzyme catalyzing a late step of ergosterol biosynthesis, impedes targeting of the tryptophan permease Tat2p to the plasma membrane, but does not promote vacuolar degradation. It is unknown whether similar features appear when other steps of ergosterol biogenesis are inhibited. We show herein that the ergosterol biosynthesis inhibitor zaragozic acid (ZA) evoked massive vacuolar degradation of Tat2p, accompanied by a decrease in tryptophan uptake. ZA inhibits squalene synthetase (SQS, EC 2.5.1.21), which catalyzes the first committed step in the formation of cholesterol/ergosterol. The degradation of Tat2p was dependent on the Rsp5p-mediated ubiquitination of Tat2p and was not suppressed by deletions of VPS1, VPS27, VPS45 or PEP12. We will discuss ZA-mediated Tat2p degradation in the context of lipid rafts.

Cholesterol (ergosterol in yeast) in conjunction with sphingolipids forms tight-packing microdomains, 'lipid rafts,' which are thought to be critical for intracellular protein sorting in eukaryotic cells. When the activity of Erg9 involved in the first step of ergosterol biogenesis, but not that of Erg6 involved in a late step, is compromised, vacuolar degradation of the tryptophan permease Tat2 is promoted. It is unknown whether this difference simply reflects the difference between the inhibition of early and late steps. Here, it is shown that the deletion in ERG2, which encodes sterol C8-C7 isomerase (the next enzymatic step after Erg6), promotes the vacuolar degradation of Tat2. It suggests that the accumulation of specific sterol intermediates may alter lipid raft structures, promoting Tat2 degradation. The erg2Delta-mediated Tat2 degradation required Tat2 ubiquitination. Lipid raft association of Tat2 is compromised in erg2Delta cells. The erg2Delta mutation showed a synthetic growth defect with the trp1 mutation, indicating that Tat2 sorting is preferentially compromised in these mutants. Consistent with this notion, the raft-associated protein Pma1 was associated with detergent-resistant membranes and sorted to the plasma membrane. This study suggests the potential for the pharmacological control of cellular nutrient uptake in humans by regulating enzymes involved in cholesterol biogenesis.

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