Cysteine import via the high-affinity GSH transporter Hgt1 rescues GSH auxotrophy in yeast.

Glutathione (GSH) is an abundant thiol-containing tripeptide that functions in redox homeostasis, protein folding, and iron (Fe) metabolism. In Saccharomyces cerevisiae, GSH depletion leads to increased sensitivity to oxidants and other toxic compounds, disruption of iron-sulfur (Fe-S) cluster biogenesis, and eventually cell death. GSH pools are supplied by intracellular biosynthesis and GSH import from the extracellular environment. Consequently, in GSH-depleted growth media, deletion of the gene encoding the first enzyme in the GSH biosynthetic pathway (GSH1) is lethal in yeast. At the other extreme, GSH overaccumulation via overexpression of the high-affinity GSH transporter Hgt1 is also toxic to cells, leading to reductive stress. Here, we engineered a yeast strain that combines gsh1 deletion with HGT1 overexpression to study the cellular effects of oscillating between GSH-deplete and -replete conditions. Surprisingly, we find that constitutive expression of HGT1 in gsh1Δ cells rescues the GSH auxotrophy of this strain. We also show that addition of cysteine or cysteine derivatives to the growth media is required for this rescue. GSH limitation in yeast causes intracellular Fe overload because of disruption of an Fe-S cluster-dependent pathway that regulates the activity of the low Fe-sensing transcription factors Aft1 and Aft2. Analysis of Fe regulation and other Fe-S cluster-dependent pathways reveals that HGT1 overexpression partially alleviates the Fe starvation-like response of gsh1Δ cells. Taken together, these results suggest that HGT1 overexpression facilitates import of cysteine or cysteine derivatives that allow limited Fe-S cluster biogenesis to sustain cell growth in the absence of GSH.