Search

Hgt1p, a high-affinity glutathione transporter from the yeast Saccharomyces cerevisiae, belongs to the structurally uncharacterized oligopeptide transporter (OPT) family. To initiate structural studies on Hgt1p, a cysteine-free (cys-free) Hgt1p was generated. This cys-free Hgt1p was nonfunctional and pointed to a critical role being played by the native cysteine residues of Hgt1p. To investigate their role, genetic and biochemical approaches were undertaken. Functional suppressors of the cys-free Hgt1p were isolated, and yielded double revertants bearing C622 and C632. Subsequent biochemical characterization of the individual C622S/A or C632S/A mutations revealed that both these cysteine residues were, in fact, individually indispensable for Hgt1p function and were required for trafficking to the plasma membrane. However, despite their essentiality, the presence of only these two native cysteines in Hgt1p generated a very weak glutathione transporter with minimal functional activity. Hence, the remaining 10 cysteines were also contributing towards Hgt1p activity, although they were not found to be singly responsible or crucial for Hgt1p functional activity. These residues, however, contributed cumulatively towards the stability and the functionality of Hgt1p, without affecting the trafficking to the cell surface. The study reveals differential roles for the cysteines of Hgt1p and provides first insights into the structural features of an OPT family member. [ABSTRACT FROM AUTHOR]

HGT1 encodes a high-affinity glutathione transporter in the yeast Saccharomyces cerevisiae that is induced under sulphur limitation. The present work demonstrates that repression by organic sulphur sources is under the control of the classic sulphur regulatory network, as seen by the absence of expression in a met4Δ background. Cysteine appeared to be the principal regulatory molecule, since elevated levels were seen in str4Δ strains (deficient in cysteine biosynthesis) that could be repressed by elevated levels of cysteine, but not by methionine or glutathione. Investigations into cis-regulatory elements revealed that the previously described motif, a 9-bp cis element, CCGCCACAC, located at the −356 to −364 region of the promoter could in fact be refined to a 7-bp CGCCACA motif that is also repeated at −333 to −340. The second copy of this motif was essential for activity, since mutations in the core region of the second copy completely abolished activity and regulation by sulphur sources. Activity, but not regulation, could be restored by reintroducing an additional copy upstream of the first copy. A third region, GCCGTCTGCAAGGCA, conserved in the HGT1 promoters of the different Saccharomyces spp, was observed at −300 to −285 but, while mutations in this region did not lead to any loss in repression, the basal and induced levels were significantly increased. In contrast to a previous report, no evidence was found for regulation by the VDE endonuclease. The strong repression at the transport level by glutathione seen in strains overexpressing HGT1 was due to a glutathione-dependent toxicity in these cells. [ABSTRACT FROM AUTHOR]

Long non coding RNAs (lncRNAs) have emerged as crucial players of several central cellular processes across eukaryotes. Target of Rapamycin (TOR) is a central regulator of myriad of fundamental cellular processes including amino acid transport under diverse environmental conditions. Here we investigated the role of lncRNA in TOR regulated amino acid uptake in S. cerevisiae. Transcription of lncRNA regulates local gene expression in eukaryotes. In silico analysis of many genome wide studies in S. cerevisiae revealed that transcriptome includes conditional expression of numerous lncRNAs in proximity to amino acid transporters (AATs). Considering regulatory role of these lncRNAs, we selected highly conserved TOR regulated locus of a pair of AATs present in tandem BAP2 and TAT1. We observed that the expression of antisense lncRNA XUT_2F-154 (TBRT) and AATs BAP2 and TAT1 depends on activities of TOR signaling pathway. The expression of TBRT is induced, while that of BAP2 TAT1 is repressed upon TOR inhibition by Torin2. Notably, upon TOR inhibition loss of TBRT contributed to enhanced activities of Bap2 and Tat1 leading to improved growth. Interestingly, nucleosome scanning assay reveal that TOR signaling pathway governs chromatin remodeling at BAP2 biphasic promoter to control the antagonism of TBRT and BAP2 expression. Further TBRT also reprograms local chromatin landscapes to decrease the transcription of TAT1. The current work demonstrates a functional correlation between lncRNA production and TOR governed amino acid uptake in yeast. Thus this work brings forth a novel avenue for identification of potential regulators for therapeutic interventions against TOR mediated diseases. Unlabelled Image • In silico analysis of available RNAseq. data revealed the occurrence of 60 long non coding RNAs (lncRNAs) at 16 AATs loci. • The expression of antisense lncRNA XUT_2F-154 (TBRT) and coexpressing pair of AATs BAP2 and TAT1 depends on TOR signaling. • Expression of lncRNA TBRT decreases Bap2 and Tat1 activity. • TOR regulates chromatin remodeling at BAP2 biphasic promoter to control the antagonism of TBRT and BAP2 expression. • TBRT also changes local chromatin environment to decrease the transcription of TAT1. [ABSTRACT FROM AUTHOR]