Bolism, mainly S-adenosylmethionine (SAM) (Kalhor and Clarke, 2003; Nau, 1976), and cysteine (Leidel et al., 2009; Noma et al., 2009). These modifications appear to become downstream on the TORC1 pathway, as yeast lacking these modifications are hypersensitive to rapamycin (Fichtner et al., 2003; Goehring et al., 2003b; Leidel et al., 2009; Nakai et al., 2008), and interactions could be detected between Uba4p and Kog1/TORC1 (Laxman and Tu, 2011). These modification pathways also play vital roles in nutrient stress-dependent dimorphic foraging yeast behavior (Abdullah and Cullen, 2009; Goehring et al., 2003b; Laxman and Tu, 2011). We reasoned that deciphering the interplay in between these modifications, nutrient availability and cellular metabolism would reveal a functional logic to their biological significance. Herein, we show that tRNA uridine thiolation abundance reflects sulfur-containing amino acid availability, and functions to regulate translational capacity and amino acid homeostasis. Uridine thiolation represents a important mechanism by which translation and growth are regulated synchronously with metabolism. These findings have substantial implications for our understanding of cellular amino acid-sensing mechanisms, and with the accompanying manuscript (Sutter et al., 2013), show how sulfur-containing amino acids serve as sentinel metabolites for cell development manage.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCell. Author manuscript; readily available in PMC 2014 July 18.Laxman et al.PageRESULTStRNA uridine thiolation amounts reflect intracellular sulfur amino acid availabilityNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptWe were intrigued by connections in between tRNA uridine modification pathways and nutrients, specifically due to the fact mutants of tRNA uridine-modifying enzymes had been hypersensitive to rapamycin (Figure S1A). We initial tested no matter whether tRNA uridine modification amounts changed in response to various nutrient environments.5-Fluorouracil To qualitatively assay tRNA uridine thiolation, tRNAs had been resolved on urea-PAGE gels containing the sulfur-coordinating mercury agent APM (Nakai et al., 2008) (Supplemental Information). We confirmed that the enzyme Uba4p is required for all tRNA thiolation (Figure S1B). Even though the majority of tRNALys (UUU), tRNAGlu (UUC) and tRNAGln (UUG) had been thiolated in cells expanding either in YPD (wealthy medium) or under continuous glucose-limitation, a fraction of these tRNAs remained unthiolated (Figure S1B), suggesting that this modification was not constitutive, and may well change in abundance below particular circumstances. We then created targeted LC-MS/MS methods to quantitatively measure amounts of thiolated, methoxycarbonylmethyl-modified (mcm5s2), or unthiolated, methoxycarbonylmethyl-modified (mcm5) tRNA uridines (Figure S1C).Fitusiran We grew cells below several nutrient conditions including wealthy (YP), or synthetic (S), minimal defined medium with either glucose (D) or lactate (L) because the carbon source (Figure 1B), and measured relative uridine modification amounts from purified tRNAs.PMID:23381601 We observed a important reduce in relative amounts of thiolated uridine in cells grown in minimal media, specifically in non-fermentable SL medium in comparison to fermentable SD medium (Figure 1C). In all samples, amounts of unthiolated (mcm5) uridines always enhanced when thiolated (mcm5s2) uridines decreased, suggesting the mcm5 modification is a lot more constitutive. Collectively, these data recommend.
