Table_1_KAE1 Allelic Variants Affect TORC1 Activation and Fermentation Kinetics in Saccharomyces cerevisiae.XLSX

The eukaryotic domain-conserved TORC1 signalling pathway connects growth with nutrient sufficiency, promoting anabolic processes such as ribosomal biogenesis and protein synthesis. In Saccharomyces cerevisiae, TORC1 is activated mainly by the nitrogen sources. Recently, this pathway has gotten renewed attention but now in the context of the alcoholic fermentation, due to its key role in nitrogen metabolism regulation. Although the distal and proximal effectors downstream TORC1 are well characterised in yeast, the mechanism by which TORC1 is activated by nitrogen sources is not fully understood. In this work, we took advantage of a previously developed microculture-based methodology, which indirectly evaluates TORC1 activation in a nitrogen upshift experiment, to identify genetic variants affecting the activation of this pathway. We used this method to phenotype a recombinant population derived from two strains (SA and WE) with different geographic origins, which show opposite phenotypes for TORC1 activation by glutamine. Using this phenotypic information, we performed a QTL mapping that allowed us to identify several QTLs for TORC1 activation. Using a reciprocal hemizygous analysis, we validated GUS1, KAE1, PIB2, and UTH1 as genes responsible for the natural variation in the TORC1 activation. We observed that reciprocal hemizygous strains for KAE1 (ATPase required for t6A tRNA modification) gene showed the greatest phenotypic differences for TORC1 activation, with the hemizygous strain carrying the SA allele (KAE1SA) showing the higher TORC1 activation. In addition, we evaluated the fermentative capacities of the hemizygous strains under low nitrogen conditions, observing an antagonistic effect for KAE1SA allele, where the hemizygous strain containing this allele presented the lower fermentation rate. Altogether, these results highlight the importance of the tRNA processing in TORC1 activation and connects this pathway with the yeasts fermentation kinetics under nitrogen-limited conditions.真核生物保守的雷帕霉素靶蛋白复合物1（TORC1）信号通路将细胞生长与营养充足状态相关联，可促进核糖体生物发生、蛋白质合成等合成代谢过程。在酿酒酵母（Saccharomyces cerevisiae）中，TORC1主要被氮源激活。近年来，该通路因在氮代谢调控中的关键作用，在酒精发酵研究领域重新受到关注。尽管酵母中TORC1下游的远端与近端效应蛋白已得到充分表征，但氮源激活TORC1的具体分子机制仍未完全阐明。本研究利用此前开发的基于微量培养的实验方法——该方法可在氮源上移实验中间接评估TORC1的激活水平——来筛选影响该通路激活的遗传变异。我们运用该方法对由两株地理起源不同、在谷氨酰胺激活TORC1表型上截然相反的菌株（SA与WE）构建的重组群体进行了表型分型。基于该表型信息，我们开展了数量性状位点（Quantitative Trait Locus，QTL）定位分析，成功鉴定出多个与TORC1激活相关的QTL。通过反向半合子分析，我们验证了GUS1、KAE1、PIB2及UTH1这四个基因是导致TORC1激活自然变异的因果基因。我们发现，KAE1基因（负责t6A tRNA修饰的ATP酶）的反向半合子菌株在TORC1激活表型上差异最为显著，其中携带SA等位基因的半合子菌株（KAE1^SA）的TORC1激活水平更高。此外，我们还在低氮条件下评估了半合子菌株的发酵能力，结果发现KAE1^SA等位基因存在拮抗效应：携带该等位基因的半合子菌株发酵速率更低。综上，本研究结果凸显了tRNA加工在TORC1激活过程中的重要性，并将该通路与氮限制条件下酵母的发酵动力学特征建立了关联。