Abstract
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.
Highlights
The TORC1 signalling pathway is conserved in eukaryotes and connects growth with nutrient sufficiency, promoting anabolic processes such as ribosomal biogenesis and protein synthesis
We developed a microculture-based methodology for indirect evaluation of TORC1 activation in a nitrogen upshift experiment (Kessi-Perez et al, 2019)
The luciferase gene is under the control of the endogenous RPL26A promoter, a downstream target of the TORC1 pathway. This new population was phenotyped in triplicate through nitrogen-upshift experiments in microculture conditions, in which cells are first grown in proline as unique nitrogen source and a pulse of glutamine is given
Summary
The TORC1 signalling pathway is conserved in eukaryotes and connects growth with nutrient sufficiency, promoting anabolic processes such as ribosomal biogenesis and protein synthesis. Deficiency of nitrogen sources in the grape must is a main problem for the wine industry, because the reduced fermentation rate leads to sluggish or stuck fermentations, generating economic losses in the industry (Taillandier et al, 2007). This occurs mainly because nitrogen sources are one of the most important factors regulating biomass during fermentation, which impacts fermentation rate (Varela et al, 2004)
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