Indirect monitoring of TORC1 signalling pathway reveals molecular diversity among different yeast strains

Abstract

Saccharomyces cerevisiae is the main species responsible for the alcoholic fermentation in wine production. One of the main problems in this process is the deficiency of nitrogen sources in the grape must, which can lead to stuck or sluggish fermentations. Currently, yeast nitrogen consumption and metabolism are under active inquiry, with emphasis on the study of the TORC1 signalling pathway, given its central role responding to nitrogen availability and influencing growth and cell metabolism. However, the mechanism by which different nitrogen sources activates TORC1 is not completely understood. Existing methods to evaluate TORC1 activation by nitrogen sources are time-consuming, making difficult the analyses of large numbers of strains. In this work, a new indirect method for monitoring TORC1 pathway was developed on the basis of the luciferase reporter gene controlled by the promoter region of RPL26A gene, a gene known to be expressed upon TORC1 activation. The method was tested in strains representative of the clean lineages described so far in S. cerevisiae. The activation of the TORC1 pathway by a proline-to-glutamine upshift was indirectly evaluated using our system and the traditional direct methods based on immunoblot (Sch9 and Rps6 phosphorylation). Regardless of the different molecular readouts obtained with both methodologies, the general results showed a wide phenotypic variation between the representative strains analysed. Altogether, this easy-to-use assay opens the possibility to study the molecular basis for the differential TORC1 pathway activation, allowing to interrogate a larger number of strains in the context of nitrogen metabolism phenotypic differences.

Disentangling the genetic bases of Saccharomyces cerevisiae nitrogen consumption and adaptation to low nitrogen environments in wine fermentation

“…One of the major actual challenges in the TORC1 field is to determine exactly how intracellular levels of nitrogen are sensed by the TORC1 complex and how this pathway differentiate between preferred and non-preferred nitrogen sources [29,30,93,94]. In this sense, a recently developed method allows the indirect monitoring of TORC1 activation for hundreds of strains, enabling the study of this phenotype by approaches like QTL mapping [95]. Using these experimental capacities, KAE1 allelic variants were identified to affect both TORC1 activation by glutamine and fermentation kinetics under nitrogen-sufficient and nitrogen-limited wine conditions [84] (Fig.…”

Role of Saccharomyces cerevisiae Nutrient Signaling Pathways During Winemaking: A Phenomics Approach

“…First, wine yeasts are mostly prototrophic and differ from the standard laboratory strains carrying various auxotrophic mutations used as selection markers. Second, some degree of genetic variation in nutrient signaling pathways, such as TORC1, has been found in natural isolates of S. cerevisiae (Kessi-Perez et al, 2019, 2020. Although they constitute a homogenous group, commercial wine starters show wide genetic variability, as reflected by their different sensitivity to inhibitors of nutrient signaling pathways (Vallejo et al, submitted).…”

Comparison of Phylogenetic Tree Topologies for Nitrogen Associated Genes Partially Reconstruct the Evolutionary History of Saccharomyces cerevisiae

“…Other strong association is observed between EAP1 and TOR2, both genes participating of the TORC1 signalling pathway (Figure 2). Interestingly, molecular diversity among the WE, NA, WA and SA strains for TORC1 activation have been recently revealed [61]. However, other TORC1 pathway associated genes (GTR1, NPR1, SAP185, SCH9 and SIT4) showed more different tree topologies, with a disperse localisation in the tree space plot representation in 2D ( Figure 2B).…”