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• STT4 has an important role in the glucose-induced activation of yeast plasma membrane H +-ATPase. • Phosphatidylinositol-4-phosphate seems to modulate of the activity of phospholipase C. • Application of different molecular approaches (pooled-segregant whole genome sequencing, QTL analysis and a new bioinformatics methodology) as tools to discover new components of signal transduction pathways. Many studies have focused on identifying the signaling pathway by which addition of glucose triggers post-translational activation of the plasma membrane H +-ATPase in yeast. They have revealed that calcium signaling is involved in the regulatory pathway, supported for instance by the phenotype of mutants in ARG82 that encodes an inositol kinase that phosphorylates inositol triphosphate (IP 3). Strong glucose-induced calcium signaling, and high glucose-induced plasma membrane H +-ATPase activation have been observed in a specific yeast strain with the PJ genetic background. In this study, we have applied pooled-segregant whole genome sequencing, QTL analysis and a new bioinformatics methodology for determining SNP frequencies to identify the cause of this discrepancy and possibly new components of the signaling pathway. This has led to the identification of an STT4 allele with 6 missense mutations as a major causative allele, further supported by the observation that deletion of STT4 in the inferior parent caused a similar increase in glucose-induced plasma membrane H +-ATPase activation. However, the effect on calcium signaling was different indicating the presence of additional relevant genetic differences between the superior and reference strains. Our results suggest that phosphatidylinositol-4-phosphate might play a role in the glucose-induced activation of plasma membrane H +-ATPase by controlling intracellular calcium release through the modulation of the activity of phospholipase C. [Display omitted] [ABSTRACT FROM AUTHOR]