Understanding the role and regulation of cation homeostasis during citric acid stress in the spolage yeast Saccharomyces cerevisiae

Citric acid is used extensively in manufactured foods and beverages as a flavour adjunct to improve taste, as a pH control agent, as a preservative to prevent microbial growth, often in conjunction with other weak-acid preservatives, and as a chelating agent to prevent food spoilage. Citric acid is a ‘sharp’-tasting, water-soluble, tri-protic organic acid with three carboxylic acid groups, and naturally occurs in the juice of lemons and other sour fruits. The deletion of genes involved in regulating cation homeostasis resulted in these strains becoming hypersensitive to citric acid. Deletion of SKY1, a protein kinase that regulates polyamine transport and tolerance to other cations such as Li+, conferred sensitivity to citric acid. Sky1p is an SR protein kinase (SRPK) that regulates the transport of mRNA from the nucleus to the cytosol and thus may play a crucial role in regulating gene expression in response to citric acid stress.
Measure changes occurring in the phospho-proteome in response to citric acid and deletion of SKY1 using affinity purification and mass spectrometry - we have quantified changes in the phosphoproteome of parent and sky1Δ strains with and without citric acid stress.
Carry out a multicopy suppressor screen of the hog1Δ and sky1Δ deletion strains to identify component genes of the signalling pathways up- or down-stream of Hog1p and Sky1p - the active site of Sky1p is necessary for citric acid resistance and citric acid stress also results in the same translocation of Hog1p to the nucleus as NaCl. The multicopy suppressor screen was performed using high (400 mM) and lower (300 mM) concentrations of citric acid for a minimum of three days to select for resistant phenotypes but unfortunately none were identified.
Create a double deletion mutant of SKY1 and HOG1 and compare the growth phenotype of this strain in the presence of citric acid to that in the single gene deletion mutants - If Hog1p and Sky1p were involved in the same citric acid resistance signalling pathway then a hog1Δ/sky1Δ double mutant would show no additional sensitivity to citric acid, whereas, an additional sensitivity would suggest they were on different signalling pathways. The data showed additional sensitivity to citric acid in the double deletion implying that the two kinases mediate tolerance to citric acid via different pathways.
Purify and identify complexes of proteins interacting with Sky1p and Hog1p using affinity chromatography with TAP-tagged Sky1p and Hog1p - one protein indirectly associated with Sky1p was Nmd5p, a carrier protein involved in the nuclear import of proteins, deletion of which results in mislocalisation of Hog1p. Deletion of NMD5 resulted in enhanced sensitivity to citric acid stress (data not shown). Thus, we hypothesise that Sky1p may be mediating tolerance to citric acid via regulation of the access of Hog1p to the nucleus.
Determination of changes in protein expression due to deletion of SKY1 in the presence of citric acid - we compared the proteomes of sky1Δ and hog1Δ to identify any proteins whose expression was co-regulated by these two protein kinases. Thus, we identified a number of proteins whose expression was regulated by both protein kinases upon exposure to citric acid stress.
Determine if the growth of hog1Δ and sky1Δ can be recovered in the presence of citric acid by supplementing the medium with extracellular cations - the principal inhibitory action of citric acid is not due to chelation of essential cations, and the protein kinases Sky1p and Hog1p mediate tolerance to citric acid via regulation of a stress response that counteracts an as yet unknown inhibitory action of this compound.