Membrane Phosphoproteomics of Yeast Early Response to Acetic Acid: Role of Hrk1 Kinase and Lipid Biosynthetic Pathways, in Particular Sphingolipids

Abstract

Saccharomyces cerevisiae response and tolerance to acetic acid is critical in industrial biotechnology and in acidic food and beverages preservation. The HRK1 gene, encoding a protein kinase of unknown function belonging to the “Npr1-family” of kinases known to be involved in the regulation of plasma membrane transporters, is an important determinant of acetic acid tolerance. This study was performed to identify the alterations occurring in yeast membrane phosphoproteome profile during the adaptive early response to acetic acid stress (following 1 h of exposure to a sub-lethal inhibitory concentration; 50 mM at pH 4.0) and the effect of HRK1 expression on the phosphoproteome. Results from mass spectrometry analysis following the prefractionation and specific enrichment of phosphorylated peptides using TiO2 beads highlight the contribution of processes related with translation, protein folding and processing, transport, and cellular homeostasis in yeast response to acetic acid stress, with particular relevance for changes in phosphorylation of transport-related proteins, found to be highly dependent on the Hrk1 kinase. Twenty different phosphoproteins known to be involved in lipid and sterol metabolism were found to be differently phosphorylated in response to acetic acid stress, including several phosphopeptides that had not previously been described as being phosphorylated. The suggested occurrence of cellular lipid composition remodeling during the short term yeast response to acetic acid was confirmed: Hrk1 kinase-independent reduction in phytoceramide levels and a reduction in phosphatidylcholine and phosphatidylinositol levels under acetic acid stress in the more susceptible hrk1Δ strain were revealed by a lipidomic analysis.

Changes in lipid metabolism convey acid tolerance in Saccharomyces cerevisiae

“…S4); the increase in VLC-FA content of the plasma membrane is probably a defense response of yeast to acid stress [ 39 ]. Indeed, VLC-FAs are the precursors of sphingolipid biosynthesis, and the accumulation of VLC-FAs is expected to increase the cellular sphingolipid content and its complexity [ 26 ]. Sphingolipids are essential structural components of cellular membranes, in particular the plasma membrane [ 40 ].…”

“…Despite these efforts, there is still a need to develop strains of S. cerevisiae tolerant to acetic acid and/or other acids. Plasma-membrane remodeling has recently been suggested to play a role in the acetic-acid adaptation of S. cerevisiae and Zygosaccharomyces bailii [ 22 – 26 ]. Particularly, sphingolipids have been shown to play an important role in acetic-acid resistance in Z. bailii [ 24 , 25 ].…”

Adaptive Response and Tolerance to Acetic Acid in Saccharomyces cerevisiae and Zygosaccharomyces bailii: A Physiological Genomics Perspective

“…The alterations occurring at the level of the membrane ph1osphoproteome during yeast early adaptive response to a sublethal concentration of acetic acid stress and the role played by the Hrk1 kinase in such response have recently been investigated ( Guerreiro et al, 2017 ). Hrk1 is a protein kinase belonging to the “Npr1-family” of kinases dedicated to the regulation of plasma membrane transporters that was identified in previous Omics approaches as a determinant of acetic acid tolerance and involved in yeast response to acetic acid stress ( Abbott et al, 2007 ; Mira et al, 2010a , b ).…”

“…Hrk1 is a protein kinase belonging to the “Npr1-family” of kinases dedicated to the regulation of plasma membrane transporters that was identified in previous Omics approaches as a determinant of acetic acid tolerance and involved in yeast response to acetic acid stress ( Abbott et al, 2007 ; Mira et al, 2010a , b ). The investigation of membrane phosphoproteome hinted toward the contribution of phosphorylation in the regulation of processes related with translation, protein folding and processing, transport, and cellular homeostasis in yeast response to acetic acid stress ( Guerreiro et al, 2017 ).…”

“…The fact that hrk1 Δ cells showed a marked increase in the intracellular concentration of radiolabeled acetic acid and that Hrk1 belongs to a family of kinases dedicated to the regulation of plasma membrane transporters, led to hypothesize that Hrk1 could be involved in the regulation of one or more plasma membrane acetate exporters, such as the MDR/MXR transporters Tpo3, Tpo2 and Aqr1, all known determinants of tolerance to this weak acid ( Mira et al, 2010a , b ). To elucidate the biological role of Hrk1, the effect of Hrk1 expression in yeast membrane associated-phosphoproteome was examined in S. cerevisiae parental and hrk1 Δ cells exposed, or not, to sublethal concentrations of acetic acid ( Guerreiro et al, 2017 ). In this study, the MDR/MXR transporters Tpo3 and Tpo4 were found to exhibit altered phosphorylation in response to acetic acid stress under the dependence of Hrk1.…”