KlHsl1 is a component of glycerol response pathways in the milk yeast Kluyveromyces lactis

Abstract

In Saccharomyces cerevisiae, HSL1 (NIK1) encodes a serine-threonine protein kinase involved in cell cycle control and morphogenesis. Deletion of its putative orthologue in Kluyveromyces lactis, KlHSL1, gives rise to sensitivity to the respiratory inhibitor antimycin A (AA). Resistance to AA on glucose (Rag + phenotype) is associated with genes (RAG) required for glucose metabolism/ glycolysis. To understand the relationship between RAG and KlHSL1, rag and Klhsl1D mutant strains were investigated. The analysis showed that all the mutants contained a phosphorylated form of Hog1 and displayed an inability to synthesize/accumulate glycerol as a compatible solute. In addition, rag mutants also showed alterations in both cell wall and membrane fatty acids. The pleiotropic defects of these strains indicate that a common pathway regulates glucose utilization and stress response mechanisms, suggesting impaired adaptation of the plasma membrane/cell wall during the respiratory-fermentative transition. KlHsl1 could be the link between these adaptive pathways and the morphogenetic checkpoint. INTRODUCTIONIn Saccharomyces cerevisiae, the cell cycle is regulated by the Cdc28 kinase and its regulatory subunits (cyclins). Swe1 kinase negatively regulates entry into mitosis through inhibitory phosphorylation of Cdc28 (Booher et al., 1993). Swe1 accumulates through the late G 1 and S phases, and is then degraded in G 2 by hyperphosphorylation (Sia et al., 1998). HSL1, also called NIK1, encodes a protein kinase, which is a negative regulator of Swe1 (Tanaka & Nojima, 1996;Ma et al., 1996). Hsl1 is a component of the septin morphogenetic checkpoint that promotes bud development and the recruitment of Swe1 at the septin ring where it is phosphorylated/degraded (McMillan et al., 1999;Lew, 2003).S. cerevisiae is a Crabtree-positive yeast in that respiration is completely repressed by glucose even under aerobic conditions, growth being supported solely by fermentation (Gancedo, 1998). In contrast, in the Crabtree-negative yeast Kluyveromyces lactis both respiratory and fermentative pathways co-exist during growth on glucose (De Deken, 1966;Breunig et al., 2000), although respiration appears to be dispensable since antimycin A (AA) does not inhibit growth on glucose (Rag + phenotype) (Goffrini et al., 1989; Wésolowski-Louvel et al., 1992a). Sensitivity to AA on glucose (Rag -) is associated with mutations in genes encoding glycolytic enzymes, glucose transporters and sensing components, as well as their regulatory factors (Goffrini et al., 1991; Wésolowski-Louvel et al., 1992b;Prior et al., 1993; Bianchi et al., 1996;Prior et al., 1996;Blaisonneau et al., 1997;Betina et al., 2001; Lemaire & Wésolowski-Louvel, 2004; Hnatova et al., 2008).During the characterization of the putative cell cycle gene KlHSL1 in K. lactis, we found that its deletion led to a Rag -phenotype, suggesting involvement in glucose metabolism. To identify the relationship between RAG and KlHSL1, rag and Klhsl1D mutant strains were further analysed. All rag Abbreviati...

Glucose 6-phosphate and alcohol dehydrogenase activities are components of dynamic macromolecular depots structures

“…This complex is specifically required during the transition from the respiratory to the fermentative growth constituting an evolutionary-conserved membrane-phospholipids adaptive system able to contrast the potential denaturing conditions determined by the accumulation of ethanol. Indeed, as previously reported, fermentative K. lactis mutants unable to accumulate ethanol and intracellular glycerol necessary to maintain redox balance show severe pleiotropic defects leading to altered lipid homeostasis with highly reduced FAS1/FAS2 expression [39][40][41] and a slightly altered Coomassie-stained pattern in native gel (Fig. 7C lanes of Δr2, r5 Δr6 vs. CBS2359).…”

Role of the Sln1‐phosphorelay pathway in the response to hyperosmotic stress in the yeast Kluyveromyces lactis

“…To determine whether glycerol accumulation occurs in K. lactis cells following hyperosmotic stress, we measured glycerol content in wild type and mutant strains and compared it to glycerol production in S. cerevisiae . We found that the glycerol content in K. lactis wild type cells increased approximately 5‐ to 7‐fold under hyperosmotic stress, which is in agreement with previous reports (Cialfi et al ., ). The glycerol content under osmotic stress in K. lactis slightly decreased in the ΔKlhog1 mutant and remained unaltered in the ΔKlsln1 and ΔKlypd1 mutants (Fig.…”

Ineffective Phosphorylation of Mitogen-Activated Protein Kinase Hog1p in Response to High Osmotic Stress in the Yeast Kluyveromyces lactis

“…It will be interesting to search for this putative alternative KlHog1p activator, one of which could be KlSte7p, since it was demonstrated that it participates in KlHog1p phosphorylation, even though it did not show an interaction with KlPbs2p (25). Additionally, it would be interesting to analyze the possible participation of the KlHsl1p kinase in KlHog1p activation in cells lacking both the SHO1 and SLN1 branches, since the null mutant is sensitive to hyperosmotic stress but shows constitutive KlHog1p phosphorylation (26).…”

“…Comparative genomic analyses have shown that K. lactis contains orthologs of most proteins of the S. cerevisiae HOG pathway (24). However, besides one genetic study (25) and the report that a mutant with a deletion of the K. lactis HSL1 (KlHSL1) gene, encoding a serine-threonine protein kinase involved in the cell cycle, was sensitive to hyperosmotic stress and showed constitutive KlHog1p phosphorylation (26), very little is known about the architecture and regulation of the K. lactis hyperosmotic signaling system (reviewed in reference 23). Here we describe some aspects of K. lactis Hog1p regulation, focusing on the role of the SHO1 branch.…”