Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ ⁹ position. We expressed two sunflower ( Helianthus annuus ) oleate Δ ¹² desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain ( trp1 ) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ ^9,12 , the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 ( TRP1 ) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp ⁺ or Trp ⁻ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae , and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.

The molecular mechanisms that enable yeast cells to detect and transmit cold signals and their physiological significance in the adaptive response to low temperatures are unknown. Here, we have demonstrated that the MAPK Hog1p is specifically activated in response to cold. Phosphorylation of Hog1p was dependent on Pbs2p, the MAPK kinase (MAPKK) of the high osmolarity glycerol (HOG) pathway, and Ssk1p, the response regulator of the two-component system Sln1p-Ypd1p. However, Sho1p was not required. Interestingly, phosphorylation of Hog1p was stimulated at 30 degrees C in cells exposed to the membrane rigidifier agent dimethyl sulfoxide. Moreover, Hog1p activation occurred specifically through the Sln1 branch. This suggests that Sln1p monitors changes in membrane fluidity caused by cold. Quite remarkably, activation of Hog1p at low temperatures affected the transcriptional response to cold shock. Indeed, the absence of Hog1p impaired the cold-instigated expression of genes for trehalose- and glycerol-synthesizing enzymes and small chaperones. Moreover, a downward transfer to 12 or 4 degrees C stimulated the overproduction of glycerol in a Hog1p-dependent manner. However, hog1Delta mutant cells showed no growth defects at 12 degrees C as compared with the wild type. On the contrary, deletion of HOG1 or GPD1 decreased tolerance to freezing of wild-type cells preincubated at a low temperature, whereas no differences could be detected in cells shifted directly from 30 to -20 degrees C. Thus, exposure to low temperatures triggered a Hog1p-dependent accumulation of glycerol, which is essential for freeze protection.

The response of yeast cells to sudden temperature downshifts has received little attention compared with other stress conditions. Like other organis

Mutations in the clk-1 gene result in slower development and increased life span in Caenorhabditis elegans. The Saccharomyces cerevisiae homologue COQ7/CAT5 is essential for several metabolic pathways including ubiquinone biosynthesis, respiration, and gluconeogenic gene activation. We show here that Coq7p/Cat5p is a mitochondrial inner membrane protein directly involved in ubiquinone biosynthesis, and that the defect in gluconeogenic gene activation in coq7/cat5 null mutants is a general consequence of a defect in respiration. These results obtained in the yeast model suggest that the effects on development and life span in C. elegans clk-1 mutants may relate to changes in the amount of ubiquinone, an essential electron transport component and a lipid soluble antioxidant.

The Cat8p zinc cluster protein is essential for growth of Saccharomyces cerevisiae with nonfermentable carbon sources. Expression of the CAT8 gene is subject to glucose repression mainly caused by Mig1p. Unexpectedly, the deletion of the Mig1p-binding motif within the CAT8 promoter did not increase CAT8 transcription; moreover, it resulted in a loss of CAT8 promoter activation. Insertion experiments with a promoter test plasmid confirmed that this regulatory 20-bp element influences glucose repression and derepression as well. This finding suggests an upstream activating function of this promoter region, which is Mig1p independent, as delta mig1 mutants are still able to derepress the CAT8 promoter. No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression. Fusions of Cat8p with the Gal4p DNA-binding domain mediated transcriptional activation. This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protein kinase, which indicated that Cat8p needs posttranslational modification to reveal its gene-activating function. Indeed, Western blot analysis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) with crude extracts from glucose-grown cells, whereas three bands (Cat8pI, -II, and -III) were identified in derepressed cells. Derepression-specific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment. Only the most extensively phosphorylated modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indicating that another protein kinase is responsible for modification form Cat8pII. The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA. Furthermore, glucose triggered the dephosphorylation of Cat8pIII, but this did not depend on the Glc7p (Cid1p) phosphatase previously described as being involved in invertase repression. These results confirm our current model that glucose derepression of gluconeogenic genes needs Cat8p phosphorylation and additionally show that a still unknown transcriptional activator is also involved.

The HXK2 gene is required for a variety of regulatory effects leading to an adaptation for fermentative metabolism inSaccharomyces cerevisiae. However, the molecular basis of the specific role of Hxk2p in these effects is still unclear. One important feature in order to understand the physiological function of hexokinase PII is that it is a phosphoprotein, since protein phosphorylation is essential in most metabolic signal transductions in eukaryotic cells. Here we show that Hxk2p exists in vivo in a dimeric-monomeric equilibrium which is affected by phosphorylation. Only the monomeric form appears phosphorylated, whereas the dimer does not. The reversible phosphorylation of Hxk2p is carbon source dependent, being more extensive on poor carbon sources such as galactose, raffinose, and ethanol. In vivo dephosphorylation of Hxk2p is promoted after addition of glucose. This effect is absent in glucose repression mutants cat80/grr1, hex2/reg1, andcid1/glc7. Treatment of a glucose crude extract fromcid1-226 (glc7-T152K) mutant cells with λ-phosphatase drastically reduces the presence of phosphoprotein, suggesting that CID1/GLC7 phosphatase together with its regulatory HEX2/REG1 subunit are involved in the dephosphorylation of the Hxk2p monomer. An HXK2 mutation encoding a serine-to-alanine change at position 15 [HXK2(S15A)] was to clarify the in vivo function of the phosphorylation of hexokinase PII. In this mutant, where the Hxk2 protein is unable to undergo phosphorylation, the cells could not provide glucose repression of invertase. Glucose induction ofHXT gene expression is also affected in cells expressing the mutated enzyme. Although we cannot rule out a defect in the metabolic state of the cell as the origin of these phenomena, our results suggest that the phosphorylation of hexokinase is essential in vivo for glucose signal transduction.