Aloys Teunissen

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras‐mediated long‐lasting cAMP increase. Addition of glucose to cells grown on a non‐fermentable carbon source or to stationary‐phase cells triggers a transient burst in the intracellular cAMP level. This glucose‐induced cAMP signal is dependent on the G alpha‐protein Gpa2. We show that the G‐protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val‐132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose‐induced loss of heat resistance, which is consistent with its lack of glucose‐induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose‐sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK‐controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE‐controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose‐sensing system must signal glucose availability for the Sch9‐dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.

Involvement of distinct G‐proteins, Gpa2 and Ras, in glucose‐ and intracellular acidification‐induced cAMP signalling in the yeast Saccharomyces cerevisiae

Adenylate cyclase activity in Saccharomyces cerevisiae is dependent on Ras proteins. Both addition of glucose to glucose‐deprived (derepressed) cells and intracellular acidification trigger an increase in the cAMP level in vivo. We show that intracellular acidification, but not glucose, causes an increase in the GTP/GDP ratio on the Ras proteins independent of Cdc25 and Sdc25. Deletion of the GTPase‐activating proteins Ira1 and Ira2, or expression of the RAS2val19 allele, causes an enhanced GTP/GDP basal ratio and abolishes the intracellular acidification‐induced increase. In the ira1Δ ira2Δ strain, intracellular acidification still triggers a cAMP increase. Glucose also did not cause an increase in the GTP/GDP ratio in a strain with reduced feedback inhibition of cAMP synthesis. Further investigation indicated that feedback inhibition by cAPK on cAMP synthesis acts independently of changes in the GTP/GDP ratio on Ras. Stimulation by glucose was dependent on the Gα‐protein Gpa2, whose deletion confers the typical phenotype associated with a reduced cAMP level: higher heat resistance, a higher level of trehalose and glycogen and elevated expression of STRE‐controlled genes. However, the typical fluctuation in these characteristics during diauxic growth on glucose was still present. Overexpression of Ras2val19 inhibited both the acidification‐ and glucose‐induced cAMP increase even in a protein kinase A‐attenuated strain. Our results suggest that intracellular acidification stimulates cAMP synthesis in vivo at least through activation of the Ras proteins, while glucose acts through the Gpa2 protein. Interaction of Ras2val19 with adenylate cyclase apparently prevents its activation by both agonists.

Aquaporin Expression Correlates with Freeze Tolerance in Baker's Yeast, and Overexpression Improves Freeze Tolerance in Industrial Strains

ABSTRACT Little information is available about the precise mechanisms and determinants of freeze resistance in bakers yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant bakers yeast strains for use in frozen dough applications.

Isolation and Characterization of a Freeze-Tolerant Diploid Derivative of an Industrial Baker's Yeast Strain and Its Use in Frozen Doughs

ABSTRACT The routine production and storage of frozen doughs are still problematic. Although commercial bakers yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial bakers yeast production and usage.

Identification of genes responsible for improved cryoresistance in fermenting yeast cells

Using repetitive freezing and thawing, different mutant industrial Saccharomyces cerevisiae strains with increased freeze resistance have been isolated. To get a better insight in the mechanisms responsible for this elevated resistance and to give us the opportunity to modify other strains so that they become more suitable for use in frozen dough preparations, we applied the microarray technology in order to identify genes that are differentially expressed in a freeze-resistant mutant when compared to a freeze-sensitive industrial yeast strain.