Mechanisms of Salt Tolerance Conferred by Overexpression of the HAL1 Gene in Saccharomyces cerevisiae

Overexpression of the HAL1 gene improves the tolerance of Saccharomyces cerevisiae to NaCl by increasing intracellular K⁺ and decreasing intracellular Na⁺. The effect of HAL1 on intracellular Na⁺ was mediated by the PMR2/ENA1 gene, corresponding to a major Na⁺ efflux system. The expression level of ENA1 was dependent on the gene dosage of HAL1 and overexpression of HAL1 suppressed the salt sensitivity of null mutants in calcineurin and Hal3p, other known regulators of ENA1 expression. The effect of HAL1 on intracellular K⁺ was independent of the TRK1 and TOK1 genes, corresponding to a major K⁺ uptake system and to a K⁺ efflux system activated by depolarization, respectively. Overexpression of HAL1 reduces K⁺ loss from the cells upon salt stress, a phenomenon mediated by an unidentified K⁺ efflux system. Overexpression of HAL1 did not increase NaCl tolerance in galactose medium. NaCl poses two types of stress, osmotic and ionic, counteracted by glycerol synthesis and sodium extrusion, respectively. As compared to glucose, with galactose as carbon source glycerol synthesis is reduced and the expression of ENA1 is increased. As a consequence, osmotic adjustment through glycerolsynthesis, a process not affected by HAL1, is the limiting factor for growth on galactose under NaCl stress. © 1997 John Wiley & Sons, Ltd.     

Low-Affinity glucose carrier gene LGT1 of Saccharomyces cerevisiae,a homologue of the Kluyveromyces lactis RAG1 gene

A low-affinity glucose transporter gene of Saccharomyces cerevisiae was cloned by complementation of the rag1 mutation in a strain of Kluyveromyces lactis defective in low-affinity glucose transport. Gene sequence and effects of null mutation in S. cerevisiae were described. Data indicated that there are multiple genes for low-affinity glucose transport.     

Effect of cinnamic acid on the growth and on plasma membrane H–ATPase activity of Saccharomyces cerevisiae

Cinnamic acid and cinnamic acid derivatives occur in plants and fruits, providing a natural protection against infections by pathogenic microorganisms. They may also inhibit wine fermentation and other fruit juice fermentations by Saccharomyces cerevisiae and raise difficulties in the biological treatment of waste water from some food industries. In the present work, it is shown that cells of S. cerevisiae YPH499 grown at pH 4 and 30°C, in the presence of concentrations of cinnamic acid (20 or 35 mg/l) that reduce the maximum specific growth rate by 46 or 53%, respectively, exhibit a more active plasma membrane H⁺–ATPase than cells grown in its absence. This stimulatory effect was detected by assaying, during yeast growth in absence or presence of cinnamic acid, both the plasma membrane ATPase activity in crude membrane extracts and its action as a proton-pump by comparing extracellular acidification as a function of culture cell density. The lag-phase of approximately 8 h observed during cultivation in the presence of 20 mg/l cinnamic acid of yeast cells previously grown in its absence was eliminated by growing the inoculum in medium supplemented with the same concentration of cinnamic acid. These cinnamic acid adapted cells exhibited a more active plasma membrane H⁺–ATPase and this phenomenon may be due to and/or be among the mechanisms underlying the adaptative response to this toxic acid in yeast.     

The SKS1 protein kinase is a multicopy suppressor of the snf3 mutation of Saccharomyces cerevisiae

Saccharomyces cerevisiae strains carrying snf3 are defective in high affinity glucose transport, and thus are unable to grow fermentatively on media with low concentrations of glucose. A multicopy suppressor of the snf3 growth defect, SKS1 (suppressor kinase of snf3), was found to encode a putative ser/thr protein kinase homologous to Ran1p, a kinase that regulates the switch between meiosis and vegetative growth in Schizosaccharomyces pombe. Overexpression of the SKS1 open reading frame is sufficient for suppression of the growth defects of snf3 mutants. Disruption of the open reading frame eliminates this suppression; as does the mutation of the consensus ATP binding site of Sks1p. A DDSE (DNA dependent snf3 suppressor element) was found to be present in the SKS1 promoter region. The suppression by this DDSE occurs in the absence of SKS1 coding region, that is, the DDSE can suppress a snf3 sks1 double null mutant which fails to grow fermentatively on low glucose as a snf3 mutant does. Both SKS1 and its DDSE can additionally suppress the growth defects of grr1 mutants, which are also impaired in high affinity glucose transport. The snf3 genomic suppressors, rgt1, RGT2 and ssn6, are also capable of suppressing snf3 associated growth defects in a strain lacking sks1.     

Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles

The existence of a K⁺/H⁺ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K⁺/H⁺ transport system, which is able to catalyse both K⁺ influx and efflux, is mainly driven by the K⁺ transmembrane gradient and can function even if the plasma membrane H⁺-ATPase is not active. Using the anionic oxonol VI and the cationic DISC₂(5) probes, it was shown that a membrane potential is not created during K⁺ fluxes. Such a dye response argues for the presence of a K⁺/H⁺ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6·8. This reversible transport system presents a high selectivity for K⁺ among other monovalent cations and a higher affinity for the K⁺ influx into the vesicles (exit from cells). The possible role of this K⁺/H⁺ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.