A gene encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD⁺) complements an osmosensitive mutant of Saccharomyces cerevisiae

Abstract

Osmoregulatory mutants of Saccharomyces cerevisiae with a defect in their capacity to readjust the cell volume/buoyant density after osmotically induced dehydration were enriched by density gradient centrifugation. Colonies derived from cells that remained dense after dehydration were screened for sensitivity to high concentrations of NaCl and defects in their osmotically induced production and intracellular accumulation of glycerol. The isolated osg (osmosensitive glycerol defective) mutants were recessive in heterozygous diploids and fell into four complementation groups (osg1-osg4). The osg1-1 mutant, described in this work, is unable to grow at low water potential and shows a decreased capacity for glycerol production and a strongly reduced activity of NAD(+)-dependent sn-glycerol 3-phosphate dehydrogenase (GPD), an enzyme in the glycerol-producing pathway. Complementation of the osg1-1 salt sensitivity defect with a low copy yeast genomic library led to the cloning of GPD1, encoding an S. cerevisiae GPD consisting of 391 amino acids and sharing 47-50% identity with GPD from other sources. Micro-sequencing of the N-terminus of purified S. cerevisiae GPD revealed a 20-amino-acid sequence that was identical to a nucleotide-deduced amino acid sequence in GPD1, but indicated that the enzyme is produced with an N-terminal extension that is removed from the functional enzyme. Subcellular fractionation does not indicate, however, that the putative pre-sequence targets GPD to any organelle; the enzyme appears to be located in the cytoplasm. Chromoblot and tetrad analysis were used to position the GPD1 gene to chromosome IV, with a distance of about 18 cM from trp1.

Dynamic Changes in the Subcellular Distribution of Gpd1p in Response to Cell Stress

“…In support of this idea, proteolytic cleavage of the N terminus of Gpd1p, which removes the PTS2 signal sequence, has been reported previously (1). However, using SDS-PAGE, we could not detect a change in the mobility of Gpd1p under conditions where its distribution shifts (i.e.…”

“…Glycerol 3-phosphate dehydrogenase (Gpd1p) is one of two NAD ϩ -dependent glycerol 3-phosphate dehydrogenases in yeast (1,2). It is classically defined as a cytosolic enzyme that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) 2 and NADH to glycerol 3-phosphate (Glycerol 3-phosphate) and NAD ϩ (2).…”

Distinct Intracellular Localization of Gpd1p and Gpd2p, the Two Yeast Isoforms of NAD+-dependent Glycerol-3-phosphate Dehydrogenase, Explains Their Different Contributions to Redox-driven Glycerol Production

“…In addition, glycerol has another important physiological function; it serves as an osmoregulator and is produced and accumulated in cells subjected to hyperosmotic stress to prevent loss of water and turgor of the cells (19 -21). The isoform of NADH-dependent glycerol-3-phosphate dehydrogenase that has a prime role in anaerobic production of glycerol is encoded by the GPD2 gene (17,18), while the other isoform of the enzyme, Gpd1p, is used for osmostressinduced glycerol production (21,22). Gpd1p also appears to be the isoform responsible for the glycerol-3-phosphate dehydrogenase activity in the G3P shuttle (8).…”

Effect of Fermentation Temperature and Culture Medium on Glycerol and Ethanol during Wine Fermentation

“…Glycerol biosynthesis is regulated by the key enzyme GPD (Gancedo et al 1968, Bradford 1976). The GPD1 and GPD2 genes, which encode the two isozymes of GPD (Larsson et al 1993), are subject to different controls; for example, the expression of GPD1 is increased in response to osmotic stress, whereas the expression of GPD2 is induced under anaerobic conditions (Albertyn et al 1994a, Pahlman et al 2001. Hyperosmotic stress also triggers the hyperosmotic glycerol (HOG) signaling pathway, which targets GPD1 (Albertyn et al 1994b) and the heat-inducible HSPI04 (Schuller et al 1994).…”