The two homologous genes GPD1 and GPD2 encode the isoenzymes of NAD-dependent glycerol 3-phosphate dehydrogenase in the yeast Saccharomyces cerevisiae. Previous studies showed that GPD1 plays a role in osmoadaptation since its expression is induced by osmotic stress and gpd1 delta mutants are osmosensitive. Here we report that GPD2 has an entirely different physiological role. Expression of GPD2 is not affected by changes in external osmolarity, but is stimulated by anoxic conditions. Mutants lacking GPD2 show poor growth under anaerobic conditions. Mutants deleted for both GPD1 and GPD2 do not produce detectable glycerol, are highly osmosensitive and fail to grow under anoxic conditions. This growth inhibition, which is accompanied by a strong intracellular accumulation of NADH, is relieved by external addition of acetaldehyde, an effective oxidizer of NADH. Thus, glycerol formation is strictly required as a redox sink for excess cytosolic NADH during anaerobic metabolism. The anaerobic induction of GPD2 is independent of the HOG pathway which controls the osmotic induction of GPD1. Expression of GPD2 is also unaffected by ROX1 and ROX3, encoding putative regulators of hypoxic and stress-controlled gene expression. In addition, GPD2 is induced under aerobic conditions by the addition of bisulfite which causes NADH accumulation by inhibiting the final, reductive step in ethanol fermentation and this induction is reversed by addition of acetaldehyde. We conclude that expression of GPD2 is controlled by a novel, oxygen-independent, signalling pathway which is required to regulate metabolism under anoxic conditions.

We have characterized the strongly homologous GPP1/RHR2 and GPP2/HOR2 genes, encoding isoforms of glycerol 3-phosphatase. Mutants lacking both GPP1 and GPP2 are devoid of glycerol 3-phosphatase activity and produce only a small amount of glycerol, confirming the essential role for this enzyme in glycerol biosynthesis. Overproduction of Gpp1p and Gpp2p did not significantly enhance glycerol production, indicating that glycerol phosphatase is not rate-limiting for glycerol production. Previous studies have shown that expression of both GPP1 and GPP2 is induced under hyperosmotic stress and that induction partially depends on the HOG (high osmolarity glycerol) pathway. We here show that expression of GPP1 is strongly decreased in strains having low protein kinase A activity, although it is still responsive to osmotic stress. The gpp1⌬/gpp2⌬ double mutant is hypersensitive to high osmolarity, whereas the single mutants remain unaffected, indicating GPP1 and GPP2 substitute well for each other. Transfer to anaerobic conditions does not affect expression of GPP2, whereas GPP1 is transiently induced, and mutants lacking GPP1 show poor anaerobic growth. All gpp mutants show increased levels of glycerol 3-phosphate, which is especially pronounced when gpp1⌬ and gpp1⌬/ gpp2⌬ mutants are transferred to anaerobic conditions. The addition of acetaldehyde, a strong oxidizer of NADH, leads to decreased glycerol 3-phosphate levels and restored anaerobic growth of the gpp1⌬/gpp2⌬ mutant, indicating that the anaerobic accumulation of NADH causes glycerol 3-phosphate to reach growth-inhibiting levels. We also found the gpp1⌬/gpp2⌬ mutant is hypersensitive to the superoxide anion generator, paraquat. Consistent with a role for glycerol 3-phosphatase in protection against oxidative stress, expression of GPP2 is induced in the presence of paraquat. This induction was only marginally affected by the general stress-response transcriptional factors Msn2p/4p or protein kinase A activity. We conclude that glycerol metabolism plays multiple roles in yeast adaptation to altered growth conditions, explaining the complex regulation of glycerol biosynthesis genes.

Duringanaerobiosis Saccharomyces cerevisiae strongly increases glycerol production to provide for non-respiratory oxidation of NADH to NAD ؉ . We here report that respiratory-deficient cells become strictly dependent on the Gpd2p isoform of the NAD ؉ -linked glycerol-3-phosphate dehydrogenase (Gpd). The growth inhibition of respiratory incompetent cox18⌬ cells lacking GPD2 is reversed by the addition of acetoin, an alternative sink for NADH oxidation. Growth is also restored by addition of lysine or glutamic acid/glutamine, the synthesis of which involves production of mitochondrial NADH. Lysine produced a stronger growth stimulating effect than glutamic acid consistent with an upregulated expression of the IDP3 gene for peroxisomal synthesis of the glutamate precursor ␣-ketoglutarate. Gpd2p is known to be a cytosolic protein but possesses a classical mitochondrial presequence, which we show is sufficient for mitochondrial targeting. A partial mitochondrial localization of Gpd2p will provide for establishment of intramitochondrial redox balance under non-respiratory conditions. Gpd1p, the other Gpd isoform, is partly cytosolic and partly peroxisomal and becomes more strictly peroxisomal in respiratory-deficient mutants. The different cellular distribution of Gpd1p and Gpd2p thus appears to be the main reason Gpd1p cannot substitute for Gpd2p in cox18⌬gpd2⌬ cells, despite similar kinetic characteristics of the two iso-enzymes.

Glycerol formation is vital for reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in the yeast Saccharomyces cerevisiae. However, relatively few studies have been made on hyperosmotic stress under anaerobic conditions. To study the combined effect of salt stress and anaerobic conditions, industrial and laboratory strains of S. cerevisiae were grown anaerobically on glucose in batch-cultures containing 40 g/l NaCl. The time needed for complete glucose conversion increased considerably, and the specific growth rates decreased by 80-90% when the cells were subjected to the hyperosmotic conditions. This was accompanied by an increased yield of glycerol and other by-products and reduced biomass yield in all strains. The slowest fermenting strain doubled its glycerol yield (from 0.072 to 0.148 g/g glucose) and a nearly fivefold increase in acetate formation was seen. In more tolerant strains, a lower increase was seen in the glycerol and in the acetate, succinate and pyruvate yields. Additionally, the NADH-producing pathway from acetaldehyde to acetate was analysed by overexpressing the stress-induced gene ALD3. However, this had no or very marginal effect on the acetate and glycerol yields. In the control experiments, the production of NADH from known sources well matched the glycerol formation. This was not the case for the salt stress experiments in which the production of NADH from known sources was insufficient to explain the formed glycerol.

Under anaerobic conditions S. cerevisiae produces glycerol to regenerate NAD + from the excess NADH produced in cell metabolism. We here report on the role of an uncharacterized protein, Yig1p (Ypl201cp), in anaerobic glycerol production. Yig1p was previously shown to interact in two-hybrid tests with the GPP1 and GPP2 encoded glycerol 3-phosphatase (Gpp), and we here demonstrate that strains overexpressing YIG1 show strongly decreased Gpp activity and content of the major phosphatase, Gpp1p. However, cells overexpressing YIG1 exhibited only slightly decreased GPP1 transcript levels, suggesting that Yig1p modulates expression on both transcriptional and post-transcriptional levels. In agreement with such a role, a GFP-tagged derivate of Yig1p was localized to both the cytosol and the nucleus. Deletion or overexpression of YIG1 did not, however, significantly affect growth yield or glycerol yield in anaerobic batch cultures, which is consistent with the previously proposed low flux control exerted at the Gpp level.