Manipulation of Reducing Power

In this previous discussion of pathways to stress tolerance we have chiefly placed emphasis on the type of protein or product that is induced. In the following, we discuss experiments indicating that the process and not so much the product may equally be important for protection. This experiment tested the long held concept that the function of accumulating metabolites is mainly or exclusively osmotic. The concept was tested in yeast, in which salinity stress leads to a dramatic increase of the simplest acyclic polyol, glycerol, to over 500 mM in the cells. Deleting the two genes encoding glycerol-1-phosphate dehydrogenase renders the cells extremely salt sensitive.69-70 We used the deletion strain lacking these two genes and incorporated genes leading to sorbitol or mannitol synthesis into the line. With sorbitol -6P DH, we achieved polyol amounts (~400 mM) nearly as high as glycerol amounts (~450 mM) in wild type. However, the sorbitol -producing yeast cells were not as resistant to salt stress as the wild type, and only marginally more tolerant than the deletion line (Shen et al, in preparation). We do not know why this is the case, but we offer one explanation. The intracellular concentration of glycerol in yeast increases from approximately 25 mM to 450 mM during stress, but much more glycerol is excreted into the medium than is retained by the cells. We estimated that approximately 95% of the glycerol synthesized is being excreted during the experiment. In contrast, the sorbitol-producing cells retained most of the sorbitol. Approximately only 20% of the sorbitol was excreted or lost to the medium. If this discrepancy is expressed in terms of reducing equivalents, yeast expends about 15 times more energy on synthesizing the glycerol that is in the medium than on sorbitol which mostly stays in the cells.71 As a hypothesis, we suggest that oxidation of NADPH—which would allow respiration to continue—may be more important than generating high amounts of osmolytes inside the cell. Possibly, the term "osmotic adjustment" does not describe the functions of osmolytes adequately.

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