Phosphoglucomutase is an in vivo lithium target in yeast.

Lithium is a drug frequently used in the treatment of manic depressive disorder. We have observed that the yeast Saccharomyces cerevisiae is very sensitive to lithium when growing in galactose medium. In this work we show that lithium inhibits with high affinity yeast (IC50 approximately 0.2 mm) and human (IC50 approximately 1.5 mm) phosphoglucomutase, the enzyme that catalyzes the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Lithium inhibits the rate of fermentation when yeast are grown in galactose and induces accumulation of glucose 1-phosphate and galactose 1-phosphate. Accumulation of these metabolites was also observed when a strain deleted of the two isoforms of phosphoglucomutase was incubated in galactose medium. In glucose-grown cells lithium reduces the steady state levels of UDP-glucose, resulting in a defect on trehalose and glycogen biosynthesis. Lithium acts as a competitive inhibitor of yeast phosphoglucomutase activity by competing with magnesium, a cofactor of the enzyme. High magnesium concentrations revert lithium inhibition of growth and phosphoglucomutase activity. Lithium stress causes an increase of the phosphoglucomutase activity due to an induction of transcription of the PGM2 gene, and its overexpression confers lithium tolerance in galactose medium. These results show that phosphoglucomutase is an important in vivo lithium target.

Regulation of monovalent ion homeostasis and pH by the Ser-Thr protein phosphatase SIT4 in Saccharomyces cerevisiae.

A gene, SIT4, was identified as corresponding to a serine/threonine protein phosphatase and when overexpressed confers lithium tolerance in galactose medium to the budding yeast Saccharomyces cerevisiae. This gene has been previously identified as a regulator of the cell cycle and involved in nitrogen sensing. It is shown that the transcription levels of SIT4 are induced by low concentrations of Li(+) in a time-dependent manner. Na(+) and K(+) at high concentrations, but not sorbitol, also induce transcription. As a response to Na(+) or Li(+) stress, yeast cells lower the intracellular K(+) content. This effect is enhanced in cells overexpressing SIT4, which also increase (86)Rb efflux after the addition of Na(+) or Li(+) to the extracellular medium. Another feature of SIT4-overexpressing cells is that they maintain a more alkaline pH of 6.64 compared with 6.17 in the wild type cells. It has been proposed that the main pathway of salt tolerance in yeast is mediated by a P-type ATPase, encoded by PMR2A/ENA1. However, our results show that in a sit4 strain, expression of ENA1 is still induced by monovalent cations, and overexpression of SIT4 does not alter the amount of ENA1 transcript. These results show that SIT4 acts in a parallel pathway not involving induction of transcription of ENA1 and suggest a novel function for SIT4 in response to salt stress.

An NH2-terminal deleted plasma membrane H+-ATPase is a dominant negative mutant and is sequestered in endoplasmic reticulum derived structures.

The NH2-terminus of the plasma membrane H+-ATPase is one of the least conserved segments of this protein among fungi. We constructed and expressed a mutant H+-ATPase from Saccharomyces cerevisiae deleted at an internal peptide within the cytoplasmic NH2-terminus (D44-F116). When the enzyme was subjected to limited trypsinolysis it was digested more rapidly than wild type H+-ATPase. Membrane fractionation experiments and immunofluorescence microscopy, using antibodies against H+-ATPase showed that the mutant ATPase is retained in the endoplasmic reticulum. The pattern observed in the immunofluorescence microscopy resembled structures similar to Russell bodies (modifications of the endoplasmic reticulum membranes) recently described in yeast. When the wild type H+-ATPase was co-expressed with the mutant, wild type H+-ATPase was also retained in the endoplasmic reticulum. Co-expression of both ATPases in a wild type yeast strain was lethal, demonstrating that this is a dominant negative mutant.