The PPZ Protein Phosphatases Are Important Determinants of Salt Tolerance in Yeast Cells

Abstract

Protein phosphatases PPZ1 and PPZ2 represent a novel form of Ser/Thr phosphatases structurally related to type 1 phosphatases and characterized by an unusual amino-terminal region. We have found that the deletion of PPZ1 gene results in increased tolerance to Na+ and Li+ cations. Simultaneous deletion of PPZ2 gene results in an additional increase in salt tolerance. After exposure to high concentration of Li+, the intracellular content of the cation was markedly decreased in ppz1 delta ppz2 delta mutants when compared to wild type cells. No significant differences were observed between both strains when the Li+ influx was measured, but ppz1 delta ppz2 delta mutants eliminated Li+ more efficiently than wild type cells. This can be explained by the fact that expression of the ENA1 gene, which encodes the major component of the efflux system for these cations, is strongly increased in ppz1 delta ppz2 delta cells. As expected, the disruption of the PPZ genes did not complement the characteristic hypersensitivity for Na+ and Li+ of a ena1 delta strain. The lack of protein phosphatase 2B (calcineurin) has been found to decrease salt resistance by reducing the expression of the ENA1 gene. We have observed that the disruption of the PPZ genes substantially enhances the resistance of the hypersensitive calcineurin-deficient mutants. Since PPZ phosphatases have been found to be functionally related to the protein kinase C/mitogen-activated kinase pathway, we have tested bck1 or mpk1/slt2 deletion mutants and found that they do not display altered salt sensitivity. However, disruption of PPZ1 fails to increase salt resistance in a mpk1/slt2 background. In conclusion, we postulate the existence in yeast of a novel PPZ-mediated pathway involved in salt homeostasis that is opposite to and independent of the recently described calcineurin-mediated pathway.

Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans

“…It is known that S. cerevisiae ppz1 tolerates saline treatment, but we found no significant differences between the parental and mutated C. albicans strains in the presence of NaCl. It was previously suggested that the tolerance to NaCl (and LiCl) of the S. cerevisiae mutant can be explained by the overexpression of the ENA1 sodium transporter (Posas et al, 1995;Ruiz et al, 2003). Sequence comparisons revealed that in C. albicans the orf19.6070 protein is an ENA1 orthologue; however, its function and regulation are not known at the moment.…”

“…We found that the null mutant was tolerant to LiCl, and was sensitive to KCl. According to these properties it behaves like the S. cerevisiae ppz1 mutant (Posas et al, 1995;Ruiz et al, 2003;Yenush et al, 2002). However, there was a clear-cut difference between the two mutants when sensitivity to NaCl was tested.…”

“…S. cerevisiae cells were grown at 28 uC in YPD medium (10 g yeast extract l 21 , 20 g peptone l 21 and 20 g glucose l 21 ) or, when carrying plasmids, in synthetic complete drop-out medium (lacking leucine). Sensitivity of yeast cells to LiCl, NaCl, caffeine (Merck) or Calcofluor White (CFW; Sigma) was evaluated by growth on agar plates (drop tests), as previously described (Posas et al, 1995). (Gutz et al, 1974) or Edinburgh minimal medium 2 (EMM2; US Biological), as plates or liquid cultures, was used with appropriate supplements (100 mg l 21 adenine, uracil, leucine).…”

“…Deletion of ScPPZ2 does not result in a detectable phenotype. However, strains lacking ScPpz1 exhibit increased tolerance to NaCl and LiCl (Posas et al, 1995), altered K + influx, and hypersensitivity to caffeine (Posas et al, 1993;Yenush et al, 2002). On the other hand, overexpression of ScPPZ1 results in slow growth and cell cycle blockage at the G1-S transition (Clotet et al, 1999).…”

“…On the other hand, overexpression of ScPPZ1 results in slow growth and cell cycle blockage at the G1-S transition (Clotet et al, 1999). The effect of the Scppz1 mutation on cation homeostasis is due to the elevated expression of the Na + -ATPase gene ENA1 (Posas et al, 1995;Ruiz et al, 2003) and to the increased influx of potassium ions mediated by the high-affinity K + -transporter Trk1 (Yenush et al, 2002). ScPPZ1 exhibits genetic interactions with SLT2, which encodes a mitogen-activated protein (MAP) kinase required for signalling in the cell wall integrity (CWI) pathway (reviewed by Levin, 2005).…”