The Toxic Effects of Ppz1 Overexpression Involve Nha1-Mediated Deregulation of K+ and H+ Homeostasis.

The alteration of the fine-tuned balance of phospho/dephosphorylation reactions in the cell often results in functional disturbance. In the yeast Saccharomyces cerevisiae, the overexpression of Ser/Thr phosphatase Ppz1 drastically blocks cell proliferation, with a profound change in the transcriptomic and phosphoproteomic profiles. While the deleterious effect on growth likely derives from the alteration of multiple targets, the precise mechanisms are still obscure. Ppz1 is a negative effector of potassium influx. However, we show that the toxic effect of Ppz1 overexpression is unrelated to the Trk1/2 high-affinity potassium importers. Cells overexpressing Ppz1 exhibit decreased K+ content, increased cytosolic acidification, and fail to properly acidify the medium. These effects, as well as the growth defect, are counteracted by the deletion of NHA1 gene, which encodes a plasma membrane Na+, K+/H+ antiporter. The beneficial effect of a lack of Nha1 on the growth vanishes as the pH of the medium approaches neutrality, is not eliminated by the expression of two non-functional Nha1 variants (D145N or D177N), and is exacerbated by a hyperactive Nha1 version (S481A). All our results show that high levels of Ppz1 overactivate Nha1, leading to an excessive entry of H+ and efflux of K+, which is detrimental for growth.

The N-Terminal Region of Yeast Protein Phosphatase Ppz1 Is a Determinant for Its Toxicity.

The Ppz enzymes are Ser/Thr protein phosphatases present only in fungi that are characterized by a highly conserved C-terminal catalytic region, related to PP1c phosphatases, and a more divergent N-terminal extension. In Saccharomyces cerevisiae, Ppz phosphatases are encoded by two paralog genes, PPZ1 and PPZ2. Ppz1 is the most toxic protein when overexpressed in budding yeast, halting cell proliferation, and this effect requires its phosphatase activity. We show here that, in spite of their conserved catalytic domain, Ppz2 was not toxic when tested under the same conditions as Ppz1, albeit Ppz2 levels were somewhat lower. Remarkably, a hybrid protein composed of the N-terminal extension of Ppz1 and the catalytic domain of Ppz2 was as toxic as Ppz1, even if its expression level was comparable to that of Ppz2. Similar amounts of yeast PP1c (Glc7) produced an intermediate effect on growth. Mutation of the Ppz1 myristoylable Gly2 to Ala avoided the localization of the phosphatase at the cell periphery but only slightly attenuated its toxicity. Therefore, the N-terminal extension of Ppz1 plays a key role in defining Ppz1 toxicity. This region is predicted to be intrinsically disordered and contains several putative folding-upon-binding regions which are absent in Ppz2 and might be relevant for toxicity.

Yeast Ppz1 protein phosphatase toxicity involves the alteration of multiple cellular targets.

Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.

Overexpression of budding yeast protein phosphatase Ppz1 impairs translation.

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.