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

Abstract

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.

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

“…We also show here that the G2A mutant retains most of the characteristic toxicity of native Ppz1 when overexpressed. This suggests that the major targets for Ppz1 toxicity do not localize at the plasma membrane, a hypothesis that fits well with our recent proposal that Ppz1 overexpression negatively impinges the protein translation process [28] and with the identification of two major cytosolic protein kinases, Snf1 and Hog1, as likely targets for the development of Ppz1 toxicity [26].…”

“…Similarly, the accumulation of unbudded cells expressing the Ppz1:2 chimera was indistinguishable from those expressing Ppz1 whereas, as mentioned above, cultures of cells bearing Ppz2 did not show alteration in the percentage of unbudded cells (Figure 2C). In addition, the recently reported beneficious effect on growth of the gcn2 [28] and hog1 [26] mutations in cells overexpressing native Ppz1 was mimicked in cells expressing the Ppz1:2 hybrid phosphatase (Figure S2). When the expression of these hybrid versions was investigated, we observed that none of them matched that of Ppz1, but while that of Ppz2:1 was very low, the expression of Ppz1:2 was similar to that observed for Ppz2.…”

“…The ppz1∆ and slt2∆ strains are kanMx4 deletant from the EUROFAN collection. The gcn2 strain is a kanMX4 deletant and the hog1 strain is a natMX4 deletant, described in [28] and [26], respectively. Strain AGS19 is a ppz1::KANMx4 ppz2::HIS3 double mutant and was described earlier [40].…”

“…Excessive Ppz1 activity is detrimental to the cell, causing reduced cell proliferation or even a total blockage in the cell cycle at the G 1 -S transition, which is accompanied by delayed expression of the G 1 phase cyclins Cln2 and Clb5 [4,15,[24][25][26]. A more recent genome-wide search using the genetic tug-of-war (gTOW) approach to identify dosage-sensitive genes demonstrated that PPZ1 is the gene for which the cell shows the lowest dosage tolerance limit [27], indicating that Ppz1 is likely the most toxic protein when overexpressed in budding yeast.…”

“…A more recent genome-wide search using the genetic tug-of-war (gTOW) approach to identify dosage-sensitive genes demonstrated that PPZ1 is the gene for which the cell shows the lowest dosage tolerance limit [27], indicating that Ppz1 is likely the most toxic protein when overexpressed in budding yeast. Very recent studies have shown that this toxicity results from the interference with diverse cellular functions, including protein translation, metabolic cell signaling and oxidative stress generation [26,28]. The investigation of the impact of Ppz1 in the cell biology might be relevant because Ppz1 has been recognized as a virulence factor in important human pathogenic fungi, such as Candida albicans [7] and Aspergillus fumigatus [29], albeit this is not a common trait for all fungal pathogens [30,31].…”