Overexpression of budding yeast protein phosphatase Ppz1 impairs translation

Calafí, Carlos; López-Malo, María; Velázquez, Diego; Zhang, Chunyi; Fernández-Fernández, José; Rodríguez-Galán, Olga; Cruz, Jesús de la; Ariño, Joaquı́n; Casamayor, Antonio

doi:10.1016/j.bbamcr.2020.118727

Cited by 13 publications

(56 citation statements)

References 74 publications

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“…In addition, we created a pCM190-derived version of both Ppz1 and Ppz2 phosphatases carrying a HA epitope tag at their C-terminus. As previously reported [ 28 ] and shown in Figure 2 A, the expression of PPZ1 from the pCM190 vector fully abolished growth. In contrast, the expression of PPZ2 from plasmid YEp352 or from the pCM190 vector did not affect growth in a significative way.…”

Section: Resultssupporting

confidence: 88%

“…Several lines of evidence suggest that such an effect is not due to the mere increase in the amount of this N-terminal polypeptide sequence but would derive from the modulation of the Ppz1 phosphatase function or/and activity. Indeed, (i) the phosphatase catalytic activity of Ppz1 is necessary for toxicity [ 28 ], whereas the overexpression of the S. cerevisiae N-terminal half alone (from a high-copy plasmid and driven by the strong GAL promoter) has no effect on cell growth [ 4 ]; and (ii) there is plenty of evidence that the modification of the N-terminal half of Ppz1 has important and specific effects in Ppz1 function. Thus, a large deletion affecting from amino acid 17 to 193 in Ppz1 resulted in the normal complementation of the LiCl tolerance phenotype but caused significant loss of the ability to restore normal caffeine sensitivity, whereas the removal of residues from 241 to 318 had the opposite effect.…”

Section: Discussionmentioning

confidence: 99%

“…Vector pCM190-PPZ1, expressing untagged Ppz1, was constructed, as described in [ 28 ], using oligonucleotides OMLM3 and OMLM4. The untagged pCM190-PPZ1 G2A version was made in the same way but using oligonucleotides OMLM9 and OMLM4 for amplification.…”

Section: Methodsmentioning

confidence: 99%

“…As mentioned above, most work has been devoted to the elucidation of Ppz1 function and regulation, while Ppz2 has received less attention. We assumed that, because Ppz1 toxicity derives from its phosphatase activity [ 28 ], the overexpression of Ppz2 might have similar effects. We show here that this is not the case.…”

Section: Introductionmentioning

confidence: 99%

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

Calafí

López-Malo

Albacar

et al. 2020

IJMS

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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.

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Section: Resultssupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Calafí

López-Malo

Albacar

et al. 2020

IJMS

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“…Changes in abundance were very limited and relatively modest: only 10 proteins were found to increase at least twofold (Yro2, Ser3, Ssa4, Nqm1, Gnd2, Rtc3, Rse1, Ald3, Ygp1, and Ypr1). This was not surprising, as it can be explained by the recently described negative effect of overexpression of Ppz1 on protein translation 26 . All of them, with the only exception of Rtc3, also displayed consistent increases in expression at the mRNA level ( Supplementary Table S3).…”

Section: Overexpression Of Ppz1 Provokes Numerous Changes In Protein mentioning

confidence: 75%

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

Velázquez

Albacar

Zhang

et al. 2020

Sci Rep

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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.

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The toxic effects of yeast Ppz1 phosphatase are counteracted by subcellular relocalization mediated by its regulatory subunit Hal3

et al. 2022

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Overexpression of Saccharomyces cerevisiae protein phosphatase Ppz1 strongly impairs cell growth. Ppz1 is negatively regulated by its subunit Hal3, and Hal3 overexpression fully counteracts the toxic effects derived from high levels of the phosphatase. We show that Ppz1 localizes at the plasma membrane, and that co‐expression of Hal3 recruits Ppz1 to internal membranes (mostly vacuolar). This effect is not observed in a catalytically impaired mutant of Ppz1. Disruption of intracellular trafficking by deletion of the ESCRT‐0 component VPS27 abolishes both Hal3‐mediated relocalization of Ppz1 and normalization of cell growth, without affecting Ppz1 protein levels. We propose that Hal3 counteracts the toxic effects caused by excess of Ppz1 not only by inhibiting its enzymatic activity but also by recruiting the phosphatase to internal structures.

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Overexpression of budding yeast protein phosphatase Ppz1 impairs translation

Cited by 13 publications

References 74 publications

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

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

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

The toxic effects of yeast Ppz1 phosphatase are counteracted by subcellular relocalization mediated by its regulatory subunit Hal3

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