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

Abstract

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.

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

“…Because most known targets for Ppz1, such as Trk1/Trk2 and Nha1 (and perhaps Pma1), are located at the plasma membrane, one might assume that removal of Ppz1 from the cell periphery should be sufficient for counteracting toxicity. However, the observation that, when expressed from plasmid pCM190 (a tetO ‐regulatable vector), mutation of Gly2 to Ala leads only to a slight attenuation of toxicity [19], indicates that very relevant targets for Ppz1 toxicity are not located at the plasma membrane. Among such targets could be diverse cytosolic protein kinases (i.e.…”

“…Generation of C‐terminally GFP‐tagged versions of Ppz1 and Ppz1 G2A expressed from plasmid YEplac195 (2‐micron, URA3 ) was described in [19]. The equivalent construct expressing the catalytically impaired R451L Ppz1 version was made by replacing the PacI and Kpn2I fragment of YEp195 Ppz1‐GFP with the equivalent one from the pCM189 Ppz1‐R451L construct (laboratory stock).…”

“…As documented in the Introduction section, previous reports described that Ppz1 was mostly associated with the peripheral membrane. The ability to associate with membranes was attributed to the myristoylable Gly at position 2 [14], and recent work demonstrated that mutation of Gly2 to Ala results in disappearance of the phosphatase from the cell's periphery and uniform distribution inside the cell [13,19]. We tested here whether the phosphatase function of Ppz1 was relevant for its subcellular localization by constructing a GFP-tagged version of Ppz1-R451L, a nearly inactive version of the phosphatase [14] whose overexpression barely affects cell growth [16].…”

“…Current evidence indicates that such toxicity derives from alteration of multiple targets affecting intracellular signalling [16,17], and the abnormal activation of the Nha1 Na + ,K + /H + antiporter [18]. Remarkably, while the phosphatase activity of Ppz1 is required for toxicity [16], the N-terminal half of Ppz1 also contributes to this effect [19].…”

“…In spite of some discrepancies among databases constructed from large-scale screening experiments (ref. [25] and https://yplp.yeastgenome.org/), it is generally accepted that Ppz1 can be detected mainly at the cell periphery, likely attached to the plasma membrane because of its conserved myristoylated Gly2 [7,13,14,19,26]. With the aim to better understand the mechanism by which Hal3 could neutralize Ppz1 toxicity, we overexpressed fluorescently tagged versions of Ppz1 and Hal3.…”