The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis

Abstract

The Ser/Thr protein phosphatase (PPase) Ppz1 is an enzyme related to the ubiquitous type-1 PPases (PP1c) but found only in fungi. It is regulated by an inhibitory subunit, Hal3, which binds to its catalytic domain. Overexpression of Ppz1 is highly toxic for yeast cells, so its de-regulation has been proposed as a target for novel antifungal therapies. While modulation of PP1c by its many regulatory subunits has been extensively characterized, the manner by which Hal3 controls Ppz1 remains unknown. We have used error-prone PCR mutagenesis to construct a library of Ppz1 variants and developed a functional assay to identify mutations affecting the binding or/and the inhibitory capacity of Hal3. We have characterized diverse Ppz1 mutated versions in vivo and in vitro and found that, although they were clearly refractory to Hal3 inhibition, none of them exhibited significant reduction in Hal3 binding. Mapping the mutations strengthened the notion that Hal3 does not interact with Ppz1 through its RVxF-like motif (found in most PP1c regulators). In contrast, the most relevant mutations mapped to a conserved α-helix region used by mammalian Inhibitor-2 to regulate PP1c. Therefore, modulation of PP1c and Ppz1 by their subunits likely differs, but could share some structural features.Protein phosphorylation is a widespread mechanism for controlling cellular processes. The phosphorylation state of a given protein results from the opposing activities of protein kinases and protein phosphatases (PPases). Yeast cells contain different kinds of PPases that are also found in other eukaryotic organisms, including humans, while they also contain fungal-specific enzymes, such as the Ppz1 Ser/Thr protein phosphatase. Ppz1 is composed of a catalytic carboxyl-terminal domain, which is approximately 60% identical to the ubiquitous catalytic subunit of protein phosphatase 1 (PP1c), and a largely unstructured NH 2 -terminal extension 1 . In budding yeast, Ppz1 is involved in the regulation of monovalent ion homeostasis, by inhibiting the expression of the Na + /K + -ATPase Ena1 2, 3 and the transport of potassium through the Trk1/Trk2 high-affinity transporters 4, 5 . Consequently, ppz1 deletion mutants are tolerant to toxic Na + and Li + cations 2 . These regulatory effects impact on diverse cellular functions, such as cell wall integrity and osmotic stability 4, 6, 7 , translational efficiency 8 , and progress through the cell cycle 9 . It is worth noting that recent work has identified Ppz1 as one of the most toxic proteins when overexpressed in budding yeast 10 , although the molecular basis for this effect is unknown. Ppz1 is regulated in vivo by Hal3/Sis2, a protein originally identified as a high-copy suppressor of the sit4 cell cycle-related growth defect 11 and by its capacity to confer halotolerance 12 . Hal3 binds to the carboxyl-terminal catalytic domain of Ppz1 and strongly inhibits its phosphatase activity, thus modulating its diverse physiological functions 13 . For that reason, cells overexpressing Hal3 are salt-tol...

Characterization of the atypical Ppz/Hal3 phosphatase system from the pathogenic fungus Cryptococcus neoformans

“…Remarkably, despite the ability to interact up to some extent with ScPpz1 or CnPpz1, the Cryptococcus Hal3‐like proteins are completely ineffective as phosphatase inhibitors in vitro . This observation supports the idea that, as previously suggested, the structural determinants of Hal3 required for binding and inhibition of Ppz1 are not the same (Munoz et al , ; Molero et al , ). The failure of CnHal3a and CnHal3b to inhibit Ppz phosphatases is not due to the different sequence within the loop in ScHal3 known to be relevant for inhibition but not necessary for interaction (Munoz et al , ), since replacement of this region in CnHal3b with the S. cerevisiae sequence did not improve inhibition in vitro (Fig.…”

“…In addition, overexpression of ScPpz1 in S. cerevisiae is highly toxic (De Nadal et al , 1998;Clotet et al , 1999;Makanae et al , 2013). These evidences have shed light on Ppz1 as a possible antifungal target Chen et al , 2016;Molero et al , 2017) and prompted us to initiate the study of this phosphatase and its putative regulatory subunits in the pathogenic fungus C. neoformans . Panel A. Ribbon representation of the structure of the monomer of CnHal3b (data accessible at PDB entry D_1200006961 (6EOA)).…”

“…In addition, overexpression of ScPpz1 in S. cerevisiae is highly toxic (De Nadal et al , ; Clotet et al , ; Makanae et al , ). These evidences have shed light on Ppz1 as a possible antifungal target (Ádám et al , ; Chen et al , ; Molero et al , ) and prompted us to initiate the study of this phosphatase and its putative regulatory subunits in the pathogenic fungus C. neoformans . To this end, we created the ppz1 deletion mutant in C. neoformans , expressed CnPpz1 in S. cerevisia e, and purified the protein from E. coli .…”

“…The fact that Ppz1 enzymes (i) are found only in fungi; (ii) they have been related to virulence in C. albicans and A. fumigatus and (iii) their regulation might differ from that of the related type‐1 protein phosphatase (an ubiquitous enzyme), allowed postulating Ppz1 enzymes as potential novel targets for antifungal therapy (Chen et al , ; Molero et al , ). Cryptococcus neoformans is an opportunistic fungal pathogen ubiquitous in the environment and the major causative agent of cryptococcosis.…”

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

“…Plasmids YEp195-PPZ1 and YEp352-PPZ2 are described in [ 46 ] and [ 3 ], respectively, and the sequence of oligonucleotides mentioned in this work can be found in Table S1 .…”