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.

Figure 1

Growth on caffeine plates of selected Ppz1 clones carrying single amino acid mutations. The indicated strains were transformed with pRS316-based plasmids (centromeric, URA3 marker) carrying the indicated versions of Ppz1. Cultures were spotted at OD₆₆₀ = 0.05 and at 1/10 dilution, and plates were grown for 72 h. Ø, empty plasmid.

Figure 2

Tolerance to Li⁺ cations conferred by the diverse variants of the Ppz1 phosphatase. (a) The indicated strains were transformed with the diverse versions of Ppz1, and cultures were spotted as in Fig. 1 and grown for 72 h in the presence of various concentrations of LiCl. (b) Protein extracts (40 μg) from the indicated strains were subjected to electrophoresis on 10% polyacrylamide-SDS gels. Proteins were transferred to membranes and probed with a polyclonal anti-GST-Ppz1 antibody. Ø, empty plasmid; WT, wild type.

Figure 3

Ppz1 variants show differences in the pattern of in vitro inhibition by Hal3. p-nitrophenyl phosphate dephosphorylation assays were carried out as described under Methods. Six-hundred ng of the different affinity-purified Ppz1 variants were pre-incubated for 5 min with increasing amount of Hal3 prior starting the assay. Values are means ± S.E. for 4 to 12 different assays, and are expressed as the percentage of phosphatase activity relative to control without inhibitor. The dashed line at 50% activity is included to facilitate comparisons between panels.

Figure 4

Interaction of Hal3 with the different Ppz1 variants. (a) Equal amounts of the indicated versions of GST-Ppz1 were immobilized on glutathione beads and used as an affinity system to recover plasmid-borne HA-tagged Hal3 from extracts of strain IM021 (ppz1 hal3). Beads were washed and processed for SDS-PAGE (8% gels) and immunoblotting using anti-HA antibodies as described in Methods. The lower panel corresponds to the Ponceau-stained membranes, to reveal the amount of Ppz1. (b) Co-expression and co-purification of Ppz1 variants and Hal3 in E. coli. Several 6xHis-tagged Ppz1-Cter versions carrying selected mutations were co-expressed together with untagged Hal3 in a polycistronic vector under the T7 promoter. The left panel shows bacterial extracts corresponding to the wild type Ppz1 as an example (−, non-induced; +, induced). The 6x-His tagged Ppz1-Cter proteins were purified by Ni-NTA agarose affinity chromatography and the protein complex subsequently eluted with buffer containing 500 mM imidazole. Samples (15 μl) were analyzed by 10% SDS-PAGE and proteins revealed with BlueSafe protein stain.

Figure 5

Phenotypic analysis and expression levels of selected Ppz1 versions carrying two amino acid changes. (a) Yeast strains were transformed with the diverse versions of Ppz1 and cultures spotted in the presence of various concentrations of caffeine or LiCl, as in Figs 1 and 2. Pictures were taken after 72 h. (b) Protein extracts (40 μg) were analyzed as described in Fig. 2b. Ø, empty plasmid; WT, wild type.

Figure 6

Hal3 binding capacity and inhibition profile of Ppz1 variants carrying two aminoacid mutations. (a) The indicated versions of GST-Ppz1 were immobilized on glutathione beads and processed as described in the legend of Fig. 4a. The upper panel reveals the amount of HA-tagged Hal3 bound to the phosphatase. The lower panel corresponds to the Ponceau-stained membranes, to show the amount of Ppz1. (b) Dephosphorylation assays were carried out as described in Fig. 3. Wild type Ppz1 (-●-); clone 120 (-□-); clone 121 (-◊-); clone 129 (-Δ-). Values are means ± S.E. for 6 to 10 different assays.

Figure 7

Alignment of the catalytic domains of Ppz1 from S. cerevisiae (ScPpz1) and C. albicans (CaPpz1), with the yeast (ScGlc7) and human (HsPP1c) catalytic subunit of type 1 protein phosphatase. The positions of the single mutations described in this work are denoted in yellow background, whereas the three relevant mutations found in clones 75, 121 and 129 are in orange. The positions of residues relevant for interaction with diverse motifs found in mammalian PP1c regulators (RVxF, SILK, NIPP-1, Arg, Inhibitor-2 helix), which are in some cases considerably conserved in Ppz1, or those forming the catalytic site, are indicated at the bottom of the sequences using a color code system. Numbering corresponds to the full length proteins. Inh-2, Inhibitor-2.

Figure 8

Mapping mutations and functional elements on a structural model of the C-terminal catalytic moiety of ScPpz1. The relevant mutations described in this work are highlighted in yellow and orange (as in Fig. 7). Residues involved in the catalytic mechanisms are pink, and the conserved hydrophobic groove, implicated in binding to RVxF-containing regulatory subunits, is denoted in green. Residues identical or similar to those involved in the interaction of mammalian PP1c with Inhibitor-2 α-helix described in the text are highlighted in white. Note that E⁵⁷⁵, V⁶⁰⁵, E⁶³⁰, and F⁶³¹, as well as diverse residues in the catalytic site, although not depicted in white, are also involved in PP1c interaction with Inhibitor-2. The correspondence between the annotated residues and the clones recovered from the screen can be found in Tables 2 and 3.