Functional mapping of the N-terminal region of the yeast moonlighting protein Sis2/Hal3 reveals crucial residues for Ppz1 regulation zquez, Marcel Albacar, Antonio Casamayor and Joaquın Arin Carlos Santolaria, Diego Vela ~o noma de Barcelona, Cerdanyola Institut de Biotecnologia i Biomedicina & Departament de Bioquımica i Biologia Molecular, Universitat Auto s, Spain del Valle Keywords The function of the Saccharomyces cerevisiae Ppz1 phosphatase is con- disordered regions; mutagenesis; protein trolled by its inhibitory subunit Hal3. Hal3 is a moonlighting protein, phosphatase; regulatory subunit; yeasts which associates with Cab3 to form a decarboxylase involved in the CoA biosynthetic pathway. Hal3 is composed by a conserved core PD region, Correspondence ~o, Institut de Biotecnologia i J. Arin required for both Ppz1 regulation and CoA biosynthesis, a long N- Biomedicina, & Departament de Bioquımica terminal extension, and an acidic C-terminal tail. Cab3 has a similar struc- i Biologia Molecular, Universitat Autonoma ture, but it is not a Ppz1 inhibitor. We show here that deletion or specific de Barcelona, 08193 Cerdanyola del Valle s, mutations in a short region of the N-terminal extension of Hal3 compro- Spain mise its function as a Ppz1 inhibitor in vivo and in vitro without negatively Tel: +34 935811315 affecting its ability to interact with the phosphatase. This study defines a E-mail: joaquin.arino@uab.es R-K-X(3)-VTFS- sequence whose presence explains the unexpected ability Present address of Cab3 (but not Hal3) to regulate Ppz1 function in Candida albicans. This Laboratory of Membrane Transport, Institute sequence is conserved in a subset of fungi and it could serve to estimate of Physiology CAS, Prague, Czech Republic the relevance of Hal3 or Cab3 proteins in regulating fungal Ppz enzymes. We also show that the removal of the motif moderately affects both Ppz1 (Received 5 April 2022, revised 15 June intracellular relocalization and counteraction of toxicity in cells overex- 2022, accepted 5 July 2022) pressing the phosphatase. Thus, our work contributes to our understanding of the regulation of Ppz phosphatases, which are determinants for virulence doi:10.1111/febs.16572 in some pathogenic fungi. Introduction Protein phosphorylation is a widespread mechanism for most functionally relevant isoform. Ppz1 is a 692- regulating cellular functions. The phosphorylation sta- residue protein composed of a C-terminal catalytic tus of a given protein is the result of the balanced action domain (about 60% identical to Glc7, the catalytic of protein kinases and protein phosphatases, often in subunit of genuine yeast PP1), and a long N-terminal response to intra or extracellular signalling cues. In segment (~ 350 residues) [3]. Ppz1 plays diverse roles addition to the well-known role of protein kinases, the in the yeast cell, but it is crucial for the maintenance relevance of protein phosphatases in signal transmission of monovalent cation homeostasis. This occurs by has become evident in the last few years [1]. decreasing potassium influx by both high-affinity Trk The Saccharomyces cerevisiae Ppz1 and Ppz2 Ser/ transporter-dependent and -independent mechanisms, Thr protein phosphatases define a subfamily of the and by downregulating the expression of the ENA1 PPP enzymes that are found only in fungi [2]. PPZ1 gene, encoding a Na+/K+-ATPase induced by high salt and PPZ2 are paralogous genes, with Ppz1 being the and alkaline pH [4–9]. Consequently, ppz1 mutants are Abbreviations CoA, coenzyme A; GFP, green fluorescent protein; MAP kinase, mitogen-activated protein kinase; PP1c, catalytic subunit of protein phosphatase 1; SGD, Saccharomyces Genome Database. The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 1 Federation of European Biochemical Societies. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Functional mapping of the Hal3 N-terminal region C. Santolaria et al. hypertolerant to Na+ and Li+ cations, as well as sensi- PPCDC activity and, hence, displays sequence identity tive to conditions or drugs affecting the integrity of with both bacterial and eukaryotic PPCDCs; and (c) a the cell wall, such as high temperature or treatment C-terminal tail, of roughly 80 residues in length, very with calcofluor white or caffeine [5,10–13]. rich in acidic residues. Previous work determined that It is known that excessive Ppz1 activity leads to a the conserved PD core of Hal3 is necessary for both total blockage in the cell cycle at the G1-S transition PPCDC activity and the regulatory role on Ppz1 [27– [11,14–17] and it has been demonstrated that PPZ1 is 29], whereas the acidic C-terminal tail is dispensable the gene for which the cell shows the lowest dosage for Hal3 function as a PPCDC, but is crucial for its tolerance limit [18], suggesting that Ppz1 is among the role as a Ppz1 inhibitor [28]. Similarly, the N-terminal most toxic proteins in yeast when overexpressed domain, although not functional by itself, might con- [17,19–21]. Consequently, Ppz1 activity must be finely tribute to stabilize the interaction between Hal3 and regulated. In S. cerevisiae Ppz phosphatases are modu- Cab3 when forming the PPCDC trimer. Interestingly, lated by two regulatory subunits, Hal3 and Vhs3 it was found that the PD and the N-terminal regions [11,22]. Both genes are paralogs, although the function were ineffective, by themselves, in inhibiting full-length of Hal3 as a Ppz regulator is more relevant in vivo Ppz1 or its catalytic C-terminal domain, but a than that of Vhs3. polypeptide containing both regions, although very Hal3 (also known as Sis2) was identified as a gene inefficient on the full-length phosphatase, was able to able to provide increased tolerance to salt stress [23], substantially inhibit the activity of the catalytic Ppz1 and to rescue cell cycle blockage in a Sit4-deficient C-ter polypeptide [28]. This evidence hinted that the mutant when overexpressed [24]. Both effects were N-terminal region of Hal3 might contribute to the subsequently explained by the ability of Hal3 to bind function of the protein as a Ppz1 regulator. to the catalytic C-terminal domain of Ppz1 and inhibit The interest in the regulation of Ppz1 activity was its activity [11,15]. In agreement with this regulatory increased upon reports that the phosphatase was a role, deletion of HAL3 renders the cell hypersensitive determinant for virulence in the pathogenic fungi Can- to NaCl or LiCl, whereas overexpression of the gene dida albicans [30] and Aspergillus fumigatus [31]. C. al- provides tolerance to these cations, decreases tolerance bicans encodes two genes resembling the Hal3/Vhs3/ to caffeine, and induces lysis in Slt2-deficient cells. Cab3 triad. The protein encoded by orf19.7378 was Overexpression of the HAL3 gene fully counteracts the designated as CaHal3 on the basis of its closer similar- toxic effects caused by high levels of Ppz1 [11,15,20], ity to S. cerevisiae Hal3/Vhs3 proteins in the PD and it has been very recently demonstrated that this domain including the presence of the typical catalytic effect requires Hal3-mediated relocalization of Ppz1 His in its conserved environment, whereas the product from the cell periphery to internal membranes [25]. of orf19.3260 was more reminiscent of Cab3 and con- In S. cerevisiae and other related fungi, Hal3 and tained the conserved catalytic Cys [32]. CaHal3 and Vhs3 are moonlighting proteins. At least one of them CaCab3 exhibit the characteristic three regions found is required for the formation of an atypical heterotri- in S. cerevisiae Hal3: a conserved PD, a long N- meric phosphopantothenoylcysteine decarboxylase terminal extension (relatively divergent), and an acidic (PPCDC) enzyme [26] and therefore, they are neces- C-terminal tail. Remarkably, although both proteins sary in the biosynthesis of coenzyme A (CoA). This behaved as their S. cerevisiae counterparts concerning trimeric structure involves a third component, Cab3 PPCDC function, only CaCab3, but not CaHal3, was (Ykl088w), which is also structurally related to Hal3 able to act as a Ppz1 inhibitor when expressed in and Vhs3, in a way that the catalytic site is formed at S. cerevisiae [32]. This unexpected result was difficult the interface of one molecule of Hal3 or Vhs3 (provid- to explain on the basis of the conservation of residues ing a key catalytic His residue) and one of Cab3 (sup- located at the PD region previously known to be rele- plying a required Cys residue). It has been shown that vant for Ppz1 regulation [27,32]. Cab3 interacts with Ppz1 but does not regulate the These open questions prompted us to undertake a phosphatase in vivo, nor inhibits the enzyme in vitro detailed structure–function analysis of the role of the [26]. N-terminal region of S. cerevisiae Hal3 concerning its The Hal3 protein (562 amino acids) can be divided function as a regulator of the Ppz1 phosphatase. We into three segments. (a) an N-terminal region (about demonstrate that this region plays an important role 250 residues), unrelated to other proteins outside in the ability of Hal3 to inhibit Ppz1 without affecting yeasts, and for which a disordered structure is pre- its binding capacity to the phosphatase, Moreover, we dicted; (b) a structured core region of similar size have been able to pinpoint the relevant region to resi- (named here PD region), which is responsible for the dues 90–105, where K90, R91 and the 95VTFS98 2 The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. C. Santolaria et al. Functional mapping of the Hal3 N-terminal region sequence are key determinants. We also show that this mediated influx attributed to the inhibition of Ppz1. region is conserved in CaCab3 but not in CaHal3, and We show in Fig. 1B (right panel) that all variants, that their exchange abolishes the ability of CaCab3 to with the exception of D67–110, D67–93 and D94–110, regulate S. cerevisiae Ppz1 function, thus providing a did block proliferation of the slt2D mutant. The con- basis to explain the apparently contradictory beha- structs producing PD-Cter (and particularly PD) viour of these proteins as Ppz1 regulators. Finally, the showed a marked loss of function in all tests. extension of this analysis to other fungi supports the These results indicate that, because all phenotypes notion that Cab3, and not Hal3, would be a moon- tested are related to the role of Hal3 as an inhibitory lighting protein in most of the species belonging to the subunit of Ppz1, the region ranging from residue 67 to CTG clade and in a few other related families. 110 must contain important determinants for such function. It can also be concluded that these determi- nants cannot be exclusively allocated to the 67–93 or Results 94–110 subregions, since both partial deletions per- fectly mimic the complete one. However, we knew Coarse mapping of the N-terminal extension of from previous work that, in this experimental setting, Hal3 reveals a defined region required for Ppz1- the levels of PD-Cter and PD could be markedly lower mediated function. than that of native Hal3, so there was the possibility As an initial attempt to identify structural determi- that the inability of our full N-terminal deletion vari- nants in the nearly 250 residues of the N-terminal ants to replace the functions of native Hal3 in overex- Hal3 extension that could be relevant for its inhibitory pression assays could be due to lower amounts of function on the Ppz1 phosphatase, we created seven protein. We, therefore, tested the levels of protein different versions bearing deletions of residues 39–51 accumulation of native Hal3 and of all nine variants. (D39–51), 67–110 (and its subdeletions D67–93 and As deduced from Fig. 1C, in contrast to the PC-Cter D94–110), 118–148 (D118–148), 209–227 (D209–227) and PD variants, which are produced clearly in lower and 248–256 (D248–256) (Fig. 1A). These specific amounts, all partial deletions are produced at levels regions were selected based on two criteria: (a) similar to those of native Hal3, ruling out the possibil- sequence conservation among different yeast species, ity that the failure to mimic the function of native and (b) the results of our recently reported chemical Hal3 could be due to insufficient levels of protein. The cross-linking experiments [33], which suggested several PC-Cter and PD variants were not included in the sub- interactions between the catalytic C-terminal domain sequent experiments because their lower levels impede of Ppz1 and diverse regions within the N-terminal the proper interpretation of the obtained results. Hal3 extension. These versions were cloned in plasmid We also took advantage of the ability of Hal3 to pWS93, to allow multicopy expression from the ADH1 counteract the dramatic growth arrest suffered by cells promoter, and were subsequently introduced into overexpressing PPZ1. Fig. 1D illustrates that, while diverse yeast strains to test for the function of these the strain ZCZ01 (which expresses Ppz1 from the Hal3 versions as Ppz phosphatase regulators. For com- strong GAL promoter) cannot grow when galactose is parison, we initially included in the study two previ- added to the medium, co-expression with native HAL3 ously characterized constructs producing a Hal3 fully restores growth. The same beneficial effect is version devoid of its N-terminal domain with (PD- observed for all Hal3 variants with the exception, once Cter) or without (PD) the acidic C-terminal tail [28]. more, of deletions in residues in the region spanning Overexpression of native Hal3 confers higher sensitiv- from 67 to 110. ity to caffeine and increased tolerance to toxic cations, We next investigated the ability of these variants to such as LiCl. As shown in Fig. 1B, all three versions regulate Ppz1 activity in vitro. To this end, the differ- bearing total or partial deletions in the region compris- ent versions were expressed in E. coli and purified as ing residues 67–110 did not decrease tolerance to caf- GST-fusions, the GST moiety removed, and the feine and barely improved tolerance to LiCl, whereas recombinant protein tested for Ppz1 inhibition. As the remaining constructs resulted in phenotypes very shown in Fig. 2, the inhibitory capacity of the D67–93, similar to those conferred by expression of native D94–110 and, in particular, the D67–110 variant, was Hal3. A second test for Hal3 function was based in clearly poorer than that of native Hal3. The D39–51 the ability of the regulator, when expressed in multi- and D118–148 deletions were indistinguishable from copy, to induce cell lysis in strains deficient in the the full-length Hal3 protein, whereas the versions with MAP kinase Slt2. As mentioned in the Introduction, deletions affecting the segments proximal to the cat- such behaviour derives from an increase in Trk1,2- alytic domain (D209–227 and D248–256) were slightly The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 3 Federation of European Biochemical Societies. Functional mapping of the Hal3 N-terminal region C. Santolaria et al. (A) 39-51 67-110 118-148 209-227 67-93 94-110 248-256 (B) Caffeine (mM) LiCl (mM) Strain Plasmid Strain 4 6 150 250 400 + Sorbitol BY4741 Ø BY4741 hal3Δ Ø Hal3 Δ39-51 Δ67-93 Δ67-110 Δ94-110 slt2Δ BY4741 Δ118-148 Δ209-227 Δ248-256 PD-Cter PD Hal3 WT Δ118-148 Δ209-227 Δ248-256 PD-Cter (C) (D) Δ67-110 Δ94-110 Δ39-51 Δ67-93 Strain Plasmid PD kDa Ø Gal 1% 100- Ø BY4741 70 - Ø 55 - Hal3 40 - Δ39-51 35 - Δ67-93 25 - ZCZ01 Δ67-110 Δ94-110 55 - Δ118-148 40 - Δ209-227 Δ248-256 Fig. 1. Coarse functional mapping of the S. cerevisiae Hal3 N-terminal extension. (A) Schematic cartoon depicting the regions deleted in the Hal3 N-terminal disordered domain. The right segment (in red) represents the start of the PD domain. (B) The wild-type BY4741 strain and its hal3D derivative were transformed with the pWS93 vector (Ø) or the same plasmid bearing the native HAL3 gene or the indicated dele- tion variants. Cultured were spotted as described in material and methods on plates containing synthetic medium lacking uracil and different concentrations of caffeine or LiCl. The same constructs were introduced into cells lacking the Slt2 MAP kinase (slt2D, right panel). For slt2D cells 10% sorbitol, which acts as osmotic support, was added to the medium as positive growth control). (C) The indicated strains were transformed with pWS93-based constructs bearing the different variants of Hal3. Protein extracts were prepared and resolved (40 lg) by SDS/PAGE. The expression levels of the various Hal3 versions were examined by immunoblot using anti-HA antibodies. The experiment was repeated twice with different protein extracts and gave coincident results. The lower panel shows the relevant section of the gel stained with red ponceau to monitor loading and transfer efficiency. Ø denotes empty plasmid. (D) Strain ZCZ01, overexpressing PPZ1 from the strong GAL1-10 promoter was transformed and cultures spotted as above. Induction of Ppz1 overexpression was achieved by inclusion of 1% galactose (GAL) in 2% raffinose containing plates. Pictures were taken in all cases after 3 days. more effective as inhibitors than native Hal3. Remark- phosphatase N-terminal extension, all Hal3 variants ably, when the different Hal3 versions were tested with were equally powerful inhibitors of the phosphatase a recombinant version of Ppz1 devoid of the activity, suggesting a functional interaction between 4 The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. C. Santolaria et al. Functional mapping of the Hal3 N-terminal region Ppz1 Ppz1 C-ter 100 Hal3 WT Δ39-51 100 Hal3 WT Δ39-51 Δ67-93 Δ67-110 Δ67-93 Δ67-110 Δ94-110 Δ118-148 Δ94-110 Δ118-148 Δ209-227 Δ248-256 Δ209-227 Δ248-256 80 Phosphatase activity (%) 80 Phosphatase activity (%) 60 60 40 40 20 20 0 0 0 1 2 3 4 0 1 2 3 4 Hal3 variants (molar excess) Hal3 variants (molar excess) Fig. 2. In vitro Ppz1 inhibitory activity of different Hal3 deletion variants. The Ppz1 phosphatase activity was measured as described in materials and methods using 2–10 pmols of full-length Ppz1 or 5 pmols of the Ppz1 C-terminal catalytic domain (Ppz1-Cter), and pNPP as substrate. The phosphatase was pre-incubated for 5 min at 30 °C with increasing amounts of native Hal3 or the indicated variants and the assay started by addition of the substrate. Values are means  SE from at least four different assays and are expressed as the percentage of phosphatase activity relative to the control without inhibitor. At least two different preparations of the phosphatases and the inhibitors were used for these assays. The specific activities of Ppz1 and Ppz1-Cter preparations were 5.05  0.11 and 5.76  0.09 nmolmin1 9 lg1 respectively. the 67–110 region of Hal3 and the N-terminal exten- functions reside within residues 67–105. Interestingly, sion of Ppz1. only the D97–110 deletion clearly failed to attenuate the toxicity derived from PPZ1 overexpression (with perhaps a minor effect of the D90–98 isoform). Immu- Fine mapping of the 67–110 region of Hal3 noblot analyses of Hal3 and ZCZ01 cells demonstrated The results described above clearly identified the 67– that the level of protein accumulation of all of these 110 segment as relevant for Hal3 function. To pinpoint variants was as high as that of native Hal3 (Fig. 3D). the specific functional determinants within this region, The ability of the new versions to inhibit Ppz1 is we generated a set of six new smaller deletions presented in Fig. 3E. As observed, the D67–91 and the (Fig. 3A) that were subjected to functional evaluation. D105–110 deletions have little or no impact on the As illustrated in Fig. 3B, only the deletion affecting inhibitory capacity of Hal3. In contrast, deletion of residues 105–110 behaved as native Hal3 in both the regions 90–98 and 97–110 were less effective in inhibit- caffeine and LiCl sensitivity tests. Interestingly, ing the activity of the phosphatase, while the other although the remaining deletions eliminated Hal3 func- variants exhibited intermediate effects. These results tion in the caffeine test, only the D90–98 and D97–110 confirmed that the relevant determinants for Ppz1 reg- changes markedly decreased the tolerance to LiCl in ulation present in the N-terminal region of Hal3 could comparison with native Hal3, while all three deletions be restricted to a segment contained within residues spanning the 67–91 region displayed a rather limited 90–105. effect. When the effect of the deletions was tested in the strain deficient in SLT2, it could be observed The 90KRVPAVTFS98 region is crucial for (Fig. 3C) than all Hal3 variants, except the one bear- regulation of Ppz1 function and activity ing the D105-110 deletion, were unable to abolish growth of the MAP kinase mutant. This pattern was To further refine our search for specific Hal3 residues similar to that observed for caffeine tolerance and sug- required for Ppz1 regulation, we introduced diverse gests that structural elements important for both mutations in the region encoding amino acids 90–105 The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 5 Federation of European Biochemical Societies. Functional mapping of the Hal3 N-terminal region C. Santolaria et al. 67TKSIMNATGTSGAVVSNTPEPGLKRVPAVTFSDLKQQQKQDSLT110 (A) 91 97 80 90 98 105 73 (B) (D) Caffeine (mM) LiCl (mM) Strain Plasmid hal3Δ 4 6 8 300 400 500 BY4741 Δ105-110 BY4741 Ø Δ97-110 Δ67-73 Δ67-80 Δ67-91 Δ90-98 hal3Δ Ø WT Hal3 Ø Ø kDa Δ67-73 Δ67-80 100 - BY4741 Δ67-91 70 - Δ90-98 Δ97-110 Δ105-110 (E) (C) Strain Plasmid Strain 100 + Sorbitol Gal 1% BY4741 Ø BY4741 Phosphatase activity (%) 80 Ø Hal3 Δ67-73 60 slt2Δ Δ67-80 ZCZ01 Δ67-91 40 Δ90-98 Hal3 WT Δ67-91 Δ97-110 20 Δ90-98 Δ97-110 Δ105-110 Δ67-73 Δ67-80 Δ105-110 0 0 1 2 3 4 Hal3 variants (molar excess) Fig. 3. Functional relevance of the Hal3 67–110 N-terminal region. (A) The sequence of the region analysed is shown. Specific deletions sub- jected to functional analysis are denoted by empty boxes. (B, C) Strains containing the indicated constructs were analysed as in Fig. 1. All pictures were taken after 3 days of incubation, except plates containing the slt2D strain (4 days). (D) Immunoblot analysis of diverse Hal3 deletions comprising residues 67–110. The indicated strains were transformed with pWS93-based constructs bearing the different variants of Hal3. The expression levels of the various Hal3 versions were monitored by immunoblot using anti-HA antibodies as in Fig. 1. Two inde- pendent sets of extracts were analysed with equivalent results. Ø denotes empty plasmid, WT, native Hal3. (E) The inhibitory capacity of the indicated Hal3 variants was determined using full-length recombinant Ppz1 as in Fig. 2. Data are mean  SE from five different assays using at least two different preparations of the phosphatase and the inhibitors. (Fig. 4A). Phenotypic testing (Fig. 4B and C) indicates the slt2 background. Finally, the triple change of 102 that mutations changing K90 and R91 to Glu or Gln QQQ104 to Ala did not alter Hal3 properties for any virtually eliminated the ability of Hal3 to induce sensi- of the phenotypes tested. These results indicated that tivity to caffeine but barely affected its ability to con- the 95VTFS98 region was important for all Ppz1- fer tolerance to LiCl. Similarly, these mutations related Hal3 functions, while mutation of K90 and eliminated the deleterious effect of Hal3 expression in R91 showed selective effects depending on the test per- SLT2-deficient cells. Interestingly, the Hal3 versions formed. In contrast, mutation of the triple Gln stretch bearing mutations that change the 95VTFS98 and had no effect. Immunoblot analysis (Fig. 4D) demon- 96 TFS98 amino acid sequences were largely ineffective strated that the level of all variants was at least as high in mimicking native Hal3 in all phenotypes tested. The as that of native Hal3. The phenotypic behaviour of combined mutation changing T96 and S98 to Ala had the Hal3 variants was largely in agreement with their no effect on Hal3 function, but the change of these ability to inhibit Ppz1 in vitro. As shown in Fig. 4E, residues to the phosphomimetic amino acids Glu and amino acid changes in the 95VTFS98 region strongly Asp affected the behaviour of Hal3 in caffeine and in decreased the ability of Hal3 to inhibit Ppz1. Changes 6 The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. C. Santolaria et al. Functional mapping of the Hal3 N-terminal region 90KRVPAVTFSDLKQQQK105 (A) (B) Caffeine (mM) LiCl (mM) (D) Strain Plasmid 6 8 200 400 BY4741 Ø hal3Δ Hal3 V95S T96A F97A S98A BY4741 Ø K90E R91E T96E F97Y S98D K90Q R91Q T96E S98D T96A S98A K90R91Q 3Q → A K90R91E hal3Δ V95S T96A F97A S98A WT T96A S98A kDa Ø Ø T96E S98D 100 - T96E F97Y S98D 70 - 3xQ A (C) (E) Strain Plasmid Hal3 WT K90E_R91E + Sorbitol K90Q_R91Q V95S_T96A_F97A_S98A T96E_F97Y_S98D 3xQ A BY4741 Ø T96A_S98A T96E_S98D Ø 100 Phosphatase activity (%) Hal3 80 K90E R91E K90Q R91Q 60 slt2Δ V95S T96A F97A S98A 40 T96A S98A T96E S98D 20 T96E F97Y S98D 0 3xQ A 0 1 2 3 4 Hal3 variants (molar excess) Fig. 4. Mapping of a functional region to Hal3 residues from 90 to 105. (A) Sequence of the region examined. Residues affected by mutations are underlined. (B, C) The phenotypic tests were performed as in Fig. 1. All pictures were taken after 3 days. (D) Immunoblot analysis of diverse changes comprising Hal3 residues 90–105. A representative experiment out of two is shown. See legend to Fig. 1 for details. (E) The ability of the different Hal3 variants to inhibit Ppz1 was tested as in Fig. 2. The mean  SE from four different assays is shown, including at least two different preparations of Ppz1 and the Hal3 variants. restricted to residues 96TFS98, those affecting K90 and strong overexpression of PPZ1 (Fig. 5B, left panel). R91, as well as the transformation of T96 and S98 in We also changed F97 to Ala and Tyr. The rather con- phosphomimetic residues, also resulted in partial loss servative F97Y change had mild or no consequences of their inhibitory activity. In contrast, mutation of on the phenotypes tested, while the mutation to Ala 102 QQQ104 or T96 and S98 to Ala had virtually no caused noticeable effects, with an intensity similar to effect on the in vitro inhibition of Ppz1 by Hal3. that observed for the mutation of V95. The level of Because the 95VTFS98 and 96TFS98 mutations pro- protein accumulation of the Hal3 variants were indis- vided rather similar phenotypes, we wanted to know tinguishable from those of the native protein (Fig. 5C). whether V95 had any relevance. Therefore, we con- Curiously, all four HAL3 mutations yielded less pow- verted V95 to Ala or Ser. As shown in Fig. 5, cells erful in vitro inhibitors of Ppz1 activity (Fig. 5D). expressing these mutated forms of Hal3 did not behave Taken together, these results indicate that the 95 as those carrying native Hal3 in the caffeine tolerance VTFS98 region, and specifically residues V95 and test nor when expressed in the slt2 background. They F97 in the N-terminal extension of Hal3, are impor- were also slightly less tolerant to LiCl and somewhat tant for the function of this protein as inhibitor of the less effective than native Hal3 in protecting cells from Ppz1 protein phosphatase. The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 7 Federation of European Biochemical Societies. Functional mapping of the Hal3 N-terminal region C. Santolaria et al. (A) (C) Caffeine (mM) LiCl (mM) Strain Plasmid 6 8 10 200 400 BY4741 BY4741 Ø hal3Δ Hal3 V95A V95S F97A F97Y WT Ø Ø kDa Ø hal3Δ V95A 100 - V95S 70 - F97A 55 - F97Y (B) (D) 100 Hal3 WT VA Strain Plasmid Gal 1% Strain Plasmid + Sorbitol 90 VS FA Phosphatase activity (%) FY 80 BY4741 Ø BY4741 Ø 70 Ø Ø 60 Hal3 Hal3 50 V95A V95A 40 ZCZ01 slt2Δ V95S V95S 30 F97A F97A 20 F97Y 10 F97Y 0 0 1 2 3 4 Hal3 variants (molar excess) Fig. 5. Identification of V95 and F97 as key residues in the N-terminal extension of Hal3. (A, B) All plates were incubated for 3 days prior documentation. (C) Immunoblot analysis of the indicated variants. Two independent set of extracts were analysed with identical results. (D) Inhibition of Ppz1 is expressed as the mean  SE from four different assays with at least two different preparations of the phosphatase and the inhibitors. The loss of functional capacity of the Hal3 Mutation of potential Ser phosphorylation sites variants is not due to lack of interaction with in the N terminus does not affect Hal3 function Ppz1 According to diverse databases, Hal3 can be phos- The loss of the ability of the Hal3 variants to inhibit phorylated in vivo at least at 18 residues, almost with- Ppz1 could be due to the inability of bound Hal3 to out exception located within the first 250 residues. block the catalytic reaction or simply to the lack of Eight of them, placed in a small cluster within resi- binding to the phosphatase. To discern among these dues 47 and 60 (S47, S50, S54, S55, S56, S57, T58 possibilities, we carried out an in vitro binding assay and S60) have been consistently identified in many using recombinant GST-Ppz1 bound to glutathione- proteome-wide experiments (see SGD at https://www. Sepharose beads and crude extracts from yeast yeastgenome.org/locus/S000001780/protein). With the expressing different HA-tagged Hal3 variants. As objective to evaluate the potential role of these phos- shown in Fig. 6, most variants exhibited a binding phorylation sites in the function of Hal3 we changed capacity similar to that of native Hal3. Only the D67– S47, S50, S54, S56 and S60 to both alanine and 110 version showed a slightly decreased interaction, aspartic residues. These versions were cloned in the but the change was not statistically significant. In con- multicopy plasmid YEp195 and expressed from the trast, variants displaying a notable incapacity to inhi- native HAL3 promoter. However, tolerance tests to bit Ppz1, such as the D90–98 deletion or the quadruple caffeine, LiCl or NaCl did not reveal any significant 95 VTFS98 mutation, demonstrated a clear increase in difference with the native Hal3 protein (not shown). binding capacity. These results indicate that the loss of We then also tested a double S54A S56A (and the Hal3 inhibitory capacity caused by these amino acid corresponding phosphomimetic version), as well as changes is not due to their incapacity to associate to the entire deletion of the 47–60 region, again with the phosphatase. negative results (not shown). Therefore, we must 8 The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. C. Santolaria et al. Functional mapping of the Hal3 N-terminal region shown in Fig. 7 (A and B), transformation with the V95S T96A F97A S98A empty vector does not lead to changes in the subcellu- lar localization of Ppz1, whereas the presence of native T96E S98D Hal3 results in recruitment of Ppz1 to internal mem- T96A S98A Hal3 WT Δ94-110 Δ67-110 Δ97-110 Δ67-93 Δ67-91 Δ90-98 branes in 80% of the cells. It should be noted that this kDa Ø value is somewhat higher than that the previously 130 - reported [25], likely because Hal3 is expressed here 100 - from a more powerful promoter. The localization of Hal3 variants the phosphatase in cells expressing the PD domain 70 - alone were indistinguishable from those transformed 130 - with the empty vector, whereas the same protein frag- 100 - GST-Ppz1 ment containing the C-terminal acidic tail (PD + CtD) 70 - provided a limited, but measurable capacity to inter- nalize Ppz1 (about 28% of cells). Remarkably, cells ** expressing the D67–93, D67–110 or D94–110 variants 4 showed a mild but significant difference when com- relative binding 3 Ppz1-Hal3 * pared to cells expressing native Hal3, ranging from 2 67% to 71% of cells with internalized Ppz1. When 1 these constructs were tested for their ability to sup- 0 press the growth defect of the MAC003 strain, we observed (Fig. 7C) that the cells expressing the D67– 93, D67–110 or D94–110 versions exhibited a mild Fig. 6. Evaluation of the physical interaction of different Hal3 growth defect when compared with native Hal3, indi- versions with Ppz1. Equal amounts (6 lg) of GST-tagged full-length cating partial compensation of Ppz1 toxicity. The ver- Ppz1 were immobilized on glutathione beads and used as bait to sion of Hal3 devoid of its N-terminal extension pull-down the different variants of Hal3 expressed from the pWS93 (PD + CtD) exhibited a very strong growth defect, plasmid in the strain IM021 (ppz1 hal3). After washing, beads were although still distinguishable from the complete lack processed for SDS/PAGE (8% gels) and native Hal3 (WT) and its of growth observed for the strain expressing only the variants immunodetected by means of their HA-tag as described in PD domain or the empty plasmid. However, this later methods (upper panel). The membrane was stained prior transfer with red ponceau to assess similar recovery of the bait in the result must be taken with caution because of the assay (middle panel). Lower panel. Quantification of the relative above-mentioned low levels of the PD + CtD and PD Hal3 vs Ppz1 amounts obtained by scanning and integration of proteins. immunoblots and recovered GST-Ppz1 signals (upper and middle panels) from at least six experiments. Data are expressed as the mean  SE. *P < 0.05, **P < 0.01 according to two-tailed Stu- Functional relevance of the N-terminal region of dent’s test. C. albicans Cab3 for Ppz1 regulation As mentioned in the Introduction, C. albicans encodes conclude that this set of phosphorylatable residues homologues of Hal3 and Cab3, both with large N- plays no role in Hal3 function. terminal extensions, although only Cab3 could regulate Ppz1. We observed that CaCab3 and CaHal3 largely differ in the N-terminal segment that we identified here The ability of Hal3 N-terminal variants to recruit to be relevant for Hal3 function in S. cerevisiae, and overexpressed Ppz1 to internal membranes that CaCab3, but not CaHal3, includes the residues We recently reported that the ability of Hal3 to rescue corresponding to R91 and the entire 95VTFS98 region the growth defect associated with PPZ1 overexpres- (Fig. 8A). We then wondered whether these structural sion involved not only the capacity to inhibit the phos- features could be a differential trait between CaCab3 phatase activity but also the ability to remove Ppz1 and CaHal3 that may contribute to the distinct beha- from the cell periphery and relocate it to internal viour of both proteins with regard to Ppz1 regulation. (mostly vacuolar) membranes. To test how the changes To test this possibility, we replaced the 89–134 region introduced in the N-terminal extension of Hal3 could of CaCab3 with the 73–90 region of CaHal3 and vice affect such ability, we transformed the MAC003 strain, versa (Fig. 8A), introduced the hybrid constructs in a ZCZ01 derivative in which the PPZ1 coding wild-type cells as well as hal3 and slt2 deletion sequence is followed by a C-terminal GFP tag. As mutants, and subjected these cells to phenotypic tests. The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 9 Federation of European Biochemical Societies. Functional mapping of the Hal3 N-terminal region C. Santolaria et al. (A) (B) Internalized Ppz1 (% of cells) 0 10 20 30 40 50 60 70 80 90 100 plasmid Ø Ø Hal3 Hal3 Δ67-93 Δ67-110 * Δ94-110 Δ90-98 ns PD+CtD *** PD *** (C) 1.4 WT Cell growth (OD600) 1.2 Δ67-93 1.0 Δ67-110 Δ94-110 0.8 0.6 0.4 Ø PD+CtD PD 0.2 0.0 0 5 10 15 20 25 30 35 40 Hours Fig. 7. The ability of Hal3 variants in recruiting overexpressed Ppz1 to internal membranes. (A) Micrographs of representative cultures of MAC003 cells (overexpressing C-terminally GFP tagged Ppz1) bearing an empty pWS93 vector (Ø) or the same vector with native Hal3. Exposition was 3 and 1.5 s respectively. Bars represent 5 lm. (B) MAC003 cultures expressing the indicated Hal3 variants were monitored after 6 h of PPZ1 overexpression and the percentage of cells showing internalized Ppz1 was determined by blind counting of a range between 293 and 813 cells. Ns, not significant *P < 0.05; ***P < 0.001, compared with native Hal3 and determined by two-tailed Student’s test. (C) MAC003 cells were grown overnight on synthetic medium lacking uracil and then transferred to YP raffinose containing 2% galac- tose at initial OD600 of 0.004. Growth was monitored every 30 min. The mean  SE from six independent cultures is shown. Ø, empty pWS93 vector. Expression of CaCab3 had little impact on caffeine function is also important in CaCab3. However, it is sensitivity but, as previously reported [32], it markedly not sufficient to transform CaHal3 into a functional improved tolerance to LiCl in a hal3D strain (Fig. 8B). regulator of ScPpz1, suggesting that additional deter- We also observed a significant effect on growth in the minants are needed. slt2D background. Interestingly, these phenotypic traits are lost in the hybrid CaCab3 version carrying the The N-terminal traits identified in Hal3 as CaHal373–90 region. However, introduction of the functionally relevant for Ppz1 regulation are CaCab389–134 segment into CaHal3 resulted in a beha- conserved only in a subset of yeast species viour identical to that of native CaHal3 (or the empty vector). In any case, the lack of effect of the hybrid The analysis of fungal genomes for the presence of proteins cannot be attributed to a deficient expression, Hal3 proteins displaying an N-terminal extension pre- since immunoblot experiments demonstrated that their ceding the PD domain revealed that this feature was, protein levels in the hal3 (Fig. 7C) or wild-type strains with very few exceptions, restricted to the order Sac- (not shown) were similar or even higher than that of charomycetales (see Discussion). However, even within native S. cerevisiae Hal3 or CaCab3. These results this subset of species the overall level of identity in this indicate that the conserved region found relevant in region was rather poor. Therefore, based on our exper- S. cerevisiae for regulation of S. cerevisiae Ppz1 imental data, we analysed the N-terminal region of 83 10 The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. C. Santolaria et al. Functional mapping of the Hal3 N-terminal region ScHal3 60 SNATTTTTKSIMNATGTSGAV----------VSNTPEPGLK-RVPAVTFSDLKQQQKQDSLTQ 111 (A) CaCab3 81 SGIAPPTKILTNSSKEVTSPVLQNSNTPILGSANTSGTNIPVRNQSVSFSDIDNQERSRRFTI 143 *. :..*. .:. .:..* :**. ..: * :*:***:.:*::. :* ScHal3 60 SNATTTTTKSIMNATGTSGAVVSNTPEPGLKRVPAVTFSDLKQQQKQDSLTQ 111 CaHal3 70 SNQPATIKLSPQLSIS-----------------------SENRETVMAGGAL 98 ** .:* . * : . . ::: . : (B) (C) CaHal3-Cab389-134 CaCab3-Hal373-90 Strain Plasmid Caffeine LiCl BY4741 Ø CaCab3 CaHal3 Ø ScHal3 ScHal3 Ø kDa CaCab3 hal3Δ 180 - CaCab3-Hal373-90 CaHal3 130 - CaHal3-Cab389-134 100 - + Sorbitol BY4741 Ø 70 - Ø 130 - ScHal3 CaCab3 100 - slt2Δ CaCab3-Hal373-90 CaHal3 70 - CaHal3-Cab389-134 Fig. 8. Exchanging relevant N-terminal regions between C. albicans CaCab3 and CaHal3. (A) Sequence comparison between the 60–111 region of S. cerevisiae Hal3 (NP_012998.1) with the corresponding regions of CaCab3 (XP_717950.1) and CaHal3 (XP_716619.1). Protein sequences were obtained from the NCBI protein databank and alignments were generated with Clustal omega. Residues found in this work to be relevant for the Hal3 regulatory function on Ppz1 are displayed in yellow background. The segments exchanged between CaCab3 and CaHal3 are highlighted in bold and the corresponding region in ScHal3 in italics. B) The different constructs were introduced into the wild- type BY4741 strain and its hal3 and slt2 derivatives and plated on synthetic medium lacking uracil and supplemented with 8 mM caffeine or 200 mM LiCl (for the hal3 background) or in the presence or absence of 10% sorbitol (for the slt2 background). Plates were incubated in all cases for 3 days. C) The indicated constructs were introduced in the hal3 strain, protein extracts prepared and the level of expression of the native and hybrid proteins was assessed by immunoblot using anti-HA antibodies. The lower panel shows red ponceau staining of the mem- brane. Two independent sets of extracts were analysed with similar results. Hal3/Vhs3 and Cab3-like proteins from 35 species for in Hal3. Similar sequences in Cab3 are also found in the presence of the R/K-R/K-X3-VTFS signature or the Phaffomycetaceae, Pichiaceae and Dipodascaceae degenerate variants (Fig. 9). Remarkably, all members families, in some cases with Val substituted by Ile. of the Saccharomycetaceae (NCBI:txid4893) and Sac- charomycodaceae (NCBI:txid34365) families examined Discussion present the canonical K-R-X3-VTFS signature in Hal3 (and in Vhs3, when the HAL3 paralog exists), whereas The fact that Ppz1 is a virulence determinant in some in Cab3 only a limited version of the consensus, pathogenic fungi [30,31], together with the unusual always lacking the basic residues, is found. In contrast, intolerance of S. cerevisiae for higher-than-normal all members of the CTG-clade examined, with the levels of this phosphatase, has stirred the interest in its exception of Meyerozyma guilliermondii, consistently regulation and, in particular, in the role played by its exhibit slightly degenerate versions of the [K-X4-V- inhibitory subunit Hal3. Previous work revealed that, (ST)-F and R/K-X3-V-(ST)-F] motifs in Cab3, but not although the isolated Hal3 N-terminal segment had no The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 11 Federation of European Biochemical Societies. Functional mapping of the Hal3 N-terminal region C. Santolaria et al. Hal3/Vhs3 Cab3 100 Ca b (0 aa) Candida albicans NQD-SNIVH VRNQSVSFS 65 Cad (0 aa) Candida dublinensis NQD-SNIVH VRNQSVSFS 100 Ct a (0 aa) Candida tropicalis NQP---D-- VRNQSVSFS 100 Cnm (0 maltosa Candida aa) NPG---LIH VRNQSVSFS 89 Shp (0 aa) passalidarum Spathaspora NNGPSRTTN * CTG clade 54 Shs (0 aa) Scheffersomyces stipitis --------- KPSQSVTFG Lodderomyces Loe (0 aa) elongisporus --------- LRHPSVSFS Cap (0 aa) Candida parapsilosis NQIPSAQSE TRNASVSFN 100 100 Came (0metapsilosis Candida aa) NQVPNSQLE TRHPSVSFN 100 100 Candida orthopsilosis Cao (0 aa) NTVPSNSSD TRNASVSFN Cll (0 aa) lusitaniae Clavispora SNSRIHTPI SRSQSVSFC 87 Mrg (0 aa) guilliermondii Meyerozyma TKKEPVAFV NDNDVMSFI Deh (0 aa) Debaryomyces hansenii NNSNNNNAT KPSQSVTFG 99 99 Mif (0 aa) farinosa Millerozyma NNSMRSQSY KAQQTVSFG Kop b (0 aa) phaffii Komagataella --------- KRANLISFD A 100 100 Pichia kudriavzevii Piu (0 aa) QHNRSVKNV KSSTSTISFQ B Yl (0 aa) lipolytica Yarrowia --------- SRKSIVSFT C 100 Cyj (0 aa) Cyberlindnera jadinii * AKYSSVSFT 100 Wa (0 aa) Wickerhamomyces anomalus --------- NKYGSVSFT A Erg (0 aa) Eremothecium gossypii KRVPTVTFS AGVAAVSFT Saccharomycetaceae 100 Kl (0 aa) Kluyveromyces lactis KRVPTVTFT LTATSVSFT 100 Kmm (0 aa) Kluyveromyces marxianus KRVPTVTFT LTGTSVSFT 69 Cgl (0 aa) Candida glabrata KRIPTVTFS SPKSTISFT Vp (0 aa) Vanderwaltozyma polyspora KRTPKVTFN ATGTVVSFT Sc c (0 aa) Saccharomyces cerevisiae KRVPAVTFS SLPSAVSFT 100 Kaa (0 aa) africana Kazachstania KRTPMVTFD AMTPSVSFC 100 Nac (0 aa) Naumovozyma castellii KRAPAVTFS STATAVSFS Tetrapisispora Ttb (0 aa) blattae KRVATVTFS NGGTVVSFT Zr (0 aa) Zygosaccharomyces rouxii KRVATVTFS GGTSAVSFT Fig. 9. Identification of the regulatory consensus sequence in diverse clades of the order Saccharomycetales. The phylogeny of the selected species was based in the alignment provided in reference [51] and represented using MEGA X software [52]. The identification of the consensus sequences was done by inspection of Clustal Omega alignments and search of FASTA files for degenerate combinations with in-house developed software. Key consensus residues are denoted in bold. The anomalous sequence in P. kudriavzevii is shaded. An asterisk denotes that the sequence found in the database appears N-terminally incomplete. (A), Phaffomycetaceae; (B), Pichiaceae; (C), Dipodascaceae. function by itself, it could contribute to the regulation detected between the catalytic C-terminal domain of of Ppz1 [28]. We show here that, indeed, this region is Ppz1 and Hal3 involve residues located within this N- vital for regulation of Ppz1. This is in keeping with the terminal extension [33]. very recent evidence obtained by chemical cross- Our results indicate an overall correlation between linking showing that the vast majority of interactions the failure of the diverse variants to mimic native Hal3 12 The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. C. Santolaria et al. Functional mapping of the Hal3 N-terminal region phenotypes in vivo and the loss of capacity to act as important functional determinants for the inhibition of Ppz1 inhibitors in vitro. We also show that the inabil- the Ppz1 phosphatase by the Hal3 protein. We also ity to inhibit Ppz1 properly is not caused by defective selected for analysis the diverse Ser residues located physical interaction between both proteins, which is within the 47–60 segment. There were several reasons actually enhanced in some cases such as the D90–98 for this choice. This region contains 5 of the 18 phos- deletion or the mutations affecting the 95VTFS98 phorylatable residues experimentally found in Hal3 (of region. The irrelevance of the R/K-R/K-X3- which 16 are located within the first 250 amino acids), VTFS motif for the interaction between Ppz1 and and these residues consistently appear as phosphory- Hal3 fits with the earlier observations that ScCab3 and lated in most public data sets. More importantly, it CaHal3, both devoid of such motif, still interact with was recently reported that Hal3 S54 and S56 were ScPpz1 or CaPpz1 [26,32]. Therefore, our data indicate specifically dephosphorylated in cells treated with that this short region is somehow involved in the inhi- acetic acid [37], suggesting that these phosphorylation bitory mechanism. In this regard, it is suggestive that sites might be responsive to alterations in the intracel- the differential in vitro effect on the inhibitory activity lular pH. Because of the known role of Ppz1 in mono- observed for the large Hal3 deletions on full-length valent cation homeostasis and intracellular pH Ppz1 (Fig. 2A) is not detected when the catalytic C- maintenance [4,5,7,21] we considered that this might terminal domain is employed. In this case, all variants be a hint for a role of this phosphosites in Ppz1 regu- behave as strong inhibitors, indistinguishable from the lation. However, the independent mutation of any of native Hal3 protein. These results suggest that specific these Ser residues to Ala (non-phosphorylatable) or interactions between the N-terminal domains of Ppz1 Asp (phosphomimetic), including the simultaneous and Hal3 might take place, and that these interactions change of S54 and S56, as well as the entire deletion could modulate the capacity of Hal3 as Ppz1 inhibitor. of this region, did not alter the properties of Hal3. This hypothesis would fit with the early notion that Therefore, we propose that this specific cluster plays the N-terminal extension of Ppz1 protects the phos- no role in the regulation of Ppz1 in vivo. phatase from excessive Hal3 inhibition [11]. It has been very recently demonstrated that the We show here that the impact of some deletions or counteraction of the toxic effects of Ppz1 overexpres- specific mutations in the Hal3 N-terminal region is dif- sion by Hal3 involves the relocation of the phos- ferent depending on the phenotype tested. Thus, all phatase from the cell periphery to internal membranes three deletions involving the 67–91 region result in a [25]. We show here that particular deletions at the N- very severe loss of function when cells are tested in caf- terminal extension of Hal3 that greatly impair both feine, but only a mild effect when challenged with LiCl the ability to inhibit Ppz1 in vitro and its function (Fig. 3B). Likewise, the T96E S98D variant behaves as in vivo, only moderately weakens their ability to relo- native Hal3 in LiCl tolerance while it is largely ineffi- cate Ppz1 and to compensate the growth defect caused cient when tested in media supplemented with caffeine. by an excess of Ppz1 (Fig. 7). This suggest that the In fact, disparate phenotypes have been previously functional region identified here has relatively little rel- reported for specific mutations affecting Hal3’s PD core evance concerning subcellular redistribution of overex- [27,34], or for the expression of the Schizosaccha- pressed Ppz1. As mentioned above, the observation romyces pombe SpHal3 protein (which lacks an N- that the versions of Hal3 devoid of the entire N- terminal extension) in budding yeast [35]. It is worth terminal region fail to promote Ppz1 internalization noting that it has been reported that specific deletions could be simply due to the markedly lower levels of of the N-terminal extension of S. cerevisiae Ppz1 dis- these variants. This might also reflect a role of the N- play a differential phenotype-specific behaviour [14]. terminal region of Hal3 in the stabilization of the This would argue in favour of a functional role of the polypeptide. In any case, our results further confirm N-terminal extension of Hal3 in modulating Ppz1 func- the relevance of intracellular redistribution in the neu- tion, as well as supporting a functional interplay tralization of the deleterious effects derived from between these N-terminal moieties. In any case, we excess of Ppz1 activity. observe here a perfect phenotypic correlation between The comparison of Hal3-like fungal proteins indi- caffeine sensitivity and the growth test performed in the cates that an N-terminal extension preceding the PD slt2D background. We interpret this as the result of the domain is found only in the genome of Saccha- described impact of caffeine on the cell wall integrity romycetes. Exceptions to this rule appear only in the pathway (see [36] and references therein). Exobasidiomycetes and Ustilaginomycetes clades (see Our experiments defined residues 90–105, highlight- below). In other ascomycota classes, the protein is ing K90 or R91 (or both), and the 95VTFS98 region as reduced to the PD domain and often lacks the acidic The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 13 Federation of European Biochemical Societies. Functional mapping of the Hal3 N-terminal region C. Santolaria et al. tail. Our work demonstrating the functional relevance (Fig. 5B). In contrast, in the case of V95, decreasing of a specific signature present in the N-terminal region the size of the residue to Ala or its hydrophobicity (by in S. cerevisiae provides an explanation as to why the mutation to Ser) resulted in a loss of function of simi- S. pombe SpHal3 protein, which lack the N-terminal lar potency. It is suggestive that this motif lies in a extension, or the C. neoformans CnHal3a or CnHal3b small segment for which a strong prediction for a dis- proteins (with a very short N-terminal region) were ordered binding region has been observed (Fig. 10). ineffective in inhibiting their own Ppz enzymes or the Such regions have been reported to be extensively S. cerevisiae Ppz1 [35,38]. Similarly, the Hal3 protein involved in regulatory and signalling functions [43]. It from the Ustilaginomycete Ustilago maydis, which could be hypothesized that the interaction of these contains a very large N-terminal extension but lacks residues with the catalytic domain of Ppz1 may con- the above-mentioned signature, cannot inhibit the tribute to the appropriate positioning of Hal3 to effec- UmPpz1 or ScPpz1 phosphatases [39]. It must be tively impair the catalytic reaction. In fact, our results stressed that the failure of these Hal3 proteins to inhi- showing that the D67–93, D94–110 and D67–110 vari- bit their corresponding Ppz1 enzymes cannot be attrib- ants could not block full-length Ppz1 activity, but that uted to structural differences in the phosphatases, they were as effective as the native Hal3 protein in because all of them were potently inhibited by ScHal3 inhibiting the C-terminal Ppz1 catalytic domain, sug- [32,35,38,39]. Taken together with the results shown gest that these interactions might involve residues of here concerning CaCab3 mutagenesis, these data rein- the Ppz1 N-terminal extension. This extension has force the notion that the R/K-R/K-X3-VTFS motif, or been also predicted as a disordered region, not only in slightly degenerated versions of it, could be an impor- S. cerevisiae but also in many other fungi [19] and, in tant structural feature for the role of Hal3 as regulator the case of S. cerevisiae, to strongly determine the of the Ppz1 phosphatase. Our comparative analysis properties of the phosphatase [14,19]. also reveals two scenarios: (1) the regulatory consensus is present in Hal3 (but not in Cab3) in the Saccha- Materials and methods romycetaceae and Saccharomycodaceae families; (2) it is found in Cab3 (but not in Hal3) not only in C. albi- Yeast strains and growth conditions cans and in some other pathogenic Candida species [40], but the vast majority of the members of the Saccharomyces cerevisiae cells were grown at 28 °C in YP CTG-clade, plus in the examples of the Phaffomyc- medium (10 gL1 yeast extract, 20 gL1 peptone) or in etaceae, Pichiaceae and Dipodascaceae families exam- synthetic medium (SC) lacking the appropriate require- ined. The genomes of all these species encode both ments when carrying plasmids for selection [44], supple- Hal3 and Cab3 proteins, so it is expected that the mented with a carbon source (20 gL1) such as glucose PPCDC enzyme will be a heterotrimer involving both (Glu, as in YPD), raffinose (Raff) or galactose (Gal), or as indicated. All yeast strains used in this work are listed in proteins. Because our work associates the presence of Table 1. Growth on plates was tested by spotting cells at the consensus with the ability to regulate Ppz1, it can an initial OD600 of 0.05, plus two 1/5 serial dilutions. be concluded that, in the first scenario, the moonlight- Growth assays in liquid media were done as in [20], except ing capacity corresponds to Hal3, whereas in the sec- that measures were made every 30 min in a Bioscreen C ond case, Cab3 is the moonlighting protein. apparatus (Thermo Labsystems, Madrid, Spain), with shak- Our results raise a key question: why is the R/K-R/ ing for 7 min before each measurement. K-X3-VTFS motif important in regulating Ppz1 func- tion? Although this sequence bears some similarity with the RVXF motif found in many PP1c regulatory DNA techniques and plasmid constructions subunits [41,42], in our case the basic residues are Escherichia coli (strain DH5a) was used as standard plas- more distant from the VXF core. In addition, the mid DNA host and was grown in LB medium at 37 °C RVXF motif has been described as necessary for the supplemented with 50 lgmL1 ampicillin when needed for interaction of the regulators with PP1c, whereas in our plasmid selection. E. coli strain BL21 (DE3) RIL was used case it is dispensable for interaction. Therefore, our for heterologous protein expression as described below. data do not support the notion that the R/K-R/K-X3- The diverse variants of S. cerevisiae Hal3 were made by VTFS motif is replacing the widespread RVXF motif, 2-step PCR, using pGEX-6P-1-ScHal3 as template and the but that it would have a Ppz1-specific role. In any oligonucleotides described in Table S1. After the overlap- case, our results show that the presence of a bulky ping PCR step the PCR products and the pGEX-6P-1- residue at position F97 is important, as the mutation ScHal3 vector were cut at the indicated restriction sites and to Ala has a stronger effect than the change to Tyr ligated (Table S2). These constructs were used to produce 14 The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. C. Santolaria et al. Functional mapping of the Hal3 N-terminal region R91 K90 95VFTS98 39-51 67-110 118-148 209-227 67-93 94-110 248-256 1.0 0.9 Anchor 0.8 IUpred2 0.7 Score 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 Residue number Fig. 10. Prediction of intrinsically disordered (IDRs) and folding upon binding regions for the N-terminal region of S. cerevisiae Hal3. Predic- tion of IDRs according the IUPred2 software is denoted in red, and prediction of disordered binding regions based in the Anchor2 software in blue. The 0.5 cut-off (discontinuous line) corresponds to 5% false positive prediction on IDRs or ordered protein segments. The cartoon on the top depicts the position of the major deletions described in our work, as well as the location of the KR-X3-VTFS motif. The N-terminal extension is shown in light blue. Table 1. Yeast strains used in this work. The C. albicans hybrid proteins were generated from the pre-existing CaHal3 and CaCab3 genes cloned in pGEX- Strain Genotype References 6P-1 [32]. These constructs were used as templates to BY4741 MAT a his3D1 leu2D0 met15D0 [53] amplify overlapping fragments with the oligonucleotides ura3D0 pairs 50 CaHal3 megaprimer/30 CaHal3 megaprimer and 50 BY4741 BY4741 hal3::KANMx [53] CaCab3 Megaprimer/30 CaCab3 Megaprimer, respectively. hal3D In a second step, the CaHal3 fragment was reamplified BY4741 BY4741 slt2::KANMx [53] using primers pGEX 50 sequencing Primer and 30 slt2D CaHal3 + 80Bsu15I, whereas for the CaCab3 fragment the ZCZ001 BY4741 pGAL1:PPZ1: kanMX6 [20] primers employed were pGEX 50 sequencing Primer and 30 MAC003 BY4741 pGAL1:PPZ1-EGFP:HISMX6 [25] CaCab3 + 100Bst1107I. Each pair of overlapping frag- IM021 MAT a ura3-52 leu2-3, 112 trp1-1 [27] his4 can-1r ppz1::KANMX hal3::LEU2 ments were amplified with oligonucleotides pGEX 50 sequencing Primer and 30 CaHal3 + 80Bsu15I (for Cahal3) or 30 CaCab3 + 100Bst1107I (for CaCab3). The former amplification fragment was inserted in pGEX-CaHal3 by in E. coli the diverse protein variants of Hal3. The con- EcoRI/Bsu15I digestion, whereas the latter was cloned in struction of vectors for expression in E. coli of GST-fused pGEX-CaCab3 by EcoRI/Bst1107I digestion. Once in full length Ppz1 and the Ppz1-Cter (D1–344) catalytic moi- pGEX-6P-1, both hybrid genes were transferred to pWS93 ety has been previously described [22]. For expression in for expression in yeast by digestion with EcoRI/KspAI. yeast, the Hal3 variants generated in pGEX-6P-1 were To perform point substitutions in HAL3 affecting phos- digested with EcoRI and XhoI, and cloned into the high phorylatable Ser in the 47–60 region, a Quickchange site- copy-number plasmid pWS93, which provide a C-terminal directed mutagenesis method [46] was carried out using 3xHA tag [45], linearized with EcoRI and SalI. Construc- YEplac195 HAL3, which contains the HAL3 gene tion of plasmid pWS93-ScHAL3 was reported earlier [26]. (2.4 Kbp) cloned at EcoRI/HindIII sites, as a template [27]. The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 15 Federation of European Biochemical Societies. Functional mapping of the Hal3 N-terminal region C. Santolaria et al. The amplification of the entire template was done with Q5 purification and removal of the GST-moiety by treatment High-Fidelity DNA Polymerase (New England Biolabs, with PreScission protease (GE Healthcare, Chicago, IL, Ipswich, MA, USA), at 1 minkb1 of extension time and USA) was done as in [34]. Quantification of the purified final reaction volume of 50 lL, with the appropriate proteins was carried out by SDS/PAGE followed by Coo- oligonucleotides described in Table S1. The PCR product massie Blue staining and integration of the relevant bands was purified with NucleoSpin Gel and PCR Clean-Up kit with the Gel Analyser software using a bovine serum albu- (Macherey-Nagel, D€ uren, Germany) and was eluted in min calibration curve as reference. 30 lL. Finally, the PCR template was digested with DpnI (FastDigest, Thermo Scientific, Waltham, MA, USA) and 1/3 of the PCR product was used to transform E. coli. Detection of Ppz1–Hal3 interactions. Deletion of the 47–60 region was made by two parallel The interaction between the diverse variants of Hal3 and PCR amplifications using YEplac195 Hal3 as template and Ppz1 was evaluated in vitro by incubating aliquots the pairs of oligonucleotides 5-Hal3-200 / 3- (  100 lL) of the glutathione-agarose beads containing Hal3delta47_60, and 5-Hal3delta47_60 / 3-Hal3+920. Both 6 lg of GST-Ppz1 with protein extracts (600 lg) prepared PCR product were diluted, mixed and used as template for from strain IM021 (ppz1 hal3) transformed with plasmid a further amplification with the 5-Hal3-200 / 3-Hal3+920 pWS93 (negative control) and with the same plasmid carry- pair of primers. The resulting fragment (about 920 bp) was ing the diverse HA-tagged version of Hal3 as in [28,34]. digested with XbaI and BamHI and cloned in these same Quantification of the level of interaction was done by deter- sites in YEplac195 HAL3. Oligonucleotides employed are mining the ratio between the immunoreactive Hal3 signal described in Table S1. and the corresponding Ponceau Red stained phosphatase The construction of pWS93-PD and pWS93-PD + CtD bait and taking the value for native ScHal3 as the unit. At can be found in [28]. least two different preparations of the phosphatase and of each Hal3 variant were used in the assays. Yeast protein extraction and immunoblot Assay of protein phosphatase activity. The appropriate strains were grown to exponential phase in SC medium lacking uracil (10 mL, OD600 = 0.8) with glu- The ability of the different versions of Hal3 to inhibit cose as carbon source (except for the ZCZ01 strain, which in vitro full-length Ppz1 or its catalytic domain was evalu- was grown on raffinose). Cells were collected by centrifuga- ated using recombinant proteins produced in E. coli as tion at 4 °C, washed twice with cold water and frozen at GST fusions after removal of the GST moiety. To this end, 20 °C until use. Protein extraction, SDS/PAGE and the Ppz1 phosphatase activity was measured with 10 mM p- immunoblot analysis were performed as in [34]. nitrophenylphosphate (pNPP) as substrate and using 2–10 pmols of full-length Ppz1 or 1–5 pmols of Ppz1-Cter essen- tially as described previously [34,48]. Fluorescence microscopy MAC003 cells carrying the plasmids of interest were grown Other methods overnight to saturation in synthetic media with glucose lacking uracil. Then, cells were diluted to OD600 = 0.004 in To select the set of Hal3-like proteins for N-terminal fresh synthetic media without uracil containing 2% raffi- comparisons, a BLASTP analysis was performed with nose, instead of glucose, and growth resumed at 28 °C until both the entire sequence and residues 1–250 of S. cere- OD600 = 0.6 was reached. At this point, galactose was visiae Hal3 (NP_012998.1) at the NCBI non-redundant added to the culture at a final concentration of 2%, to protein database, excluding Saccharomyces cerevisiae induce Ppz1 overexpression. Cell monitoring was per- (taxid:4932). The full sequences of fungal Hal3-like pro- formed using a l-Slide 8 Well (Ibidi GmbH, Gr€afelfing, teins were aligned using the Clustal Omega algorithm of Germany) as described in [25] after 6 h of Ppz1 overexpres- the EMBL-EBI server [49]. Assignation to homologues of sion. Pictures were taken using a Nikon Eclipse TE-2000E ScHal3/ScVhs3 or ScCab3 was done according to the p- fluorescence microscope. values of the BLASTP analysis followed by identification in Cab3 of the conserved Cys residue in the substrate recognition clamp as well as the canonical PXMNXXMW Recombinant expression of proteins in E. coli motif [26]. The prediction of intrinsically disordered All recombinant proteins were expressed in E. coli BL21 regions (IDRs) was done with the IUPRED2 software (with (DE3) RIL cells as described for Ppz1 in [47] using Terrific the “short disordered regions” setting) and disordered Broth (tryptone 12 gL1, 24 gL1 yeast extract, 4 mLL1 binding regions were identified using the ANCHOR2 pre- glycerol, 17 mM KH2PO4, 72 mM K2HPO4). Affinity- diction algorithm [50]. 16 The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. C. Santolaria et al. Functional mapping of the Hal3 N-terminal region Acknowledgements 5 Posas F, Camps M, Ari~ no J. The PPZ protein phosphatases are important determinants of salt The technical assistance of Montserrat Robledo is tolerance in yeast cells. J Biol Chem. 1995;270:13036–41. acknowledged. We thank J. Pi~ nol for allowing access 6 Ruiz A, Yenush L, Ari~ no J. Regulation of ENA1 Na+- to microscopy facilities and L. Yenush for critical ATPase gene expression by the Ppz1 protein review of the manuscript. We also thank Prof I. Sa phosphatase is mediated by the calcineurin pathway. Correia (Univ. of Lisbon) for sharing with us informa- Eukaryot Cell. 2003;2:937–48. tion about acetic acid-induced phosphorylation 7 Yenush L, Mulet JM, Ari~ no J, Serrano R. The Ppz changes in Hal3 before publication. This research was protein phosphatases are key regulators of K+ and pH funded by the Ministerio de Economıa, Industria y homeostasis: implications for salt tolerance, cell wall Competitividad, Spain, grant number BFU2017-82574- integrity and cell cycle progression. EMBO J. P, to JA and AC. MA, CS and DV were recipients of 2002;21:920–9. PIF fellowships from the Universitat Aut onoma de 8 Ruiz A, Ruiz MC, Sanchez-Garrido MA, Arino J, Barcelona, Spain. The funding source(s) was not Ramos J. 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The yeast halotolerance determinant Hal3p is most experiments; MA and DV performed some an inhibitory subunit of the Ppz1p ser/Thr protein experiments and contributed with reagents. JA, AC, phosphatase. Proc Natl Acad Sci USA. 1998;95:7357– CS and MA analysed the data; JA wrote the paper, 62. and all authors contributed to its edition. 12 Merchan S, Bernal D, Serrano R, Yenush L. Response of the Saccharomyces cerevisiae Mpk1 mitogen- activated protein kinase pathway to increases in internal Data availability statement turgor pressure caused by loss of Ppz protein The data that supports the findings of this study are avail- phosphatases. Eukaryot Cell. 2004;3:100–7. able in Figures 1 to 10, and Table1 and in the supplemen- 13 Posas F, Casamayor A, Ari~ no J. The PPZ protein tary material of this article. Any other data or aditional phosphatases are involved in the maintenance of information are available from the corresponding author osmotic stability of yeast cells. FEBS Lett. 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Supporting information Molecular characterization of Ypi1, a novel Additional supporting information may be found Saccharomyces cerevisiae type 1 protein phosphatase online in the Supporting Information section at the end inhibitor. J Biol Chem. 2003;278:47744–52. of the article. 49 Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, et al. The EMBL-EBI search and Table S1. Oligonucleotides used in this work. sequence analysis tools APIs in 2019. Nucleic Acids Res. Table S2. PCR amplifications and cloning strategies. 2019;47:W636–41. The FEBS Journal (2022) Ó 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 19 Federation of European Biochemical Societies.