International Journal of Molecular Sciences Article Comparative Analysis of Type 1 and Type Z Protein Phosphatases Reveals D615 as a Key Residue for Ppz1 Regulation Antonio Casamayor 1 , Diego Velázquez 1 , Carlos Santolaria 1 , Marcel Albacar 1 , Morten I. Rasmussen 2 , Peter Højrup 2 and Joaquín Ariño 1, * 1 Institut de Biotecnologia i Biomedicina & Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; Antonio.Casamayor@uab.cat (A.C.); diego.velazquez91@icloud.com (D.V.); Carlos.Santolaria@uab.cat (C.S.); Marcel.Albacar@uab.cat (M.A.) 2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark; morras@massai.dk (M.I.R.); php@bmb.sdu.dk (P.H.) * Correspondence: joaquin.arino@uab.es Abstract: Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We  explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are  distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes Citation: Casamayor, A.; are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties Velázquez, D.; Santolaria, C.; that are specific for each kind of phosphatase. In some cases, these differences likely affect the Albacar, M.; Rasmussen, M.I.; distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the Højrup, P.; Ariño, J. Comparative PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo Analysis of Type 1 and Type Z co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking Protein Phosphatases Reveals D615 as a Key Residue for Ppz1 and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two Regulation. Int. J. Mol. Sci. 2022, 23, major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 1327. https://doi.org/10.3390/ in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to ijms23031327 Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi. Academic Editor: David Magne Received: 23 November 2021 Keywords: protein phosphatase; Ppz1; PP1c; sequence comparisons; cross-linking proteomics; Accepted: 21 January 2022 enzyme inhibition; fungi Published: 25 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Analysis of the Saccharomyces cerevisiae genome allows the identification of at least 19 proteins with predicted or experimentally tested Ser/Thr protein phosphatase activity. Twelve of these can be classified within the PPP (phosphoprotein phosphatase) superfamily, which includes homologs of the broadly distributed type 1 (PP1), 2A (PP2A) and 2B Copyright: © 2022 by the authors. (PP2B) phosphatases, accounting for most of the serine/threonine phosphatase activity Licensee MDPI, Basel, Switzerland. in eukaryotic cells. From a structural point of view, they all share a conserved central β This article is an open access article sandwich that coordinates two metal ions [1,2]. In S. cerevisiae, three different proteins distributed under the terms and represent type 1 phosphatases: Glc7, Ppz1/2, and Ppq1. GLC7 is an essential gene that conditions of the Creative Commons Attribution (CC BY) license (https:// encodes a 312-residue protein, mostly comprising the canonical catalytic PPP domain. It creativecommons.org/licenses/by/ can be considered the yeast homolog of the ubiquitous eukaryotic type 1 catalytic subunit 4.0/). (PP1c) and shows a high level of sequence conservation (>80%), even among distant species. Int. J. Mol. Sci. 2022, 23, 1327. https://doi.org/10.3390/ijms23031327 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 1327 2 of 18 Ppz and Ppq1 enzymes contain a conserved C-terminal PPP domain preceded by an N- terminal extension that is structurally unrelated. In contrast to Glc7, phosphatases Ppz1/2 and Ppq1 are found only in fungi. However, while Ppz enzymes can be found in virtually any fungal species, Ppq1 has a much more restricted distribution, as it is only found in some of the families of Saccharomycetales, such as Saccharomycetaceae, Pichiaceae or Debaryomycetaceae (but it is absent in Dipodascaceae and Trichomonascaceae), and little is known about its function and regulation. For these reasons, the Ppq protein family is not included in this study. Glc7 plays a myriad of cellular roles, including glucose repression, translation, tran- scription, cation homeostasis, and cell cycle regulation. The enormous functional diversity of eukaryotic type 1 enzymes is explained by its interaction with a plethora of regulatory subunits that modulate activity, localization, or substrate selection of the catalytic sub- unit. The association of Glc7 with not less than 30 such proteins was reported [1–3]. The characterization of the structural basis for the interaction between PP1c and its regulatory subunits was carried out mostly with mammalian enzymes [4], and it involves docking motifs, that is, short sequences (about 4–8 residues) present in the regulatory subunits that, in combination, create a larger interaction surface for PP1c. About 10 known distinct PP1-docking motifs were identified in the regulatory subunits in mammals, although not all of them are found in yeast [5]. The most general and conserved motif is the “RVxF” consensus sequence, which interacts with the hydrophobic groove of PP1c. As expected for such finely regulated activity, the overexpression of Glc7 is detrimental for the cell [6,7]. In S. cerevisiae, Ppz proteins are represented by two paralogs, Ppz1 and Ppz2 (692 and 710 residues, respectively), although most fungi are endowed with a single Ppz-encoding gene. As mentioned above, the ScPpz1 catalytic PPP domain occupies the C-terminal half of the protein (about 340 residues), which is about 60% identical to Glc7, and it is preceded by a nearly 350-residue-long, much more divergent N-terminal region [8,9]. Ppz phosphatases were characterized in some detail in several fungi [10–15], and were, in some cases, identified as a virulence determinant [16–18]. As a rule, the catalytic domain appears quite conserved, whereas the N-terminal extension is highly variable or almost nonexistent in some cases. It is generally accepted that Ppz phosphatases play an important role in monovalent cation homeostasis. In S. cerevisiae, Ppz1 is also involved in the regulation of ubiquitin, dephosphorylation of the ubiquitin ligase adaptor Art1, and translation initiation (see [2] for references). In contrast to Glc7, only two regulatory subunits, Hal3 and Vhs3, were determined for ScPpz1, Hal3 being the major in vivo regulator. Both proteins bind to the catalytic domain of the phosphatase and inhibit its phosphatase activity [19–23]. It must be noted that in S. cerevisiae and other related fungi, Hal3 and Vhs3 are moonlighting proteins involved in the biosynthesis of coenzyme A (CoA). This occurs by interaction with a third structurally re- lated protein (Cab3), thus forming an atypical heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme [24]. The evidence suggests that the regulation of Ppz1 activity must be exceptionally effective, since even moderately higher than normal levels of the phosphatase cause a dramatic cell growth arrest [19,25]. Genetic approaches indicate that, when overexpressed, Ppz1 could be the most toxic protein in budding yeast [26], and recent evidence suggests that such toxicity derives from the alteration of multiple targets that are likely different from those of Glc7 [27,28]. An important question is how PP1 and PPZ regulation (and therefore cellular functions) can be insulated. In fact, such insulation is likely not perfect, since it is known that Ppz1 can interact with certain Glc7 subunits endowed with the RVxF interaction motif, such as Glc8 and Ypi1 [29,30]. In addition, Ppz1 has a moderate sensitivity to mammalian inhibitor-2, a well-known regulatory component for PP1c [31,32]. Although there is evidence suggesting that the N-terminal extension of Ppz phosphatases could influence their function and affect interaction with the Hal3 inhibitor [13,19,25,33,34], the fact that Hal3 mainly interacts with the catalytic C-terminal domain suggests that this region should contain specific structural determinants for regulation. Important Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 19 Int. J. Mol. Sci. 2022, 23, 1327 3 of 18 suggests that this region should contain specific structural determinants for regulation. Important insights were derived from the 3D structure of the catalytic domain of C. albi- insights were derived from the 3D structure of the catalytic domain of C. albicans Ppz1 cans Ppz1 (CaPpz1) [32], revealing structural determinants that could allow regulation of (CaPpz1) [32], revealing structural determinants that could allow regulation of this enzyme bythis enzyme byregulators PP1c-specific PP1c-specific and anregulators additional and an additional structured structured C-terminal C-terminal α-helix not presentα-helix in not present PP1c enzymes. in PP1c enzymes. Most Most of of ourour current current knowledge knowledge onon this this field field derives derives from from thethe study study of of a very a very limited limited number of species (generally S. cerevisiae and C. albicans). Therefore, we number of species (generally S. cerevisiae and C. albicans). Therefore, we considered that considered that extension extension of of thethe analysis analysis across across thethe fungal fungal kingdom kingdom may may provide provide a set a set of of common common andand distinctive characteristics from which more general rules on the differential regulation of of distinctive characteristics from which more general rules on the differential regulation fungal fungal PP1PP1 andandPPZPPZ phosphatases phosphatases couldcould be derived. be derived. SuchSuch studystudy hastoled has led thetoidentification the identifica- of a strongly conserved residue (D615) that is most likely relevant for specificspecific tion of a strongly conserved residue (D615) that is most likely relevant for func- functions tions of Ppz1. of Ppz1. 2. 2. Results Results andand Discussion Discussion WeWe selected selected 57 57 species species as representatives as representatives of three of three mainmain different different fungi fungi clades clades (Figure (Figure 1) according to a to 1) according proteome-based a proteome-based classification [35].[35]. classification TheThenumber numberandanddistribution distributionof of PP1PP1 and Ppz and proteins Ppz proteinsanalyzed analyzed areare shown shown in in Table Table1, and thethe 1, and identities of of identities thethe selected species selected species areare presented presented inin Table TableS1.S1.Most Mostofofthese theseorganisms organismspossess possessoneonePP1 PP1andandoneonePpz1, Ppz1, but but a a few few exceptions observed. Thus, exceptions are observed. Thus,several severalmembers membersofof thethe Saccharomycetaceae Saccharomycetaceae family, family, cor- corresponding to the post-WGD lineage (S. cerevisiae, C. glabrata and K. lactis) responding to the post-WGD lineage (S. cerevisiae, C. glabrata and K. lactis) have two genes have two genes coding coding for PPZ for PPZ proteins, proteins, andand about about seven seven veryvery similar similar PPZ PPZ proteins proteins areare annotatedininthe annotated theZygomycota Rhizopusdelemar ZygomycotaRhizopus delemargenome. genome. Figure 1. Simplified Figure phylogenetical 1. Simplified treetree phylogenetical of the ofdifferent cladesclades the different covered in thisinstudy. covered Branches this study. labeledla- Branches as beled A, B and M Bdenote as A, and Mthe Ascomycota, denote Basidiomycota the Ascomycota, and “Monokarya” Basidiomycota fungi clades. and “Monokarya” fungiThe figure clades. The was constructed figure based on based was constructed data from reference on data [35]. from reference [35]. Table Table 1. 1. Distributionofofselected Distribution selectedorganisms organismsand andtheir theiranalyzed analyzed sequences sequences among among different fungal fun- galclades. clades. Phylum PhylumSpecies Species PP1 PP1 Ppz Ppz Saccharomycotina Saccharomycotina 15 1515 15 18 18 Ascomycota Pezizomycotina Ascomycota Pezizomycotina 13 1313 13 13 13 Taphrinomycotina 7 Taphrinomycotina 7 11 11 7 7 Agaricomycotina Agaricomycotina 10 1011 11 10 10 Wallemiomycetes Wallemiomycetes Basidiomycota Basidiomycota Ustilagomycotina 5 Ustilagomycotina 55 5 5 5 Pucciniomycotina Pucciniomycotina Zygomycota Zygomycota “Monokaryotic” Chytridiomycota 7 9 14 “Monokaryotic” Chytridiomycota Cryptomycota 7 9 14 Cryptomycota Total number 57 64 67 Total number 57 64 67 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 19 Int. J. Mol. Sci. 2022, 23, 1327 4 of 18 Concerning PP1, two proteins are found in a set of species included in the Taphrino- mycotina subphylum Concerning thattwo PP1, correspond to the proteins are Schizosaccharomycetaceae found in a set of species includedfamilyin(the the fission Taphrino- mycotina yeast S. pombe,subphylum S. japonicus, that correspond S. cryophilus to S. and Schizosaccharomycetaceae theoctosporus). In addition to family (the fission the “standard” PP1, theseS.organisms yeast pombe, S. japonicus, possess a S. cryophilus and S. octosporus). “non-conventional” PP1, namedInSds21,addition whichto the “standard” differs in a fewPP1, these organisms structural aspects from possess a “non-conventional” the canonical PP1 proteins. The PP1, named Sds21, which Agaricomycotina differs Calocera vis- in a few structural aspects from the canonical PP1 proteins. The Agaricomycotina cosa and the Chytridiomycota Gonapodya prolifera and Allomyces macrogynus also possess Calocera twoviscosa and the Chytridiomycota Gonapodya prolifera and Allomyces macrogynus also possess PP1 proteins. two PP1 proteins. 2.1. Sequence Comparison of Catalytic Domains across Fungi Identifies Conserved Ppz-Specific 2.1. Sequence Comparison of Catalytic Domains across Fungi Identifies Conserved Residues Ppz-Specific Residues Alignment of the selected 131 sequences corresponding to the catalytic domain of Alignment of the selected 131 sequences corresponding to the catalytic domain of PP1 PP1 and PPZ proteins using Clustal Omega revealed that 141 residues are conserved in and PPZ proteins using Clustal Omega revealed that 141 residues are conserved in PP1, PP1, whereas only 50 are maintained in PPZ proteins (Figure S1). Forty-three of these res- whereas only 50 are maintained in PPZ proteins (Figure S1). Forty-three of these residues idues are shared between both types of phosphatases. However, a closer inspection of the are shared between both types of phosphatases. However, a closer inspection of the data data clearly indicates that this relatively low conservation level (mainly affecting PPZ en- clearly indicates that this relatively low conservation level (mainly affecting PPZ enzymes) zymes) is caused is caused by theby inclusion the inclusion of “Monokaryotic” of “Monokaryotic” fungifungi in the in the analysis. analysis. Indeed, Indeed, when when these these organisms are excluded, 171 residues (56.7%) are conserved in PP1, organisms are excluded, 171 residues (56.7%) are conserved in PP1, and 137 (41.0%) in and 137 (41.0%) in PPZ PPZenzymes. enzymes.Of Ofthese, these,104104are common are common toto both PP1 both PP1 and PPZ and proteins. PPZ proteins.In addition, In addition,the the phylogenetic tree generated from the above-mentioned Clustal Omega phylogenetic tree generated from the above-mentioned Clustal Omega analysis revealed analysis revealed thatthat the the sequences sequences of the PP1PP1 of the proteins proteinsareare much muchmoremore homogeneous homogeneous than thethe than sequences sequences of the catalytic region of the analyzed PPZs. In fact, the most of the catalytic region of the analyzed PPZs. In fact, the most divergent PP1 divergent PP1 sequences sequences correspond correspond to the Sds21 to the Sds21proteins, proteins,thethe second second type typeofofPP1PP1phosphatase phosphatasefoundfoundspecifically specifically in in fission fissionyeasts yeasts(Figure (Figure2). 2).These Theseresults resultsindicate indicatethat thatthethestructure structureofofPP1 PP1enzymes enzymesshowsshows a a higher higherlevel levelofofconservation conservationacross acrossfungi fungithan thanthat thatofofPPZ PPZenzymes. enzymes. Figure Figure 2. PP1 2. PP1 enzymes enzymes are more are more conserved conserved thanacross than PPZ PPZ across fungi. fungi. Phylogenetic Phylogenetic tree containing tree containing the the sequences sequences of PP1 of PP1 and the and the catalytic catalytic regions regions of PPZ proteins. of PPZ proteins. Phylogenetic Phylogenetic tree data tree data generated generated from Clustal fromOmega Clustalalignments was visualized Omega alignments using the Dendroscope was visualized software (version using the Dendroscope software3.7.3) as rectan- (version 3.7.3) as gular phylogram rectangular [36]. Pez: [36]. phylogram Pezizomycotina. Sac: Saccharomycotina. Pez: Pezizomycotina. Tap: Taphrinomycotina. Sac: Saccharomycotina. BAS: Tap: Taphrinomycotina. Basidiomycota. MON: “Monokaryotic” BAS: Basidiomycota. fungi. fungi. MON: “Monokaryotic” Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 19 Int. J. Mol. Sci. 2022, 23, 1327 5 of 18 Most Mostdifferences differences between between PP1 and PPZ PP1 and PPZcatalytic catalyticdomains domainsare arelisted listedinin Table Table S2 S2 andand can can be grouped into four categories. In the first one (class A), PP1 and PPZ be grouped into four categories. In the first one (class A), PP1 and PPZ differ in 26 residues differ in 26 residues whoseis nature whose nature is or invariant invariant or largely maintained largely maintained in both types inofboth types Twenty-seven enzymes. of enzymes. Twenty-seven residues are virtually constant in PP1 but present a substantial residues are virtually constant in PP1 but present a substantial variation in PPZs variation (classinB). PPZs (class B). The third group (C) is composed of residues found to The third group (C) is composed of residues found to be constant in PPZ but variable be constant in PPZin but PP1variable (C) andinisPP1 (C) and issmaller substantially substantially smaller (only three (onlyAthree cases). cases). fourth groupA(D),fourth group (D), containing five containing five instances, corresponds to differences between both enzymes instances, corresponds to differences between both enzymes that are only found in specific that are only found clades.inAs specific can be clades. observedAsincan be observed Figure in Figure 3, when models 3, when for ScGlc7 and models ScPpz1for areScGlc7 and constructed, ScPpz1 are constructed, many residues from these groups map onto many residues from these groups map onto the surface of both proteins. Interestingly, the surface of both proteins. several ofInterestingly, several of the the class A differences, such class A differences, as N484, T486, V488 such as N484, or M604, map T486, nearV488 or the PPZ M604, mapsite. catalytic nearWetheconsidered PPZ catalyticthissite. lastWe considered group this lastrelevant as particularly group astoparticularly rele- predict possible vant to predictfunctional PPZ-specific possible PPZ-specific features. functional features. Figure Figure3.3.Mapping Mappingof relevant surface of relevant residues surface that that residues differ in Ppz1 differ and Glc7. in Ppz1 The upper and Glc7. The panel upperiden- panel tifies identifies class B residues on the surface of a Glc7 model (orange). In the lower panel, in class B residues on the surface of a Glc7 model (orange). In the lower panel, residues class residues Ain(raspberry) or C (blue) class A (raspberry) orare mapped C (blue) areon the surface mapped on theofsurface the S. cerevisiae of the S.Ppz1 model cerevisiae for model Ppz1 its catalytic for its region. Residues forming the catalytic site are shown in yellow. The transition between images cor- catalytic region. Residues forming the catalytic site are shown in yellow. The transition between responds to a rotation (anticlockwise) of 90⁰ on the Y-axis. images corresponds to a rotation (anticlockwise) of 900 on the Y-axis. Because Becauseofofthethepossible possible relevance relevance of of charged chargedresidues residuesininthe theinteraction interactionwith with regula- regulatory tory proteins, we were interested in their distribution on the surface of proteins, we were interested in their distribution on the surface of both types of enzymes. both types of en- To zymes. To this end, we identified those positions for which a change this end, we identified those positions for which a change in charge could be predicted forin charge could be predicted all speciesfor (orall at species least for(or at least specific for specific orders orders or families, or families, Tables S2 and S3). Tables S2 and S3). We identified We 30 such identified 30 such changes, of which 23 can be considered quite constant. changes, of which 23 can be considered quite constant. In 14 of those, changes were from In 14 of those, changes acidic inwere PP1 tofrom acidic neutral in PP1intoPPZ residues neutral residues (or vice versa),inwhereas PPZ (or7 vice versa), implied whereas 7 the substitution implied the substitution of a basic residue by a neutral one (or vice versa). of a basic residue by a neutral one (or vice versa). Two remarkable modifications were PP1c Two remarkable modifications K210 and K259, were PP1cwere which K210 and K259, changed to Dwhich in thewere PPZ ofchanged all fungitoevaluated D in the PPZ (D566of all andfungi D615 evaluated in ScPpz1). (D566 These and D615 in residues wereScPpz1). mapped These on theresidues surfacewere mapped of models on the surface constructed of for ScGlc7 models constructed for ScGlc7 and ScPpz1 (Figure 4 and see below). As and ScPpz1 (Figure 4 and see below). As it can be seen, the distribution of charges is not it can be seen, the distribution too differentofoncharges is notsurrounding the surface too differentthe on extremely the surfaceconserved surrounding the extremely catalytic site (panelscon-A), served catalytic site (panels A), but it is appreciable but it is appreciable in many other areas of both molecules. in many other areas of both molecules. Int. Int. J.J. Mol. Mol. Sci. Sci. 2022, 2022, 23, 23, x1327 FOR PEER REVIEW 66 of of 19 18 Figure 4. Figure 4. Distribution Distributionofofchanges changesaffecting charged affecting chargedresidues on the residues on surface of both the surface of Glc7 both(upper panel) Glc7 (upper and theand panel) catalytic region region the catalytic of Ppz1of(lower Mapped panel).panel). Ppz1 (lower residues Mapped represent residues potential represent alterations potential in altera- tions surfacein surface charges charges in PP1 orin PPZ PP1 or PPZ enzymes. enzymes. AcidicAcidic residues residues are in are redinand redbasic and basic ones ones in in dark dark blue. blue. BoxedBoxed residue residue names names are changes are changes present present in S. cerevisiae in S. cerevisiae Glc7 Glc7 or Ppz1or Ppz1 butnecessarily but not not necessarily in in most most other species. The C-terminal modeled residue of Glc7 and Ppz1 are depicted in other species. The C-terminal modeled residue of Glc7 and Ppz1 are depicted in cyan to facilitate cyan to facil- itate identification. identification. A to A D to D denotes denotes the positions the positions after after 90⁰ anticlockwise 900 anticlockwise rotation rotation onY-axis. on the the Y-axis. 2.2. The PP1 Structural Features Features Required RequiredforforModulation Modulationby byRegulatory RegulatorySubunits SubunitsAppear AppearLargely Conserved Largely in Fungiin Fungi Conserved Most regulatory Most regulatory subunits subunits of of mammalian mammalian PP1 PP1 possess possess aa conserved conserved “RVxF” “RVxF” motifmotif that that interacts with interacts with thethe hydrophobic hydrophobic groove groove of PP1c, which of PP1c, which is is fully fully conserved conserved inin fungal fungal PP1 PP1 and and strongly maintained in PPZ proteins (Figure S2). Such conservation strongly maintained in PPZ proteins (Figure S2). Such conservation most likely explainsmost likely explains the observed the observed interactions interactions between between ScPpz1 ScPpz1 and and diverse diverse Glc7 Glc7 regulatory regulatory subunits, subunits, such such asas Glc8 (the homolog of mammalian inhibitor-2), Ypi1, Gip2 or Sla1 [29,30], Glc8 (the homolog of mammalian inhibitor-2), Ypi1, Gip2 or Sla1 [29,30], as well as the as well as the sensitivity of sensitivity of ScPpz1 ScPpz1 andand CaPpz CaPpz to to inhibitor-2 inhibitor-2 [32,37]. [32,37]. It It also also suggests suggests that that these theseregulatory regulatory interactions are maintained in most fungi, if not all. Mammalian inhibitor-2 interactions are maintained in most fungi, if not all. Mammalian inhibitor-2 binds to binds to PP1c PP1c through the RVxF and the SILK motifs plus a long helix, which expands through the RVxF and the SILK motifs plus a long helix, which expands across the PP1 across the PP1 active site [38]. While the SILK motif is conserved in all fungal PP1 (only a conservative active site [38]. While the SILK motif is conserved in all fungal PP1 (only a conservative change of I for L in position 58 in a few species), it is not conserved in PPZ. In contrast, the change of I for L in position 58 in a few species), it is not conserved in PPZ. In contrast, long helix is strongly conserved in both PP1 and PPZ enzymes (Figure S2). Since it has the long helix is strongly conserved in both PP1 and PPZ enzymes (Figure S2). Since it has been proposed that Hal3 blocks the activity of the phosphatase by interacting with this been proposed that Hal3 blocks the activity of the phosphatase by interacting with this region [39], this would explain why ScHal3 was able to inhibit in vitro all PPZ tested so far. region [39], this would explain why ScHal3 was able to inhibit in vitro all PPZ tested so Recent work by Choy and coworkers [40] identified the residues in PP1c required for far. interaction with the regulatory subunit Sds22, which lacks the RVxF motif and is composed Recent work by Choy and coworkers [40] identified the residues in PP1c required for of a short unstructured N-terminal region followed by a folded leucine-rich repeat (LRR) interaction with the regulatory subunit Sds22, which lacks the RVxF motif and is com- domain. Sds22 is conserved from yeast to human. In S. cerevisiae, Sds22 is an essential posed of a short unstructured N-terminal region followed by a folded leucine-rich repeat protein important for Glc7 nuclear targeting [41,42]. The PP1c region required for Sds22 (LRR) domain. Sds22 is conserved from yeast to human. In S. cerevisiae, Sds22 is an essen- interaction involves a relatively large number of amino acids (26 residues) and they are tial quiteprotein conservedimportant in PPZfor Glc7 nuclear phosphatases (73% targeting identity,[41,42]. The PP1c region 88% conservation), exceptrequired those more for Sds22 interaction involves a relatively large number of amino acids (26 N-terminally located (379, 380, 391 and 395 in ScPpz1). The equivalent residues in PP1c residues) and they are are quite mostly conserved involvedininPPZ the phosphatases interaction with (73%theidentity, first LRR 88% conservation), [40]. Interestingly, except Sds22those was Int. J. Mol. Sci. 2022, 23, 1327 7 of 18 found to physically interact with Ppz1 in diverse large-scale screenings [43–45]. However, it is unknown whether Sds22 could affect Ppz1 activity and function. The same pattern of conservation can be found in the Ppq1 phosphatase, for which interaction with Sds22 was also described [44]. These observations suggest that the first LRR of Sds22 could not be involved in the interaction with PPZ proteins. Conservation of other recognized motifs in mammalian PP1c, such as the MyPhone (myosin phosphatase N-terminal element) commonly located N-terminally to the RVxF motif, is relatively high in fungal PP1. However, the conservation is only partial in the case of PPZs in Saccharomycetales and even minimal in other fungi (Figure S2). The ΦΦ motif appears strongly conserved in all fungal PP1, but not in the PPZ enzymes. Finally, the NIPP1-helix interaction pocket is well conserved in fungal PP1, but clearly less so in PPZ. It is worth noting that fungi do not seem to encode homologs of mammalian NIPP-1 proteins. In conclusion, the capacity to interact with (and perhaps be regulated by) a subset of PP1 regulatory subunits could be conserved in PPZ among most fungal species. 2.3. The C-Terminal Extra Alpha-Helix of CaPpz1 Is Likely Present in Most Ppz Enzymes Chen and coworkers [32] presented evidence that CaPpz1 contains a C-terminal extra α-helix that is not present in mammalian PP1c(α). This feature is related to another characteristic difference located at the N-terminal region of the phosphatase. In CaPpz1, the N-terminal helix A’ extends an extra turn in comparison with mammalian PP1c(α), and the conformation of the loop connecting helix A’ and A (L1) is different in both phosphatases. These authors attributed a primary role for this difference to the fact that Y144 in PP1c(α) helix C is substituted by a smaller residue (C309 in CaPpz1). Remarkably, the equivalent position in ScGlc7 (Y143) is conserved in most fungal PP1 enzymes (in the few exceptions, F, an equally bulky and hydrophobic residue, is present). This same position in Ppz is also a C in all Saccharomycetales, in most Pezyzomycetes; it is also common in other clades, and, when not present, it is usually substituted by a small side chain residue (T or A). Since the authors proposed that this change results in the widening of the pocket defined by helix A’, L1, and helix B, it can be concluded that such a structural feature would be shared by virtually any fungal Ppz enzyme. According to Chen and coworkers, the mentioned widened pocket can interact with the C-terminal end of CaPpz1 and induces its restructuring into an extra α-helix that is not present in mammalian PP1c(α). The hydrophobic CaPpz1 residues L464, L469, V472, and M473 are the key to such interactions. They are very different in PP1c(α) (and in fungal PP1 enzymes, see Table S2). These positions are fully conserved in ScPpz1 and correspond to L654, L659, V662 and M663. As observed in Figure 5, except for V662, these positions are strongly conserved in all fungi (and when different, they are conservative changes), except for the “Monokaryotic” ones. ScPpz1 Val662 is largely conserved in other Saccharomycetales (except for Yarrowia lipolytica, where it is an E). Interestingly, this position is mostly occupied by non-hydrophobic residues (mainly H and E) in the rest of the fungi. Therefore, it seems likely that the extra C-terminal α-helix described for CaPpz1 will be present in most (if not all) Saccharomycetales (and possibly in most fungi), but not in “Monokaryotic” species. Mol. Sci. 2022, Int.23, x FOR J. Mol. PEER Sci. 2022,REVIEW 23, 1327 8 of 19 8 of 18 Figure 5. Conservation Figure 5.ofConservation the C-terminal extra of the alpha-helix C-terminal in alpha-helix extra PPZ proteins. The proteins. in PPZ abundance Theofabundance spe- of specific cific residues at the C-terminus of PPZ proteins is depicted as logos grouping the different clades residues at the C-terminus of PPZ proteins is depicted as logos grouping the different clades indicated indicated on the left. Numbering corresponds to the S. cerevisiae Ppz1 enzyme. The residues dis- on the left. Numbering corresponds to the S. cerevisiae Ppz1 enzyme. The residues discussed in the cussed in the text are shaded in grey. text are shaded in grey. 2.4. Chemical Cross-Link of Co-Purified 2.4. Chemical Cross-LinkPpz1 and Hal3 Ppz1 of Co-Purified from S. andcerevisiae Hal3 fromPoints to Possible S. cerevisiae Rel-to Possible Points evant Ppz-Specific Conserved Relevant Residues Ppz-Specific Conserved Residues The interaction between The Ppz1between interaction and Hal3Ppz1 can and occurHal3 in vivo when in can occur both proteins vivo are proteins are when both simultaneously expressed in E.expressed simultaneously coli [39]. We in E.took coli advantage [39]. We tookof this fact andofco-expressed advantage this fact and co-expressed an N-terminallyan 6xHis-tagged version of the version N-terminally 6xHis-tagged catalyticofdomain of Ppz1 the catalytic (Ppz1of domain the Cter ) and the ) and(Ppz1 CterPpz1 native full-length Hal3 native protein by full-length using Hal3 a pDuet-based protein by using avector. His-tagged pDuet-based Ppz1 vector. was thenPpz1 was then His-tagged purified purified by metal by metal affinity and, asaffinity shownand, as shown in Figure in Figure 6A, Hal3 was 6A, alsoHal3 was also recovered withrecovered the with the phosphatase in a roughly 1:1 ratio. The mixture was subjected phosphatase in a roughly 1:1 ratio. The mixture was subjected to chemical cross-linking to chemical cross-linking with BS3 (bis(sulfosuccinimidyl)suberate), with BS3 (bis(sulfosuccinimidyl)suberate), which react which react with covalently covalently lysinewith lysine residues, and residues, the analyzed and the products products by analyzed SDS-PAGE.by SDS-PAGE. As shown As shown6B, in Figure in treatment Figure 6B,with treatment BS3 ledwith BS3 led to theofformation to the formation very highofmolecular very highmassmolecular massthat structures structures barely that barely entered theentered the resolving gel. resolving These gel. These bands werebands wereand excised excised and processed processed for LC-MS/MSfor LC-MS/MS analysis as analysis as described described in Sec- in Section 4. tion 4. Roughly 40% of the lysines identified by MS correspond to Hal3–Hal3 links, which is not surprising if we consider the ability of this protein to trimerize in vitro [22,46]. A number of cross-linked lysine residues corresponding to interprotein Ppz1–Hal3 cross- links were observed (Table 2), and Figure 6C displays these as a bidimensional map. It is worth noting that whereas in the case of Ppz1Cter the 16 cross-linked reactive lysines are widely distributed along the polypeptide; in the case of Hal3, the links are mostly restricted to the N-terminal half and the first third of the conserved PD domain (the part that shares similarity with known PPCDC enzymes). This is interesting because previous work reported that the N-terminal extension of Hal3 is, by itself, unable to interact in vitro with Ppz1 (neither with the full-length protein nor with its catalytic domain), but it is relevant for the inhibitory function [22]. This suggests that initial interactions could involve residues located within the PD region that would be reinforced by further interactions with the N-terminal section of Hal3 (which is predicted to be highly disordered). In this regard, it is worth noting that K315, 316 and 323 within this region present consistent interactions with diverse regions of Ppz1 (Figure 6C). This scenario would agree with the notion that the N-terminal extension of Hal3 contributes to its function as a regulator of Ppz1. Mol. Sci. 2022, Int.23, x FOR J. Mol. PEER Sci. 2022,REVIEW 23, 1327 9 of 19 9 of 18 Figure Figure 6. Chemical 6. Chemical cross-link between cross-link between the catalytic domainthe of catalytic Ppz1 anddomain Hal3. of(A)Ppz1 The and Hal3.do- catalytic (A) The catalytic main of Ppz1 tagged domain with a 6xHis of Ppz1 sequence tagged with a at the sequence 6xHis N-terminus (6xHisPpz1 at the N-terminus Cter) (6xHisPpz1 Cter ) was co-expressed was co-expressed with full-lengthwith Hal3.full-length Upon metal-affinity Hal3. Uponpurification and metal-affinity change ofand purification the change buffer by filtration, of the buffer sam- by filtration, samples were electrophoresed and stained with Coomassie blue. (B) Samples of the Ppz1 Hal3 ples were electrophoresed and stained with Coomassie blue. (B) Samples of the Ppz1 Cter plusCter plus Hal3 mixture mixture were incubated with BS3 were incubated with(10× BS3excess) as described (10× excess) and electrophoresed as described and electrophoresed in 6% polyacryla- in 6% polyacrylamide gels mide gels prior staining. Arrows indicate the position of the monomeric proteins and the asterisk prior staining. Arrows indicate the position of the monomeric proteins and the asterisk (*) the high (*) the high molecular mass complex excised for MS determination. “Stck” denotes the position of molecular mass complex excised for MS determination. “Stck” denotes the position of the stacking gel the stacking gel (not removed prior imaging). (C) Bidimensional map showing the relative positions of cross-linked K(not in removed prior Ppz1Cter and imaging). Hal3. (C) K Relevant Bidimensional map showing residues discussed the are in the text relative positionsinof cross-linked K highlighted in Ppz1 Cter color. (D) Mapping of K584andandHal3. K589 Relevant K residues in a surface model ofdiscussed in the text Ppz1. Residues thatare highlighted differ betweenin color. (D) Mapping Ppz1 of K584 and Glc7 are in red, andand K589residues catalytic in a surface are model of Ppz1. in yellow. PurpleResidues residues that differ between correspond to aminoPpz1 and Glc7 are in red, acids previously identified as relevant and catalytic for Ppz1 residues inhibition are in [39]. See yellow. Purple main text residues for discussion. correspond to amino acids previously identified as relevant for Ppz1 inhibition [39]. See main text for discussion. Roughly 40% of the lysines identified by MS correspond to Hal3–Hal3 links, which is not surprising if we Table consider 2. Relevant the residues lysine ability of thiscatalytic in the proteindomain to trimerize in that of ScPpz1 vitro [22,46].with cross-link A Hal3 residues. number of cross-linked lysine residues corresponding to interprotein Ppz1–Hal3 cross- links were observed (Table 2), andPpz1 Figure 6C displays these as a bidimensionalHal3 map. It is worth noting that whereas in the356/359 case of Ppz1Cter the 16 cross-linked reactive lysines 39 are widely distributed along the polypeptide; 358 in the case of Hal3, the links are 29,mostly 41, 315 re- stricted to the N-terminal half and378 the first third of the conserved PD domain 113, 197,(the 315,part 316 381 210, 483 that shares similarity with known PPCDC enzymes). This is interesting because previous 396 236 work reported that the N-terminal extension 433 of Hal3 is, by itself, unable to interact 210in vitro with Ppz1 (neither with the full-length 453 protein nor with its catalytic domain), but it is 280 relevant for the inhibitory function468 [22]. This suggests that initial interactions 201 could in- volve residues located within the496/502 PD region that would be reinforced by further 315 interac- 584 tions with the N-terminal section of Hal3 (which is predicted to be highly disordered). 90, 113, 323 In 589 90 this regard, it is worth noting that K315, 316 and 323 within this region present consistent 664 113, 210, 483 interactions with diverse regions of Ppz1 (Figure 6C). This scenario would agree with the Note: Based on the MS/MS fragmentation spectra it has not been possible to differentiate between K356/K359 notion that theandN-terminal extension of Hal3 contributes to its function as a regulator of between K496/K502. Ppz1. Int. J. Mol. Sci. 2022, 23, 1327 10 of 18 Although Hal3 does contain an “RVxF”-like motif (263 KLHVLF268 ), it is known that such a motif is not relevant for interaction with Ppz1 [47]. Although it has been described that inhibition of Ppz1 by Hal3 could happen by occlusion of the catalytic site, similar to the way inhibitor-2 inhibits PP1c, the structural features defining the interaction between Hal3 and Ppz1 are, in fact, still elusive. Our data show that Ppz1 K584 cross-links with Hal3 K90, a conserved residue in the N-terminal extension of Saccharomycetaceae (Figure 6C). Note that when K584 is mapped in our Ppz1 model, it lies very close to D566 and D615 (Figure 6D). These two residues align with Glc7 K210 and K259 and, therefore, impose a drastic change in the charge of this specific region surface. Such major changes differentiate ScPpz1 from Glc7, and they are strongly conserved in all fungi examined. It is known that Hal3 does not inhibit [30] and barely interacts with Glc7 (our own data). In contrast, the Hal3 protein from S. cerevisiae binds and acts as a potent inhibitor of all Ppz phosphatases (even from very distant fungi) so far examined [14,15,46,48], suggesting that all Ppz phos- phatases retain key elements for interaction with ScHal3. These observations point to ScPpz1 D566 and D615 residues as possible structural elements involved in the interaction with Hal3, likely by establishing electrostatic interactions. Ppz1 K589 is relatively close to K584 and shows some interactions with Hal3 K90 too. Such interactions could also be of interest, as they lie near a cluster of three residues that appear systematically different in PP1 and PPZs (Table S2). While the first change is rather conservative (Ppz1 V520 vs. Glc7 I164), the two next residues are negative charged in PP1 (usually D in position 165 and E in position 166) but they are always neutral in PPZs (A521 and G522 in Ppz1). 2.5. Mutagenesis Analysis Reveals a Key Role of Conserved D615 in Ppz1 Regulation and Function To test the functional role of D566 and D615 in Ppz1, we changed these amino acids to K, the residues always present at the equivalent positions in PP1c. The modified phos- phatases were expressed in S. cerevisiae from its own promoter in a centrometric plasmid to carry out in vivo functional studies. As shown in Figure 7A, immunoblot analysis of cell extracts showed that all versions of Ppz1 were expressed in yeast at similar levels. It has been known for many years that the mutation of PPZ1 increases tolerance to lithium cations [37]. As shown in Figure 7B (left panel), reintroduction of the native phosphatase normalizes lithium tolerance. In comparison with the native phosphatase, cells expressing the D566K variant were slightly more lithium tolerant, whereas those expressing the D615K version showed a slight decrease in tolerance. The strain expressing the double mutation displayed an intermediate phenotype. This could be compatible with the D566K mutation negatively affecting Ppz1 function and the D615K change having a positive effect, perhaps due to deregulation of the enzyme. To further test this possibility, we introduced native Ppz1 and its variants in Slt2-deficient cells (strain JC10). Cells lacking the Slt2 MAP kinase are very sensitive to caffeine, and this sensitivity is attenuated by a moderate increase in Ppz1 activity [9,39], as well as by the deletion of Hal3 [20]. As shown in Figure 7B (right panel), the expression of native Ppz1 improved the growth of slt2∆ cells in the presence of 2 mM caffeine. An effect of similar potency was observed for the D566K and D566K D615K versions. In contrast, cells expressing the D615K variant displayed a more vigorous growth, approaching that of slt2∆ hal3∆ cells (strain CCV186). Thus, our results suggest that the D615K mutation renders Ppz1 abnormally active, whereas that of D566 to K may have the opposite effect. erate increase in Ppz1 activity [9,39], as well as by the deletion of Hal3 [20]. As shown in Figure 7B (right panel), the expression of native Ppz1 improved the growth of slt2Δ cells in the presence of 2 mM caffeine. An effect of similar potency was observed for the D566K and D566K D615K versions. In contrast, cells expressing the D615K variant displayed a more vigorous growth, approaching that of slt2Δ hal3Δ cells (strain CCV186). Thus, our Int. J. Mol. Sci. 2022, 23, 1327 results suggest that the D615K mutation renders Ppz1 abnormally active, whereas that 11 of of18 D566 to K may have the opposite effect. Figure Figure 7. 7.Functional Functional characterization characterization of of thethe D566K D566K andandD615K D615K mutations mutations in in Ppz1. Ppz1.(A)(A) Forty Fortymi-mi- crograms of protein extracts from the wild-type strain BY4741 and ppz1Δ cells crograms of protein extracts from the wild-type strain BY4741 and ppz1∆ cells bearing the different bearing the different variants variants ofof Ppz1 Ppz1 (Ø, (Ø,empty empty plasmid) plasmid) were were subjected subjected toto SDS-PAGE SDS-PAGE (10% (10% polyacrylamide polyacrylamide gels) and gels) and were transferred to membranes. The Ppz1 versions were detected with a were transferred to membranes. The Ppz1 versions were detected with a polyclonal anti-GST-Ppz1 polyclonal anti-GST-Ppz1 antibody. (B) Left panel. Wild-type and ppz1Δ cells were transformed with the diverse versions of antibody. (B) Left panel. Wild-type and ppz1∆ cells were transformed with the diverse versions of Ppz1 and cultures spotted (OD660 = 0.05 and at 1/5 serial dilution) on synthetic media (lacking uracil) Ppz1 and cultures spotted (OD = 0.05 and at 1/5 serial dilution) on synthetic media (lacking uracil) plates with or without 100 mM 660 LiCl. Pictures were taken after six days. Ø, empty plasmid. Right plates panel. with or Strains without JA100 (wild100 mM type, LiCl. open Pictures bar), were taken JC10 (slt2Δ, after and grey bars) six days. CCV186 Ø, empty plasmid. (slt2Δ hal3Δ, Right striped panel. bars) were Strains JA100 (wild transformed withtype, open bar), the indicated JC10 (slt2∆, plasmids grey bars) and growth and CCV186 in liquid in the hal3∆, culture (slt2∆ presencestriped of bars)caffeine 2 mM were transformed (initial OD600with the indicated = 0.004) as in [33]. plasmids Growthand wasgrowth in liquid monitored everyculture 30 minininthe presence of a BioScreen C 2apparatus mM caffeine (Thermo (initial Fisher OD600 = Sci., Waltham, 0.004) MA, as in [33]. USA)was Growth andmonitored a representative every 30result min inafter 24 h is C a BioScreen shown. apparatusData (Thermo are meanFisher ± SEMSci., from three independent Waltham, MA, USA)clones determined at least and a representative resultby duplicate. after *, p < 24 h is shown. 0.01; **, p < 0.001; ***, p < 0.0001 (C) Interaction of the different versions of Data are mean ± SEM from three independent clones determined at least by duplicate. *, p < 0.01; Ppz1 with Hal3. Equal amounts of the***, **, p < 0.001; indicated p < 0.0001versions of GST-tagged (C) Interaction phosphatases of the different were versions immobilized of Ppz1 with Hal3. onEqual glutathione amounts beads and incubated with protein extracts of strain IM021 (ppz1Δ hal3Δ) expressing a HA-tagged of the indicated versions of GST-tagged phosphatases were immobilized on glutathione beads and version of Hal3. Beads were washed and processed for SDS-PAGE (8% polyacrylamide gels) and incubated with protein extracts of strain IM021 (ppz1∆ hal3∆) expressing a HA-tagged version of immunoblotting using anti-HA antibodies as described in Materials and Methods. Ponceau staining Hal3.the shows Beads wereofwashed amount GST-Ppz1 andin processed the assay.for(D)SDS-PAGE (8% versions The different polyacrylamide of Ppz1 gels) and immunoblot- (○, native; ■, D566K; •, ting D615K; using▲,anti-HA antibodies D566K D615K) wereasincubated describedwith in Materials increasing and Methods. amounts Ponceau staining of bacterially expressed shows Hal3. the amount of GST-Ppz1 in the assay. (D) The different versions of Ppz1 (#, native; , D566K; •, D615K; N, D566K D615K) were incubated with increasing amounts of bacterially expressed Hal3. Data are represented as the percentage of phosphatase activity in the absence of inhibitors and correspond to the mean ± SEM from at least three independent determinations using two different preparations of each protein. To obtain further insight into the effect of the mutations introduced in Ppz1, the three variants plus the native enzyme were expressed and purified from E. coli as GST-fusion proteins by mean of glutathione beads. The immobilized phosphatases were then used as an affinity system to pull down HA-tagged Hal3 from yeast extracts prepared from strain IM021, and the material retained by the beads analyzed by SDS-PAGE and immunoblot against the HA epitope. As can be observed in Figure 7C, none of the mutations affected the ability of Ppz1 to interact with Hal3, indicating that the mutated residues were either Int. J. Mol. Sci. 2022, 23, 1327 12 of 18 not relevant for the interaction or that the change was insufficient to block the interaction between the regulatory subunit and the phosphatase. We then removed the GST moiety from the Ppz1 versions by protease treatment and used the enzyme preparations for in vitro testing of its capacity to be inhibited by Hal3. As shown in Figure 7D, the D566K version of Ppz1 was inhibited by Hal3 as potently as the native form of the phosphatase. In contrast, the D615K version was largely refractory to the presence of the inhibitor. Notably, the double mutant displayed an intermediate behavior. This result indicates that D615 could be a key structural element mediating Hal3 inhibition and would agree with the increase in function deduced from the phenotypic results (Figure 7B). In contrast, the D566K version, which is inhibited in vitro by Hal3 as much as the native phosphatase, showed some decrease in function in vivo. We determined the specific activity of the different variants expressed in E. coli and found that, compared to the native recombinant Ppz1, the D566K version systematically appeared slightly less active (81.5% ± 8.2, n = 3), whereas the D615K variant was indistinguishable from the native enzyme (99.3 ± 1.3, n = 3). Therefore, a lesser catalytic activity of the D566K version may explain the in vivo results. The intermediate behavior in vivo of the double mutated version may be explained by a compensatory effect of both mutations. The observation that the D615K mutation abolishes the ability of Hal3 to inhibit Ppz1 suggests that electrostatic interactions may play a role in the inhibitory mechanism. The scenario would fit with the early proposal that the in vivo interaction between Ppz1 and Hal3 is dependent on the intracellular pH [49]. It must be noted that previous work [39] revealed two acidic residues (E575 and E630) whose mutation to G resulted in a marked decrease in the ability of Hal3 to inhibit Ppz1 without significant change in the interaction. This could imply that the interaction between Ppz1 and Hal3 involves diverse structural determinants that are redundant, so elimination of one of them has little or no effect in the interaction. These residues are also near the catalytic site (in purple in Figure 6D). However, in contrast to E615, E575 and E630 are conserved not only in Glc7 (D219 and E274, respectively), but also in the vast majority of fungal species analyzed in this work (see Figure S1). All these results indicate that the conserved D615 in Ppz1 is an important structural determinant that contributes to the specificity of the regulation of Ppz and PP1c enzymes in fungi. 3. Conclusions We analyzed the sequences of PP1 and the catalytic domains of PPZ proteins of multiple species representative of all three main fungi clades. Our data indicate that Ppz1 proteins are more diverse than PP1 proteins, and this is much more evident if we exclude the Sds21 family of PP1. Characteristic residues of each type of phosphatases mainly mapped on the surface of the modeled proteins and, in some cases, involved a change in the net charge. This fact could explain, at least in part, the specificity of some regulatory subunits for binding to PP1 or PPZ. Our data show, for example, that several of the motifs required for the binding of PP1 to its regulatory subunits are not present or only partially conserved in PPZ1 proteins. We also propose specific structural features, such as an extra C-terminal α-helix, to be differential landmarks between Ppz and PP1c enzymes. Finally, comparative analysis of dozens of PPZ and fungal PP1 proteins allowed the identification of a strongly conserved (and differential) residue (D615 in ScPpz1) that appears crucial for inhibition by Hal3. 4. Materials and Methods 4.1. Yeast Strains Yeast cells were incubated in YP medium (1% yeast extract, 2% peptone) or in synthetic medium (SC) lacking uracil [50], supplemented with glucose at 2% as a carbon source, at 28 ◦ C. Plates contained 2% agar. The ppz1∆ mutant is a kanMX deletant derived from strain BY4741 (MATa his3∆1 leu2∆ met15∆ ura3∆) [51]. Strains JC010 (slt2∆::LEU2) and CCV186 (slt2∆::LEU2 hal3∆::KanMX) are JA100 (MATa ura3-52 leu2-3,112 trp1-1 his4 can-1r) deriva- Int. J. Mol. Sci. 2022, 23, 1327 13 of 18 tives and are reported in Refs. [11] and [48], respectively. Strain IM021 (ppz1∆::KanMx4 hal3∆::LEU2) is reported in [47]. 4.2. Plasmid Construction Escherichia coli DH5α cells, employed as plasmid DNA host, were grown at 37 ◦ C in LB medium supplemented with 50 µg/mL ampicillin when carrying plasmids. Transforma- tions of S. cerevisiae and E. coli, and standard recombinant DNA techniques were performed as described [52]. Mutation of Ppz1 D566 to K was done as follows. Two partially overlap- ping fragments were PCR amplified from vector pRS316-Ppz1 [39] and oligonucleotides Ppz1_50 _seq_cat/3_Ppz1_D566K (fragment 1), and M13rev/5Ppz1_D566K (fragment 2). Both fragments were mixed and subjected to a second round of amplification with primers Ppz1_50 _seq_cat/M13rev. The amplification fragment was digested with PacI and HindIII and used to replace the equivalent fragment in pRS316-Ppz1. The mutation of D615 to K was done in the same way but using primers 5_Ppz1_D615K and 3_Ppz1_D615K. The double mutant was constructed by introducing the D615K change in the pRS316-Ppz1D566K construct. The mutated versions were introduced in pGEX6P-1 for expression in E. coli by digestion of the pRS-based construct with Kpn2I and Bsp1407I and replacement of the same region in pGEX6P-1-Ppz1 [47]. Plasmid pGEX6P-Hal3 was used to express Hal3 fused to GST in E. coli [30]. The sequence of oligonucleotides mentioned in this work can be found in Table S1. 4.3. Bioinformatics Analyses Using the whole-proteome-based fungi phylogeny as a reference [35], we selected 57 species representative of the Ascomycota (35), Basidiomycota (15) and Monokaryotic fungi (7) and identified their Ppz and PP1 proteins. For this purpose, we used the Ppz1 and Glc7 protein sequences from S. cerevisiae to perform BLAST [53] searches in the selected species at the NCBI server (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 April 2021). Top hits were submitted to BLAST search against the S. cerevisiae proteome at the SGD webpage [54] (https://www.yeastgenome.org/blast-sgd, accessed on 20 April 2021) and classified as PP1 or Ppz1 accordingly. Because the conserved catalytic domain of Ppz proteins does not start in the first residues as it does in PP1 proteins, we removed the non-catalytic N-terminal residues of Ppz for further sequence alignment and comparison with PP1. To this end, Ppz proteins were scanned for the localization of the SM000156 Smart domain [55], which begins at residue 385 in S. cerevisiae Ppz1. However, it was clear that the similarity between PP1 and Ppz extends further toward the N-terminus. Therefore, we found that the protein phosphatase domain PTHR11668, predicted by the Panther evolutionary classification tool [56], which initiates at residue 359 in ScPpz1, was more suitable to our purposes. Unless otherwise stated, the numbering of residues relates to ScGlc7 and ScPpz1 sequences, respectively. The Clustal Omega tool available at the EMBL-EBI server [57] was used to create the sequence alignments. Shading of residues according to their conservation across species was done with pyBoxshade, a desktop updated version of the Boxshade WEB interface (https://embnet.vital-it.ch/software/BOX_form.html, downloaded 3 May 2021). Proteomes were obtained from Uniprot (https://www.uniprot.org/, accessed on 20 May 2021), and the number of Ser and Thr for each species was obtained with a script developed in Visual Basic. Theoretical pI were calculated using the ProtParam tool available at the Expasy server [58]. Models of S. cerevisiae Glc7 and Ppz1 were generated at the Swiss Model Server (https://swissmodel.expasy.org/, accessed on 23 December 2020) [59]. The numbering corresponds to the full-length proteins. Sequence logos were generated with the Seq2Logo generator (https://services.healthtech.dtu.dk/, accessed on 10 June 2021), as described in [60]. The Shannon logo type was selected; clustering method was set to none, and “Weight on prior” was set to 0. The coloring scheme was the default, except that Phe was denoted in yellow. Int. J. Mol. Sci. 2022, 23, 1327 14 of 18 4.4. Chemical Cross-Linking of the Ppz1 Catalytic Domain and Hal3 Co-purification of the Ppz1-Cter/Hal3 complex was accomplished upon co-expression of both proteins in E. coli BL21 DE3 RIL cells transformed with a pET-Duet1-based plasmid bearing the C-terminal half (∆1–344) of Ppz1 (N-terminally fused to a 6xHis tag) and the entire Hal3, as described in [23]. Bacterial cultures were grown in a shaker at 37 ◦ C to an OD600 of 0.6–0.8. Recombinant protein expression was initiated by addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) at 0.1 mM and continued for 16 h at 21 ◦ C. Cultures were centrifuged at 6500× g for 20 min at 4 ◦ C, and pellets were resuspended in lysis buffer (50 mM NaH2 PO4 pH 7.5, 150 mM NaCl, 20 mM Imidazole, 0.1% Triton X-100) containing Complete EDTA-free protease inhibitor cocktail (Roche, Sant Cugat del Vallès, Spain) plus 2 mM PMSF (approximately 3 mL of buffer per gram of pellet). Samples were placed on ice and sonicated using a Vibra- cell (Sonics and Materials Inc.) equipment for 5–6 min (10 s on, 10 s off) at 30% amplitude. Samples were then centrifuged at 4 ◦ C (12,400× g for 30 min) to remove cellular debris. The soluble fraction was incubated with Ni-NTA Agarose beads (Qiagen® , Hilden, Germany) following the manufacturer’s specifications and loaded in a 10 mL column. The mixture was washed with about 5 bed volumes of lysis buffer (without Triton X-100) until the eluate was free of proteins (monitored by the Bradford’s reagent). The 6x-His tagged Ppz1-Cter/Hal3 complex was recovered by incubating the beads for 10 min with 1.5 bed volume of elution buffer (washing buffer containing 150 mM imidazole). Finally, imidazole was eliminated by exchanging the elution buffer with cross-linking buffer (50 mM NaH2 P04 pH 7.5, 150 mM NaCl) using an Amicon® Ultra Centrifugal filter (size 30 kDa, Millipore, Burlington, MA, USA). The amounts of the recovered recombinant proteins were determined by SDS-PAGE followed by Coomassie-blue staining and scanning of the gels with an Epson Perfection V500 Photo Scanner. Quantification was done with the Gel Analyzer software (version 2010a) and different amounts of bovine serum albumin (BSA) as standards. The purified Ppz1C-ter/Hal3 protein complex was adjusted to 0.2–1 µg/µL with cross-linking buffer. Forty micrograms of the complex was used in each experiment. The proteins were incubated for 1 h at room temperature with 5, 10 and 20 weight excesses of BS3 (bis(sulfosuccinimidyl)suberate) (Covachem LLC, Loves Park, IL, USA). Cross linking was then quenched by addition of NH4 CO3 at 100 mM. Then, samples were analyzed by SDS-PAGE (6% polyacrylamide gels) and stained. The relevant bands were excised from the gel into individual low-binding polypropylene tubes. In-gel tryptic digestion was performed essentially as described [61]. Each sample was desalted using a Rainin pipette tip packed with Poros R2 reversed phase material (Applied Biosystems, Foster City, CA, USA). 4.5. LC-MS/MS Analysis of Cross-Linked Proteins The digested peptides were dissolved in 0.1% TFA, and approximately 1µg peptides were analyzed using an EASY-nanoLC 1000 system (Thermo Scientific, Dreieich, Germany) coupled to a Q-Exactive HF mass spectrometer. The peptides were loaded onto a 2.5 cm in-house packed Reprosil-Pur 120 C18-AQ (5 µm: Dr. Maisch GmbH, Germany) precolumn with an internal diameter of 100 µm and eluted directly onto a 19 cm in-house packed Reprosil-Pur C18-AQ column (3 µm; Dr. Maisch GmbH, Ammerbuch, Germany) with an internal diameter of 75 µm. A 104 min HPLC gradient with a flow rate of 250 nL/min was used, with an increasing concentration of Solvent B (95% ACN, 0.1% FA) in the following increments: 1–3% for 3 min, 3–25% for 80 min, 25–45% for 10 min, 45–100% for 3 min and 100% for the final 8 min. Solvent A was 0.1% FA. The mass spectrometer was operated in data-dependent acquisition mode, and each analysis started with an MS1 scan detected in the Orbitrap (resolution: 120.000, scan range: 300–1600 m/z, AGC target: 1e6, maximum injection time: 100 ms, RF lens: 30%). For each MS1 spectrum, the 12 most intense peaks were selected for MS2. Monoisotopic peak determination was set to peptide, and dynamic exclusion was switched on (exclude after 1 time, exclusion duration: 7 s, mass tolerance: ±10ppm, exclude isotopes). The selected Int. J. Mol. Sci. 2022, 23, 1327 15 of 18 MS2 precursors were isolated in the quadrupole at 1.2 Th (m/z) and fragmented using HCD at a collision energy of 32%. MS2 fragments were detected in the Orbitrap (resolution: 30.000, first mass: 110 m/z, AGC target: 1e4). Raw data files were converted to mgf format by Rawconverter 1.2.0.1 (Scripps Research Institute, La Jolla, CA, USA). The data were further filtered using an “MGF filter” (www. massai.dk, Stenstrup, Denmark) using the following settings: Only scans with charge states between +3 and +9 were retained to maximize the number of scans containing highly charged cross-links. Only scans containing signature peaks for tryptic digests (y1 ions) and at least one signature peak from the cross-linker itself were kept. The sequence of ScPpz1 (P26570) and Hal3 (P36024) were acquired from the UniProt database (www.UniProt.org, accessed on 1 April 2021). The sequences were used for examining BS3 crosslinks by the MassAI search engine using the parameters: 10 ppm MS accuracy, 0.10 Da MS/MS accuracy, trypsin for the enzyme, four allowed missed cleavages, carbamidomethylation of cysteine as fixed modification and oxidation of methionine, BS3-water and BS3-ammonia dead-end of lysine residues and the protein N-terminus as variable modifications. Cross links were manually validated, and cross-link spectra were only approved if b- and/or y-ions from both involved peptides were present in the spectrum. Representative MS/MS scans are shown in Figure S3. 4.6. Preparation of Yeast Extracts and Immunoblotting Protein extracts for Ppz1 detection were prepared from 10 mL of cultures and subjected to SDS-PAGE and immunoblot as described [62,63]. Ppz1 was detected with polyclonal anti-Ppz1 antibodies (1:250 dilution) followed by secondary anti-rabbit IgG-horseradish peroxidase antibodies (GE Healthcare, 1:20,000 dilution) and the ECL Prime Western blotting detection kit. For detection of HA-tagged Hal3, yeast extracts containing HA- tagged Hal3 were prepared from strain IM021 (ppz1∆ hal3∆) as described [15], except that 50 mL cultures were grown and 500 µL of lysis buffer was used for cell disruption. After cell lysis, 125 µL of the lysis buffer was added to increase the extraction volume. Immunodetection of HA-Hal3 was done as in [15], except that the anti-HA antibody was from Biolegend. 4.7. Hal3 Pull down Experiments GST-Ppz1, Ppz1D566K , Ppz1D615K , and Ppz1D566K, D615K were expressed in BL21 (DE3) RIL E. coli cells and purified as reported [22] with the modifications described in [14]. Quantification of the expressed phosphatase versions was done as in [15]. The interac- tion between the Ppz phosphatases and Hal3 was determined by in vitro binding assays performed as previously described [14,22] with minor modifications. Thus, aliquots of glu- tathione agarose beads containing 8 µg of the different Ppz1 versions were incubated with protein extracts (0.6 mg of proteins) prepared from strain IM021 (ppz1∆ hal3∆). The beads were finally resuspended in 120 µL of 2x_sample buffer and, after boiling for 5 min, 40 µL of each sample was analyzed by SDS-PAGE and HA-Hal3 immunodetection performed as described above. 4.8. Ppz1 Inhibition Assays For Ppz1 inhibition assays, all recombinant proteins were expressed in E. coli BL21(DE3) RIL cells as described in [39,62], except that Terrific Broth was used instead of LB medium. Removal of the GST moiety from recombinant Ppz1 versions and Hal3 was done essentially as described in [62]. Phosphatase activity was determined using p-nitrophenylphosphate as substrate and five picomoles of the different phosphatases. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms23031327/s1. Author Contributions: Conceptualization, A.C., P.H. and J.A.; validation, A.C., P.H. and J.A.; formal analysis, A.C., P.H. and J.A.; investigation, A.C., M.A., C.S., D.V., M.I.R., P.H. and J.A.; data curation, Int. J. Mol. Sci. 2022, 23, 1327 16 of 18 A.C., M.I.R., P.H. and J.A.; writing—original draft preparation, A.C. and J.A.; writing—review and editing, A.C., P.H. and J.A.; supervision, A.C., P.H. and J.A.; funding acquisition, A.C., P.H. and J.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Ministerio de Economía, Industria y Competitividad, Spain, grant number BFU2017-82574-P, to JA and AC. MA, CS and DV were recipients of PIF fellowships from the Universitat Autònoma de Barcelona, Spain. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data supporting the reported results are presented in this manuscript and its supplemental material. Acknowledgments: The technical assistance of Montserrat Robledo is acknowledged. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Offley, S.R.; Schmidt, M.C. Protein phosphatases of Saccharomyces cerevisiae. Curr. Genet. 2018, 65, 41–55. [CrossRef] [PubMed] 2. Ariño, J.; Velázquez, D.; Casamayor, A. Ser/thr protein phosphatases in fungi: Structure, regulation and function. Microb. Cell 2019, 6, 217–256. [CrossRef] [PubMed] 3. Cannon, J.F. 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