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MINIREVIEW Novel protein phosphatases in yeast An update Joaquı ´ n Arin ˜ o Department de Bioquı ´ mica i Biologia Molecular, Facultat de Veterina ` ria, Universitat Auto ` noma de Barcelona, Bellaterra Spain During the last decade several novel yeast genes encoding proteins related to the PPP family of Ser/Thr protein phos- phatases have been discovered and th eir functional charac- terization initiated. Most of these novel phosphatases display intriguing structural features and/or are involved in a number of impo rtant functions, such as cell cycle regulation, protein synthesis and maintenance of cellular integrity. While in some cases these genes a ppear to be restricted to fungi, in others similar proteins can be found in higher eukaryotes. This r eview will summarize the l atest a dvances in our understanding about how these phosphatases are regu- lated and fulfil their func tions in the yeast cell. Keywords: Ser/Thr protein phosphatases; cell cycle; cell integrity; cation tolerance; protein synthesis; yeast. INTRODUCTION In a splendid cartoon that appeared in Trends in Biochemical Sciences a few years ago [1], it was pointed out that, by comparison to the relatively uniform protein kinases, protein phosphatases could be considered to be ÔeccentricÕ. This review will deal with the most eccentric members of this family of eccentric proteins, and will focus on yeast cells. In addition to their relevance in biotechnological processes, yeasts, and in particular the budding yeast Saccharomyces cerevisiae, represent a very important model for basic research in biology. The biochemistry of this eukaryotic organism is well known, and S. cerevisiae allows very powerful and relatively simple experimental approaches based on both classical a nd molecu lar genetics. Its complete genomic sequence has been available since 1996 (it was the first eukaryotic genome fully sequ enced) and th is knowledge has been an important reference for research in other model systems. Although a relatively simple o rganism, S. cerevisiae contains at least 30 different proteins with verified or likely protein phosphatase catalytic activity, as well as a large number (still growing) of regulatory components that, in many cases, have very close structural and functional counterparts in plants and animals. These protein phos- phatase activities include Ser/Thr phosphatases, Tyr phos- phatases and members of the dual phosphatase subfamily, able to dephosphorylate both Ser/Thr and Tyr residues. Ser/Thr proteins phosphatases are commonly classified i nto two groups: PPP, that includes the homologs of the type 1, 2A and 2B phosphatases identified by biochemical approa- ches in the early 1980s, and PPM, consisting of the type 2C phosphatases. This review will focus on those members of the P PP family that, w hile in most c ases related to the type 1 and 2A proteins in their primary structure, are functionally different from those well characterized phosphatases (Table 1, Fig. 1). R ecent i n sights i nto t he function and regulation o f the se a typical phosphatases have unveiled most interesting aspects of the yeast biology. Because the limitation of s pace we will focus m ainly in the latest findings in this field and direct the reader for additional background information to several excellent reviews that have appeared within the last few years [1–3]. PPZ1/PPZ2 This group is composed by two S. cerevisiae genes, PPZ1 and PPZ2 [4–6], and similar genes identified in Schizosac- charomyces pombe and Neurospra crassa [7,8]. These proteins display C-terminal catalytic domains of about 300 residues that are 75–90% identical each other, a nd all of them share about 60% identity with the catalytic subunit of protein phosphatase-1 (Fig. 1). Their N-terminal moieties are much less closely related, although common structural features are retained within the first 40 amino acids, including a conserved Gly2 that, in the case of Ppz1, has been shown to be myristoylated in vivo [9]. Deletion of PPZ2 in an otherwise wild-type background does not result in a readily d etectable phenotype. Strains lacking Ppz1 are prone to cell lysis under certain circum- stances, such as the presence of low c oncentrations of caffeine and this effect, as for other phenotypes described below, is aggravated by deletion of PPZ2 [10]. Ppz1 functionally interacts with the protein kinase C-activated mitogen activated protein (MAP) kinase pathway, involved in maintenance of cellular integrity, that results in the activation of the Slt2/Mpk1 MAP kinase (reviewed in [11]), as suggested by the observations that overexpression of Ppz1 or Ppz2 suppresses the lytic phenotype of a mpk1 mutant, whereas deletion of the PPZ1 gene or inhibition of Correspondence to J. Arin ˜ o, Department de Bioquı ´ mica i Biologia Molecular, Facultat de Vete rina ` ria, Universitat Auto ` noma de Barcelona, Bellaterra 08193, Ba rcelona, Spain. E-mail: Joaquin.Arino@uab.es Abbreviations: MAP kinase, mitogen activated protein kinase; TOR, target of rapamycin; PP1, type 1 protein phosphatase; PP2A, type 2A protein phosphatase; TPR, tetratricopeptide repeats. (Received 6 August 2001, revised 3 October 2001, accepted 5 October 2001) Eur. J. Biochem. 269, 1072–1077 (2002) Ó FEBS 2002 the phosphatase activity resulted in a phenotype additive to that of t he mpk 1D mutant [ 5,12]. The molecular basis of this functional interaction is unknown, but recent evidence indicates that both Ppz1 and Sit4 phosphatases can modulate, in an opposite fashion, the phosphorylation state and activity of the Slt2/Mpk1 kinase (see below). Strains lacking Ppz1 display a strong phenotype o f hypertolerance to sodium or lithium cations, which is enhanced by additional deletion of PPZ2 [13]. This is, at least in part, the result of an increase in the expression of the ENA1 gene, encoding a P-type Na + -ATPase which repre- sents the major determinant for sodium tolerance in budding yeast. The effect on ENA1 expression is independ- ent and opposite to the effect described for the Ser/Thr phosphatase calcineurin, a positive effector of the ATPase gene [13,14]. However, an Ena1-independent role of Ppz1 in salt tolerance cannot be discarded, because it has been recently reported that overexpression of the Sky1 protein kinase increases sensitivity to LiCl in a m anner that i s dependent on the function of PPZ1 but not of ENA1 [15]. Very recent genetic and biochemical evidence points to the Trk1 potassium transporter system as a target of Ppz action (L. Yenush, J. Arin ˜ o & R. Serrano, unpublished results). The identification of Ppz1 as an important element in sodium tolerance a llowed t he establishment of a link between t his phosphatase and t he HAL3/SIS 2 gene product (see also below), because HAL3 was identified as a gene able, when overexpressed, to increase tolerance to sodium cations in a calcineurin-independent manner [16]. Genetic and biochemical evidence supports the proposal that Hal3 acts as a negative regulatory subunit of Ppz1 and inhibits the activity of the phosphatase by binding to its C-terminal catalytic moiety [12]. A remarkable feature of Hal3 is that this protein appears to regulate most of (if not all) Ppz1 functions [12]. For instance, high-copy number expression of HAL3 in a slt2/ mpk1 background mimics the effect of deletion of PPZ1, indicating that Hal3 regulates the function(s) of Ppz1 related to the cell integrity pathway. Furthermore, it is known that overexpression of Ppz1 leads to slow growth [9], a pheno- type thatis suppressedby h igh-copy expressionof HAL 3 [12]. This situation appears rather different from the one described for the GLC7 gene, encoding the single catalytic subunit of type 1 protein phosphatase (PP1) in S. cerevisiae. In this case, deletion of GLC7 results in lethality whereas the absence of regulatory components y ields less dramatic phenotypes, suggesting that the diverse cellular roles attrib- uted to Glc7 are the result of specific interactions of the catalytic subunit with different regulatory subunits. It must be noted, however, that Glc7 and Ppz phosphatases might share s ome common features. For instance, PPZ1 and PPZ2 display genetic interactions with GLC7, as deduced from the different growth defects observed in cells carrying mutant alleles of GLC7 in combination with null alleles of the PPZ phosphatases [17]. Furthermore, interactions between Ppz1 and several known or putative regulatory subunits of Glc7 (including Glc8, encoding a protein with similarity to the mammalian inhibitor-2) have been docu- mented by two-hybrid analysis [17]. These evidence suggests that Glc7 and Ppz functions might overlap up to some extent, and that Ppz1 shares a s ubset of Glc7 regulatory subunits. HAL3 is an allele of SIS2, a gene found as a multicopy suppressor of the growth defect of sit4 mutants [18]. The link between Hal3/Sis2 and Ppz1, together with the observation that overexpression of PPZ1 resulted in slow growth due to slow passage through G 1 /S [9] suggests a functional connection between Ppz1 and Sit4 in the regulation of cell cycle [19]. It was demonstrated that deletion of PPZ1 partially rescued the growth defect of a sit4 mutant, thus mimicking the effect of overexpression of HAL3.Furthermore,lackofPPZ1 suppressed the s ynthetic lethality of the sit4 hal3 and sit4 cln3 mutations. Therefore, Table 1. Novel yeast Ser/Thr protein phosphatases. SN refers to the systematic gene name after the yeast gen omic sequencing pro gram. Size is expressed in number of amino acids. Subfamily SN Gene name Size Function PP1 YML016c PPZ1 692 Regulates salt tolerance and cell cycle YDR436w PPZ2 710 Regulates salt tolerance YPL179w PPQ1/SAL6 549 Involved in protein synthesis PP2A YDR075w PPH3 308 Supply some PP2A-like function but also has specific roles YDL047w SIT4/PPH1 311 Regulates G 1 /S cell cycle transition YNR032w PPG1 368 Modulates glycogen synthesis YGR123c PPT1 513 Unknown, similar in sequence to mammalian PP5 Fig. 1. Schematic depiction of the structure of yeast Ser/Thr protein phosphatases described in this review. Black boxes refers to the con- served catalytic domain common to the PPP family of Ser/Thr phos- phatases. The asterisks denote the conserved motif for N-terminal myristoylation. Ó FEBS 2002 Novel protein phosphatases in yeast (Eur. J. Biochem. 269) 1073 Ppz1 appears as a novel regulatory component of the yeast cell cycle, acting in an opposite way to Sit4. The Ppz phosphatases are also involved in regulation of protein translation. Two dimensional electrophoretic ana- lysis of 32 P-labeled yeast cells revealed a prominent phos- phoprotein that was shifted to more acidic regions in cells lacking ppz1 ppz2 or in wild-type cells overexpressing HAL3 [20]. This protein was identified as the translation elongation factor 1Ba (Tef5), the GTP–GDP exchan ging factor for translation elongation factor 1. Tef5 appeared to be phosphorylated in vivo at the conserved Ser86 and lack of the Ppz phosphatases increased phosphorylation at this site. Although it is not clear whether the translation factor is a direct substrate for the phosphatase, it is evident that regulation of the phosphorylation state of Tef5 by Ppz results in modification of translation accuracy, as deduced from the observation that ppz mutants display increased tolerance to the drug paromomycine and increased read through at nonsense codons [20]. The link between the Ppz phosphatases a nd protein synthesis is reinforced by the observation that affinity purified Ppz1 preparations contain bound Ssb1 and/or Ssb2 (E. De Nadal, T. Haystead & J. Arin ˜ o, unpublished observations). These proteins are members of the HSP70 family [21] involved in the passage of nascent chain through ribosome. Interestingly, although t he double mutant ssb1 ssb2 is viable, it grows slowly at high NaCl concentrations and exhibits hypersensitivity to paromomycin, just t he opposite behaviour to that d isplayed by ppz mutants. As mentioned above, the fission yeast S. pombe and the filamentous fungi N. crassa encode Ppz-like phosphatases [7,8]. Comparison of yeast Ppz sequences with those deposited in data banks suggest that this type of enzyme might be restricted to fungi. This is somewhat surprising, because homologs of HAL3 have been found in both plants and animals [22]. Given the high sequence divergence at the N-terminal half even between different fungi Ppz enzymes, it might not be easy to identify an equivalent gene product by sequence i dentity s earch in databanks from other organ- isms. Deletion of S. pombe pzh1 + results in cells hypertol- erant t o Na + but hypersensitive to potassium ions [7], pointing out that Pzh1 was involved in cation homeostasis. However, a number of studies indicate that the mechanisms of action of Pzh1 in S. pombe is different from that observed for Ppz1 in budding yeast. For instance, cells lacking pzh1 do not show altered sodium or lithium efflux, but they display decreased influx for t hese cations, a s well as reduced K + efflux [23]. Furthermore, Pzh1 was unable to restore wild-type tolerance to sodium cations in a S. cerevisiae ppz1 strain [24]. In contrast, expression of the N. crassa PZL-1 phosphatase from the PPZ1 promoter in S. cerevisiae ppz1 cells restored wild-type sensitivity to caffeine and s odium ions. Furthermore, overexpression of PZL-1 induced growth arrest in wild-type budding yeast and alleviated the lytic phenotype of a slt2/mpk1 MAP kinase mutant, suggesting that despite the marked divergence within their N-terminal sequences, PZL-1 appears t o fulfil most of the Ppz1 functions [24]. THE SIT4/PPH1 PHOSPHATASE The gene SIT4 was i nitially cloned in a n screening for restoration of HIS4 expression in strains lacking Bas1, B as2 and Gcn4 [25] and subsequently found to be necessary for proper progression for the G 1 /S cell cycle transition [26,27]. The phenotype of sit4 cells depends on the polymorphic SSD1 locus and results either in unviable cells (absence of SSD1 or presence of ssd1-d alleles) or viable cells that show a slow growth phenotype [26]. Sit4 is required in late G 1 for progression into S phase and for expression of the CLN1 and CLN2 cyclins, as well as of the SWI4 transcrip tion factor, in a pathway additive to that of CLN3. In addition, bud emergence is also compromised in sit4 mutants, and this defect appears to be independent of cyclin expression [28]. O verexpression of human PP6 or Drosophila PP V complements the cell growth defect of a sit4 mutant, suggesting that they are functional homologs [27,29]. Sit4 associates w ith several proteins, known collectively as SAP (for sit4-associated proteins), such as Sap155, Sap185, Sap190 and p erhaps S ap4, in a cell cycle-dependent fashion. Loss of all four SAP is equivalent to the loss of Sit4. All of them function positively with Sit4, although they asso ciate with the phosphatase in separate complexes and probably have distinct functions [30]. It is not clear whether the Sap proteins are modulator of Sit4 activity or substrates of the phosphatase. Sit4 also associates, in a SAP-independent fashion to Tap42, an essential protein [31] that is phosphorylated by the target of rapamycin (TOR) kinases [32]. Therefore, Sit4 appears involved in a pathway that links nutrient sensing and cell growth [32,33] and that also involves the type 2A protein phosphatase (PP2A) catalytic subunits Pph21 and Pph22. Recent evidence suggests that Sit4 also interacts physically and is regulated by the phosphotyrosyl phospha- tase activators (PTPA) Ncs1/Rrd1 and Noh1/Rrd2 [34]. These proteins are also requ ired for regulation of a subset of PP2A functions [35,36], supporting the proposal that Sit4 is not the only target for these activators [34]. I n any case, t hey must play a pivotal role in controlling progression through the G 1 phase of the y east cell cycle as shown by t he fact that deletion of both genes results in a lethal phenotype [34,36,37]. As mentioned above, Sit4 and Ppz1 phosphatases play opposite roles in regulating the G 1 /S transition. As a r esult, a sit4 hal3 mutant (which lacks sit4 and presents an hyperac- tivated Ppz1) is a rrested at G 1 and c annot grow [18,38]. This situation has been exploited to find further components involved in cell cycle progression by c onstructing a condi- tional sit4 hal3 mutant [38] and searching for high-copy suppressors (designated as VHS for viable sit4 hal3). Both Pph21 and Pph22 phosphatases have been found among the suppressor genes (Mun ˜ oz I., Simo ´ n, E., Arin ˜ o, J. and Herrero, E., unpublished r esults), supporting the notion that these PP2A phosphatases and Sit4 can share partially overlapping function(s) [26]. Interestingly, a type 2C phos- phatase, PTC2, can also support growth of the conditional sit4 hal3 mutant. Other members of the yeast type 2C family,suchasPTC1 and PTC3 also behave as VHS clones, although with somewhat less potency. This screening has also uncover a role for the Na + ,K + /H + Nha1 a ntiporter i n cell cycle, because high-copy expression of NHA1 allows growth of a sit4 hal3 mutant. Despite the observation that, under certain circumstances, Sit4 can influence monovalent cation homeostasis and pH [39], the effect of Nha1 is most probably independent of its a ntiporter a ctivity and suggest a novel function for this protein [38]. 1074 J. Arin ˜ o(Eur. J. Biochem. 269) Ó FEBS 2002 The functional connection between Sit4 and Ppz1 can also be extended to the Pkc1/M pk1 pathway. Mpk1 is phosphorylated and activated in response to several signals related to cell integrity, such as cell wall stress (reviewed in [11]). sit4 mutants display increased Mpk1 phosphorylation under basal conditions, as well as under situations that activate the pathway (De la Torre, M. A., Torres, J., Arin ˜ o, J. and Herrero, E., u npublished results). This effect appears to be independent of the role of Msg5 a nd Ptp2, two phosphatases known to dephosphorylate Mpk1. An increase in Mpk1 phosphorylation was also observed in cells overexpressing Ppz1, while mutation of this phospha- tase resulted in decreased Mpk1 phosphorylation. Evidence has been gathered that the function of Sit4 might be upstream of Pkc1, negatively regulating the activity of the kinase (De la Torre, M . A., Torres, J., Arin ˜ o, J. and Herrero, E., unpublished results). SIT4 has been also cloned and characterized in the budding yeast K luy verom yces l ac tis [40]. The protein is highly similar to that of S. cerevisiae (93% identity) and it has a bro ad role i n modulating multidrug resistance, both positively and negatively. Recent evidence suggests that the Pdr5 multidrug transporter can be a target for the phosphatase [41]. PPH3 The PPH3 gene encodes a protein related t o t ype 2A catalytic subunits (52% and 58% identity, respectively). Although null mutants are viable, PPH3 gene function is required for survival in the absence of PPH21 and PPH22 [42], suggesting that this protein has some biological activity overlapping with that of PP2A. However, Pph3 function(s) probably differs from that of PP2A, because its overproduction does not suppress the growth defect of pph22 ts mutants at 37 °C [43] and it has been described for Pph3 a number of c atalytic features distinct from th ose of PP2A, PPX or PP1 enzymes [44]. In addition, unlike sit4 cla4 mutants, which are nonviable, pph3 cla4 mutants grow even at 37 °C, suggesting t hat despite the relatively high level of sequence similarity, Pph3 and Sit4 are functionally unrelated. However, recent evidence have linked the Pph3 phosphatase with the TOR signalling pathway t hat regulates nitrogen catabolite r epression through t he G ATA-type transcription factor Gln3 [45]. TOR k inase activity is required f or phosphorylation of Gln3, thus inhibiting nuclear translocation of the t ran- scription factor and maintaining repression of Gln3- dependent genes. Expression of one of su ch genes, GAP1, is strongly reduced after rapamycin treatment in cells lacking Pph3, suggesting that this p hosphatase might be directly or indirectly involved in dephosphorylation and activation of Gln3. I t must be noted that, a lthough a Tap42–Sit4 complex has been claimed as crucial for the activation of Gln3 [33], controversial reports on this issue can be found in the literature [46]. PPG1 The PPG1 gene was cloned by virtue of its sequence similarity with other Ser/Thr phosphatases [47], although the encoded polypeptide shows distinctive features, such a C-terminal extension of about 50 residues directly following the conserved catalytic domain. This extension has no similarity with the C-terminal extension of type 2B catalytic subunits and ends with the highly conserved DYFL sequence found in type 2A phosphatases. In addition, an internal insertion of 10 residues (from amino acids 205–215) is found w hen comparing with PP2A or PP1 catalytic subunits. D eletion of the PPG1 gene yields v iable cells whose only known phenotype is a decrease in glycogen accumulation [47]. The state of activation of glycogen synthase was not modified by the absence of Ppg1 (in contrast to that reported fo r other phosphatases known to regulate glycogen metabolism, such as PP1 or PP2A) but its amount was significantly r educed in ppg1 cells, in agreement with the low glycogen levels. Interestingly, a recent large- scale yeast two -hybrid screen for protein–protein interac- tions [48] has revealed the possibility t hat Ppg1 m ay interact with another Ser/Thr phosphatase, Ppt1 (see below). The biological significance of this finding is unknown. PPQ1/SAL6 The PPQ1 gene encodes a type1-related phosphatase characterized by an N-terminal extension rich in Ser and Asn, that is not related in sequence to those found in Ppz1 or Ppz2 [49]. Null ppq1 mutants are viable, altho ugh they display reduced growth rate in different media, exhibit mild defects in protein synthesis a nd are sensitive to several protein synthesis inhibitors [49]. Multiple copies of SAL6 cause antisuppression of nonsense mutations [50,51] and lack of Sal6 acts as an allosuppressor in suppressor strain backgrounds (that is, enhances efficiency of translational suppressors). These findings suggest a role for Sal6p in the regulation of the fidelity of translation. It should be noted that this situation is somewhat different from the one described for the Ppz phosphatases, because ppz mutations result in changes in t ranslational accuracy in an otherwise wild-type background [20]. It has been reported that a ppq1 mutation do not show genetic interactions with ppz1 ppz2 or diverse glc7 mutations [17]. PPT1 The protein encoded by PPT1 contains two distinct domains: an N-terminal extension of almost 200 residues, characterized by four tetratricopeptides repeats (TPR), and a C-terminal domain that displays the typical f eatures of the PPP family [52]. Proteins s imilar to S. cerevisiae PPT1 have been found in S. pombe (clone SPBC3F6.01c) and N. crassa [53]. However, Ppt1 is equally distant from PP2A and PP1 enzymes and, in fact, its closest relative is PP5, an ubiquitous phosphatase in eukaryotic cells that also displays several TPR motifs [52–55]. The TPR motif is a p rotein–protein interaction structural element, often found in multiple c opies in a number of functionally different proteins that f acilitates specific interactions w ith partner protein(s). Most TPR- containing proteins are associated with multiprotein com- plexes; TPR motifs appear to be important for the function of different protein families, such as chapero nes, transcrip- tion, cell-c ycle and protein transport complexes (reviewed in [56]). Mammalian PP5 has been implicated in several signal transduction pathways, cell cycle regulation at G 1 and M phases and, perhaps, regulation of potassium channels (reviewed in [57]). In contrast, our knowledge on yeast Ppt1 Ó FEBS 2002 Novel protein phosphatases in yeast (Eur. J. Biochem. 269) 1075 has experienced little increase in the last few years. Deletion of PPT1 results in no obvious change in phenotype . A recent genomic t wo-hybrid screen reported interactions between Ppt1 and two other proteins: the Ppg1 phosphatase, described in this review, and Adr1, a zinc-finger transcrip- tion factor required for glycerol metabolism and peroxisome biogenesis. As in may other cases, the functional relevance of these interactions remains to be elucidated. CONCLUDING REMARKS From the evidence outlined above, it is clear that these ÔnovelÕ phosphatases play relevant roles in the biology of the yeast (cell integrity, cell c ycle regulation, cation homeostasis, etc) and that they interact with several key signalling pathways. In some cases, such as Ppz1/Ppz2 and Sit4, our knowledge on the regulation and function of these enzymes has inc reased substantially within the last few years, while in other cases, as Ppg1, many aspects still remain to be elucidated. We should expect in the next few years that the combination of classical approaches and g enome-wide research (genomic microarrays, large-scale two hybrid analysis, proteomics.) will give a powerful boost to t he research in this field and p rovide new i nsight into the regulation and function of these remarkable proteins. 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