Báo cáo Y học: Novel protein phosphatases in yeast

Eur. J. Biochem. 269, 1072–1077 (2002) (cid:211) FEBS 2002

M I N I R E V I E W

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

fungi, in others similar proteins can be found in higher eukaryotes. This review will summarize the latest advances in our understanding about how these phosphatases are regu- lated and fulfil their functions in the yeast cell.

Keywords: Ser/Thr protein phosphatases; cell cycle; cell integrity; cation tolerance; protein synthesis; yeast.

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 their functional charac- terization initiated. Most of these novel phosphatases display intriguing structural features and/or are involved in a number of important functions, such as cell cycle regulation, protein synthesis and maintenance of cellular integrity. While in some cases these genes appear to be restricted to

I N T R O D U C T I O N

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 PPP family that, while in most cases related to the type 1 and 2A proteins in their primary structure, are functionally different from those well characterized phosphatases (Table 1, Fig. 1). Recent insights into the function and regulation of these atypical phosphatases have unveiled most interesting aspects of the yeast biology. Because the limitation of space we will focus mainly 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].

P P Z 1 / P P Z 2

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, and 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].

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 (cid:212)eccentric(cid:213). 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 and molecular genetics. Its complete genomic sequence has been available since 1996 (it was the first eukaryotic genome fully sequenced) and this knowledge has been an important reference for research in other model systems. Although a relatively simple organism, 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 into two groups: PPP, that includes the homologs of the type 1,

Deletion of PPZ2 in an otherwise wild-type background does not result in a readily detectable phenotype. Strains lacking Ppz1 are prone to cell lysis under certain circum- stances, such as the presence of low concentrations of caffeine and this effect, as for other phenotypes described is aggravated by deletion of PPZ2 [10]. Ppz1 below, 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 Veterina` ria, Universitat Auto` noma de Barcelona, Bellaterra 08193, Barcelona, 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)

Novel protein phosphatases in yeast (Eur. J. Biochem. 269) 1073

(cid:211) FEBS 2002

Table 1. Novel yeast Ser/Thr protein phosphatases. SN refers to the systematic gene name after the yeast genomic sequencing program. Size is expressed in number of amino acids.

Subfamily SN Gene name Size Function

PP1

PPZ1 PPZ2 PPQ1/SAL6 YML016c YDR436w YPL179w 692 710 549 Regulates salt tolerance and cell cycle Regulates salt tolerance Involved in protein synthesis PP2A

(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].

PPH3 SIT4/PPH1 PPG1 PPT1 YDR075w YDL047w YNR032w YGR123c 308 311 368 513 Supply some PP2A-like function but also has specific roles Regulates G1/S cell cycle transition Modulates glycogen synthesis Unknown, similar in sequence to mammalian PP5

the phosphatase activity resulted in a phenotype additive to that of the mpk1D 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).

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 that is suppressed by high-copy expression of HAL3 [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 yields 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 some 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 subset of Glc7 regulatory subunits.

Strains lacking Ppz1 display a strong phenotype of 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 manner that is 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 allowed the establishment of a link between this phosphatase and the HAL3/SIS2 gene product

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 G1/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, lack of PPZ1 suppressed the synthetic lethality of the sit4 hal3 and sit4 cln3 mutations. Therefore,

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.

1074 J. Arin˜ o (Eur. J. Biochem. 269)

(cid:211) FEBS 2002

Ppz1 appears as a novel regulatory component of the yeast cell cycle, acting in an opposite way to Sit4.

and Gcn4 [25] and subsequently found to be necessary for proper progression for the G1/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 G1 for progression into S phase and for expression of the CLN1 and CLN2 cyclins, as well as of the SWI4 transcription 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]. Overexpression of human PP6 or Drosophila PPV complements the cell growth defect of a sit4 mutant, suggesting that they are functional homologs [27,29].

Sit4 associates with several proteins, known collectively as SAP (for sit4-associated proteins), such as Sap155, Sap185, Sap190 and perhaps Sap4, 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 associate 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.

The Ppz phosphatases are also involved in regulation of protein translation. Two dimensional electrophoretic ana- lysis of 32P-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 exchanging 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 and 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 the double mutant ssb1 ssb2 is viable, it grows slowly at high NaCl concentrations and exhibits hypersensitivity to paromomycin, just the opposite behaviour to that displayed by ppz mutants.

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 required for regulation of a subset of PP2A functions [35,36], supporting the proposal that Sit4 is not the only target for these activators [34]. In any case, they must play a pivotal role in controlling progression through the G1 phase of the yeast cell cycle as shown by the fact that deletion of both genes results in a lethal phenotype [34,36,37].

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 identity search in databanks from other organ- isms. Deletion of S. pombe pzh1+ results in cells hypertol- erant to 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 these cations, as 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 sodium 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 to fulfil most of the Ppz1 functions [24].

T H E S I T 4 / P P H 1 P H O S P H A T A S E

As mentioned above, Sit4 and Ppz1 phosphatases play opposite roles in regulating the G1/S transition. As a result, a sit4 hal3 mutant (which lacks sit4 and presents an hyperac- tivated Ppz1) is arrested at G1 and cannot grow [18,38]. This situation has been exploited to find further components involved in cell cycle progression by constructing 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 results), 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, such as PTC1 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 antiporter in 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 antiporter activity and suggest a novel function for this protein [38].

The gene SIT4 was initially cloned in an screening for restoration of HIS4 expression in strains lacking Bas1, Bas2

Novel protein phosphatases in yeast (Eur. J. Biochem. 269) 1075

(cid:211) FEBS 2002

The functional connection between Sit4 and Ppz1 can also be extended to the Pkc1/Mpk1 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., unpublished results). This effect appears to be independent of the role of Msg5 and 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).

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 when comparing with PP2A or PP1 catalytic subunits. Deletion of the PPG1 gene yields viable 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 for other phosphatases known to regulate glycogen metabolism, such as PP1 or PP2A) but its amount was significantly reduced 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 that Ppg1 may interact with another Ser/Thr phosphatase, Ppt1 (see below). The biological significance of this finding is unknown.

P P Q 1 / S A L 6

SIT4 has been also cloned and characterized in the budding yeast Kluyveromyces lactis [40]. The protein is highly similar to that of S. cerevisiae (93% identity) and it has a broad role in modulating multidrug resistance, both positively and negatively. Recent evidence suggests that the Pdr5 multidrug transporter can be a target for the phosphatase [41].

P P H 3

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, although they display reduced growth rate in different media, exhibit mild defects in protein synthesis and 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 translational 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].

P P T 1

The PPH3 gene encodes a protein related to type 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 pph22ts mutants at 37 (cid:176)C [43] and it has been described for Pph3 a number of catalytic features distinct from those of PP2A, PPX or PP1 enzymes [44]. In addition, unlike sit4 cla4 mutants, which are nonviable, pph3 cla4 mutants grow even at 37 (cid:176)C, suggesting that 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 that regulates nitrogen catabolite repression through the GATA-type transcription factor Gln3 [45]. TOR kinase activity is required for phosphorylation of Gln3, thus inhibiting nuclear translocation of the tran- scription factor and maintaining repression of Gln3- dependent genes. Expression of one of such genes, GAP1, is strongly reduced after rapamycin treatment in cells lacking Pph3, suggesting that this phosphatase might be directly or indirectly involved in dephosphorylation and activation of Gln3. It must be noted that, although 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].

P P G 1

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 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 features of the PPP family [52]. Proteins similar 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 protein–protein interaction structural element, often found in multiple copies in a number of functionally different proteins that facilitates specific interactions with 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 chaperones, transcrip- tion, cell-cycle and protein transport complexes (reviewed in [56]). Mammalian PP5 has been implicated in several signal transduction pathways, cell cycle regulation at G1 and M phases and, perhaps, regulation of potassium channels (reviewed in [57]). In contrast, our knowledge on yeast Ppt1

1076 J. Arin˜ o (Eur. J. Biochem. 269)

(cid:211) FEBS 2002

10. Posas, F., Casamayor, A. & Arin˜ o, J. (1993) The PPZ protein phosphatases are involved in the maintenance of osmotic stability of yeast cells. FEBS Lett. 318, 282–286.

has experienced little increase in the last few years. Deletion of PPT1 results in no obvious change in phenotype. A recent genomic two-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.

11. Heinisch, J.J., Lorberg, A., Schmitz, H.P. & Jacoby, J.J. (1999) The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae. Mol. Microbiol. 32, 671–680.

12. De Nadal, E., Clotet, J., Posas, F., Serrano, R., Gomez, N. & Arin˜ o, J. (1998) The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase. Proc. Natl Acad. Sci. USA 95, 7357–7362.

C O N C L U D I N G R E M A R K S

13. Posas, F., Camps, M. & Arin˜ o, J. (1995) The PPZ protein phos- phatases are important determinants of salt tolerance in yeast cells. J. Biol. Chem. 270, 13036–13041.

14. Mendoza, I., Rubio, F., Rodrı´ guez-Navarro, A. & Pardo, J.M. (1994) The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae. J. Biol. Chem. 269, 8792– 8796.

From the evidence outlined above, it is clear that these (cid:212)novel(cid:213) phosphatases play relevant roles in the biology of the yeast (cell integrity, cell cycle 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 increased 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 genome-wide research (genomic microarrays, large-scale two hybrid analysis, proteomics.) will give a powerful boost to the research in this field and provide new insight into the regulation and function of these remarkable proteins.

15. Erez, O. & Kahana, C. (2001) Screening for modulators of sper- mine tolerance identifies Sky1, the SR protein kinase of Saccharo- myces cerevisiae, as a regulator of polyamine transport and ion homeostasis. Mol. Cell. Biol. 21, 175–184.

16. Ferrando, A., Kron, S.J., Rios, G., Fink, G.R. & Serrano, R. (1995) Regulation of cation transport in Saccharomyces cerevisiae by the salt tolerance gene Hal3. Mol. Cell. Biol. 15, 5470–5481. 17. Venturi, G.M., Bloecher, A., Williams-Hart, T. & Tatchell, K. (2000) Genetic interactions between GLC7, PPZ1 and PPZ2 Saccharomyces cerevisiae. Genetics 155, 69–83.

A C K N O W L E D G E M E N T S

18. Di Como, C.J., Bose, R. & Arndt, K.T. (1995) Overexpression of SIS2, which contains an extremely acidic region, increases the expression of SWI4, CLN1 and CLN2 sit4 mutants. Genetics 139, 95–107.

Thanks are given to all colleagues that shared unpublished observa- tions. Research in the author’s laboratory has been supported by grants PB98-0565-C04-02 (MCYT, Spain) and 1999-SGR00100 (CIRIT, Generalitat de Catalunya). 19. Clotet, J., Garı´ , E., Aldea, M. & Arin˜ o, J. (1999) The yeast Ser/Thr phosphatases Sit4 and Ppz1 play opposite roles in regulation of the cell cycle. Mol. Cell. Biol. 19, 2408–2415.

R E F E R E N C E S

20. De Nadal, E., Fadden, R.P., Ruiz, A., Haystead, T. & Arin˜ o, J. (2001) A role for the Ppz Ser/Thr protein phosphatases in the regulation of translation elongation factor 1Ba. J. Biol. Chem. 276, 14829–14834.

1. Cohen, P.T. (1997) Novel protein serine/threonine phosphatases: variety is the spice of life. Trends Biochem. Sci. 22, 245–251. 2. Stark, M.J., Black, S., Sneddon, A.A. & Andrews, P.D. (1994) Genetic analyses of yeast protein serine/threonine phosphatases. FEMS Microbiol. Lett. 117, 121–130. 3. Stark, M.J. (1996) Yeast protein serine/threonine phosphatases: 21. Pfund, C., Lopez-Hoyo, N., Ziegelhoffer, T., Schilke, B.A., Lo´ pez- Buesa, P., Walter, W.A., Wiedmann, M. & Craig, E.A. (1998) The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome-nascent chain complex. EMBO J. 17, 3981–3989. multiple roles and diverse regulation. Yeast 12, 1647–1675.

22. Espinosa-Ruiz,A.,Belle´ s,J.M.,Serrano,R.&Culia´ nez-Macia´ ,F.A. (1999) Arabidopsis thaliana AtHAL3: a flavoprotein related to salt and osmotic tolerance and plant growth. Plant J. 20, 529–539. 23. Balcells, L., Calero, F., Go´ mez, N., Ramos, J. & Arin˜ o, J. (1999) The Schizosaccharomyces pombe Pzh1 protein phosphatase regu- lates sodium influx in a Trk1-independent fashion. Eur. J. Bio- chem. 260, 31–37. 4. Posas, F., Casamayor, A., Morral, N. & Arin˜ o, J. (1992) Mole- cular cloning and analysis of a yeast protein phosphatase with an unusual amino-terminal region. J. Biol. Chem. 267, 11734–11740. 5. Lee, K.S., Hines, L.K. & Levin, D.E. (1993) A pair of functionally redundant yeast genes (PPZ1 and PPZ2) encoding type 1-related protein phosphatases function within the PKC1-mediated path- way. Mol. Cell. Biol. 13, 5843–5853.

24. Vissi,E.,Clotet,J.,deNadal,E.,Barcelo,A.,Bako,E.E.,Gergely,P., Dombradi, V. & Arin˜ o, J. (2001) Functional analysis of the Neurospora crassa PZL-1 protein phosphatase by expression in budding and fission yeast. Yeast 18, 115–124. 6. Hughes, V., Muller, A., Stark, M.J. & Cohen, P.T. (1993) Both isoforms of protein phosphatase Z are essential for the main- tenance of cell size and integrity in Saccharomyces cerevisiae response to osmotic stress. Eur. J. Biochem. 216, 269–279.

25. Arndt, K.T., Styles, C.A. & Fink, G.R. (1989) A suppressor of a HIS4 transcriptional defect encodes a protein with homology to the catalytic subunit of protein phosphatases. Cell 56, 527–537. 7. Balcells, L., Gomez, N., Casamayor, A., Clotet, J. & Arin˜ o, J. (1997) Regulation of salt tolerance in fission yeast by a protein- phosphatase-Z-like Ser/Thr protein phosphatase. Eur. J. Biochem. 250, 476–483.

26. Sutton, A., Immanuel, D. & Arndt, K.T. (1991) The SIT4 protein phosphatase functions in late G1 for progression into S phase. Mol. Cell. Biol. 11, 2133–2148.

8. Szoor, B., Feher, Z., Zeke, T., Gergely, P., Yatzkan, E., Yarden, O. & Dombradi, V. (1998) pzl-1 encodes a novel protein phosphatase- Z-like Ser/Thr protein phosphatase in Neurospora crassa. Biochim. Biophys. Acta. 1388, 260–266.

27. Mann, D.J., Dombradi, V. & Cohen, P.T. (1993) Drosophila protein phosphatase V functionally complements a SIT4 mutant in Saccharomyces cerevisiae and its amino-terminal region can confer this complementation to a heterologous phosphatase cata- lytic domain. EMBO J. 12, 4833–4842. 9. Clotet, J., Posas, F., de Nadal, E. & Arin˜ o, J. (1996) The NH2- terminal extension of protein phosphatase PPZ1 has an essential functional role. J. Biol. Chem. 271, 26349–26355.

Novel protein phosphatases in yeast (Eur. J. Biochem. 269) 1077

(cid:211) FEBS 2002

42. Ronne, H., Carlberg, M., Hu, G.Z. & Nehlin, J.O. (1991) Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis. Mol. Cell. Biol. 11, 4876–4884. 28. Ferna´ ndez-Sarabia, M.J., Sutton, A., Zhong, T. & Arndt, K.T. (1992) SIT4 protein phosphatase is required for the normal accumulation of SWI4, CLN1, CLN2, and HCS26 RNAs during late G1. Genes Dev. 6, 2417–2428.

43. Evans, D.R. & Stark, M.J. (1997) Mutations in the Saccharomyces cerevisiae type 2A protein phosphatase catalytic subunit reveal roles in cell wall integrity, actin cytoskeleton organization and mitosis. Genetics 145, 227–241. 29. Bastians, H. & Ponstingl, H. (1996) The novel human protein serine/threonine phosphatase 6 is a functional homologue of budding yeast Sit4p and fission yeast ppe1, which are involved in cell cycle regulation. J. Cell Sci. 109, 2865–2874.

44. Hoffmann, R., Jung, S., Ehrmann, M. & Hofer, H.W. (1994) The Saccharomyces cerevisiae gene PPH3 encodes a protein phospha- tase with properties different from PPX, PP1 and PP2A. Yeast 10, 567–578. 30. Luke, M.M., Della Seta, F., Di Como, C.J., Sugimoto, H., Kobayashi, R. & Arndt, K.T. (1996) The SAP, a new family of proteins, associate and function positively with the SIT4 phos- phatase. Mol. Cell. Biol. 16, 2744–2755.

31. Di Como, C.J. & Arndt, K.T. (1996) Nutrients, via the Tor pro- teins, stimulate the association of Tap42 with type 2A phospha- tases. Genes Dev. 10, 1904–1916.

32. Jian, Y. & Broach, J.R. (1999) Tor proteins and protein phos- phatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J. 18, 2782–2792.

33. Beck, T. & Hall, M.N. (1999) The TOR signalling pathway con- trols nuclear localization of nutrient-regulated transcription fac- tors. Nature 402, 689–692. 45. Bertram,P.G.,Choi,J.H.,Carvalho,J.,Ai,W.,Zeng,C.,Chan,T.F. & Zheng, X.F. (2000) Tripartite regulation of Gln3p by TOR, Ure2p, and phosphatases. J. Biol. Chem. 275, 35727–35733. 46. Cardenas, M.E., Cutler, N.S., Lorenz, M.C., Di Como, C.J. & Heitman, J. (1999) The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev. 13, 3271–3279. 47. Posas, F., Clotet, J., Muns, M.T., Corominas, J., Casamayor, A. & Arin˜ o, J. (1993) The gene PPG encodes a novel yeast protein phosphatase involved in glycogen accumulation. J. Biol. Chem. 268, 1349–1354.

34. Mitchell, D.A. & Sprague, G.F. Jr (2001) The phosphotyrosyl phosphatase activator, Ncs1p (Rrd1p), functions with Cla4p To regulate the G(2)/M transition in Saccharomyces cerevisiae. Mol. Cell. Biol. 21, 488–500. 48. Uetz, P., Giot, L., Cagney, G., Mansfield, T.A., Judson, R.S., Knight, J.R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P. et al. (2000) A comprehensive analysis of protein– protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627.

49. Chen, M.X., Chen, Y.H. & Cohen, P.T. (1993) PPQ, a novel protein phosphatase containing a Ser + Asn-rich amino-terminal domain, is involved in the regulation of protein synthesis. Eur. J. Biochem. 218, 689–699. 35. Van Hoof, C., Janssens, V., De Baere, I., de Winde, J.H., Winderickx, J., Dumortier, F., Thevelein, J.M., Merlevede, W. & Goris, J. (2000) The Saccharomyces cerevisiae homologue YPA1 of the mammalian phosphotyrosyl phosphatase activator of pro- tein phosphatase 2A controls progression through the G1 phase of the yeast cell cycle. J. Mol. Biol. 302, 103–120.

50. Vincent, A., Newnam, G. & Liebman, S.W. (1994) The yeast translational allosuppressor, SAL6: a new member of the PP1-like phosphatase family with a long serine-rich N-terminal extension. Genetics 138, 597–608.

36. Van Hoof, C., Janssens, V., De Baere, I., Stark, M.J., de Winde, J.H., Winderickx, J., Thevelein, J.M., Merlevede, W. & Goris, J. (2001) The Saccharomyces cerevisiae phosphotyrosyl phosphatase activator proteins are required for a subset of the functions disrupted by protein phosphatase 2A mutations. Exp. Cell Res. 264, 372–387.

51. Benko, A.L., Vaduva, G., Martin, N.C. & Hopper, A.K. (2000) Competition between a sterol biosynthetic enzyme and tRNA modification in addition to changes in the protein synthesis machinery causes altered nonsense suppression. Proc. Natl Acad. Sci. USA 97, 61–66.

37. Rempola, B., Kaniak, A., Migdalski, A., Rytka, J., Slonimski, P.P. & di Rago, J.P. (2000) Functional analysis of RRD1 (YIL153w) and RRD2 (YPL152w), which encode two putative activators of the phosphotyrosyl phosphatase activity of PP2A in Saccharomyces cerevisiae. Mol. General Genet. 262, 1081– 1092.

52. Chen, M.X., McPartlin, A.E., Brown, L., Chen, Y.H., Barker, H.M. & Cohen, P.T. (1994) A novel human protein serine/thre- onine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus. EMBO J. 13, 4278–4290. 53. Yatzkan, E. & Yarden, O. (1997) ppt-1, a Neurospora crassa PPT/ PP5 subfamily serine/threonine protein phosphatase. Biochim. Biophys. Acta. 1353, 18–22. 38. Simo´ n, E., Clotet, J., Calero, F., Ramos, J. & Arin˜ o, J. (2001) A screening for high-copy suppressors of the sit4 hal3 synthetically lethal phenotype reveals a role for the yeast Nha1 antiporter in cell cycle regulation. J. Biol. Chem. 276, 29740–29747.

54. Becker, W., Kentrup, H., Klumpp, S., Schultz, J.E. & Joost, H.G. (1994) Molecular cloning of a protein serine/threonine phospha- tase containing a putative regulatory tetratricopeptide repeat domain. J. Biol. Chem. 269, 22586–22592. 39. Masuda, C.A., Ramı´ rez, J., Pen˜ a, A. & Montero-Lomeli, M. (2000) Regulation of monovalent ion homeostasis and pH by the Ser-Thr protein phosphatase SIT4 in Saccharomyces cerevisiae. J. Biol. Chem. 275, 30957–30961.

55. Becker, W., Buttini, M., Limonta, S., Boddeke, H. & Joost, H.G. (1996) Distribution of the mRNA for protein phosphatase T in rat brain. Mol. Brain Res. 36, 23–28.

40. Chen, X.J., Bauer, B.E., Kuchler, K. & Clark-Walker, G.D. (2000) Positive and negative control of multidrug resistance by the Sit4 protein phosphatase in Kluyveromyces lactis. J. Biol. Chem. 275, 14865–14872. 56. Blatch, G.L. & Lassle, M. (1999) The tetratricopeptide repeat: a structural motif mediating protein–protein interactions. Bioessays 21, 932–939. 57. Chinkers, M. (2001) Protein phosphatase 5 in signal transduction. 41. Chen, X.J. (2001) Activity of the Kluyveromyces lactis Pdr5 multidrug transporter is modulated by the Sit4 protein phospha- tase. J. Bacteriol. 183, 3939–3948. Trends Endocrinol. Metab. 12, 28–32.