M I N I R E V I E WNovel 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 Barcel
Trang 1M 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
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
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
I N T R O D U C T I O N
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 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
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,
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]
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 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 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)
Trang 2the 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)
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
ENA1gene, 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
(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/ mpk1background 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 PPZ2display 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
HAL3is 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 sit4mutant, 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,
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.
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.
Trang 3Ppz1 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 of32P-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
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
The gene SIT4 was initially cloned in an screening for
restoration of HIS4 expression in strains lacking Bas1, Bas2
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 SSD1locus and results either in unviable cells (absence of SSD1or presence of ssd1-d alleles) or viable cells that show
a slow growth phenotype [26] Sit4 is required in late G1for 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
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 G1phase 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, Sit4 and Ppz1 phosphatases play opposite roles in regulating the G1/S transition As a result, a sit4 hal3mutant (which lacks sit4 and presents an hyperac-tivated Ppz1) is arrested at G1and 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]
Trang 4The 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)
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 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
pph22tsmutants at 37°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 cla4mutants, which are nonviable, pph3 cla4 mutants
grow even at 37°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 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
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 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 G1and M phases and, perhaps, regulation of potassium channels (reviewed in [57]) In contrast, our knowledge on yeast Ppt1
Trang 5has 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
C O N C L U D I N G R E M A R K S
From the evidence outlined above, it is clear that these
ÔnovelÕ 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
A C K N O W L E D G E M E N T S
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).
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