From the Department of Molecular Genetics and the
Graduate Program in Genes and Development, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 and the ¶ Department
of Biochemistry and Molecular Biology, University of Miami, School of
Medicine, Miami, Florida 33136-1015
Received for publication, February 20, 2001
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ABSTRACT |
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The p21-activated kinase, Shk1, is
required for cell viability, establishment and maintenance of cell
polarity, and proper mating response in the fission yeast,
Schizosaccharomyces pombe. Previous genetic studies
suggested that a presumptive protein methyltransferase, Skb1, functions
as a positive modulator of Shk1. However, unlike Shk1, Skb1 is not
required for viability or mating of S. pombe cells and
contributes only modestly to the regulation of cell morphology under
normal growth conditions. Here we demonstrate that Skb1 plays a more
significant role in regulating cell growth and polarity under
conditions of hyperosmotic stress. We provide evidence that the
inability of skb1 Skb1/Hsl7-related proteins have been highly conserved through
evolution, with homologs identified in eukaryotes from yeast to human
(1-3). The fission yeast Skb1 protein was identified from a two-hybrid
screen for proteins that interact with the p21-activated kinase
(PAK)1 homolog, Shk1 (also
known as Pak1 and Orb2; Refs. 1, 4, 5). Shk1 is essential for cell
viability, establishment and maintenance of cell polarity, and normal
mating response in fission yeast (1, 2, 4-6). Genetic and molecular
data suggest that Shk1 is a critical effector for the Rho-type p21
GTPase Cdc42, which like Shk1 is required for viability, morphological
polarity, and normal mating of Schizosaccharomyces pombe
cells (4-6). Cdc42 and Shk1 interact functionally with Ras1, the
single known fission yeast homolog of the mammalian Ras p21 GTPase (1,
4, 5, 7-9). Ras1 is required for normal morphology and mating of
S. pombe cells, but unlike Cdc42 and Shk1, it is not
essential for cell viability (7, 8). Skb1 interacts with the
amino-terminal regulatory domain of Shk1, and co-overexpression of the
two proteins suppresses the morphological defect of ras1 The budding yeast homolog of the skb1 gene, HSL7,
was discovered from a screen for mutations that are synthetically
lethal in combination with a deletion of the amino terminus of histone H3 (3). The same screen resulted in the identification of mutations in
genes encoding budding yeast homologs of the fission yeast cyclin-dependent kinase Cdc2 (Cdc28) and the Wee1
inhibitory kinase Nim1 (Hsl1). Loss-of-function of Hsl7 results in a
delay in G2/M progression, suggesting that Hsl7, in
contrast to Skb1, functions as a mitotic inducer (3). The
G2/M delay of the hsl7 mutant can be suppressed
by a null mutation in the swe1 gene, suggesting that Hsl7 is
an inhibitor of Swe1 (3). This role for Hsl7 is supported by the
findings of McMillan et al. (10), who provided evidence that
Hsl7 acts in concert with Hsl1 to target Swe1 for degradation in
S. cerevisiae, and by those of Shulewitz et al. (11), who showed that phosphorylation and ubiquitinylation of Swe1,
modifications that target Swe1 for degradation, are substantially reduced in cells lacking Hsl7.
The cellular functions of metazoan Skb1/Hsl7-related proteins have yet
to be defined, however, a human Skb1/Hsl7 homolog, Skb1Hs (also known
as IBP72 (13) and JBP1 (14)), can substitute for Skb1 in fission yeast,
suggesting that Skb1 protein function has been substantially conserved
through evolution (2). Skb1Hs has been shown to associate with several
different proteins in mammalian cells, including the tyrosine kinase
JAK2 (14), the subtype 1 somatostatin receptor (15), and a protein of
unclarified function, pICln (13). The biological significance of the
interactions between Skb1Hs and these various proteins has not yet been established.
Skb1/Hsl7-related proteins lack significant structural homology to any
other characterized proteins. However, Pollack et al. (14)
noted that among the proteins with which Skb1Hs exhibits relatively
weak homology (E >10 In this report, we show that Skb1, which is largely dispensable for the
regulation of cell morphology under normal conditions, is a
hyperosmotic shock stimulated enzyme required for normal cell viability
and morphological polarity under conditions of hyperosmotic stress. We
also provide molecular evidence for evolutionary conservation of a role
for Skb1/Hsl7-related proteins as mediators of hyperosmotic stress
response, as well as Skb1/Hsl7 regulatory mechanisms in eukaryotic
organisms. Our results provide important new insights into the cellular
roles of this novel class of protein methyltransferases.
Yeast Strains, Manipulation, and Plasmids--
S.
pombe strains used in this study were SP870
(h90 ade6-M210 leu1-32 ura4-D18) (D. Beach), SKB1U (h90 ade6-M210 leu1-32
ura4-D18 skb1::ura4) (1), CHP428
(h+ ade6-M210 his7-366 leu1-32
ura4-D18) (from C. Hoffman), and SMMG100 (h+ ade6-M210 his7-366 leu1-32 ura4-D18
skb1::ura4) (1). S. pombe cultures
were grown in either rich medium (YEA) or in synthetic minimal medium
(EMM) with appropriate auxotrophic supplements (16). The plasmids
pAAUGST (2), pAAUGST-Skb1 (2), pART1CM (4), pART1CMSkb1 (2), and
pART1CMSkb1Hs (2) have been described. pREPUHA-Skb1 was constructed by
cloning a 2.4-kilobase BamHI-SacI fragment of the
Skb1 protein coding sequence isolated from pART1CMSkb1 into the
ura4-based plasmid, pREP4XHA (a gift from E. Chang). This
plasmid allows for expression of a triple HA epitope-tagged Skb1
protein from the nmt1 promoter (17).
F-actin Staining and Indirect Immunofluorescence
Microscopy--
F-actin was visualized using rhodamine-phalloidin
as described (16). Indirect immunofluorescence microscopy of HA-Skb1
was performed using mouse monoclonal anti-HA antibody 12CA5 (19) and
goat anti-mouse fluorescein isothiocyanate-conjugated secondary antibody (Pierce), essentially as described (18).
Protein Methylation Assays--
S. pombe cells
transformed with the plasmids pART1CM, pART1CMSkb1, or pART1CMSkb1Hs
were grown in 200 ml of EMM to about 107 cells/ml prior to
harvesting of cells by centrifugation. Cultures were diluted with an
equal volume of either EMM or EMM containing 3 M KCl and
grown for 15-60 min prior to harvesting. Cell lysates were prepared as
described (6). Immunoprecipitations were performed by incubating
extract volumes containing 2 mg of protein with 5 µl of anti-c-Myc
monoclonal antibody 9E10 ascites (20) and 25 µl of protein A-agarose
beads (Roche Molecular Biochemicals) for 2 h at 4 °C. Immune
complexes were pelleted by centrifugation and washed three times with 1 ml of yeast lysis buffer and then twice with 1 ml of methylation assay
buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA). Samples were divided into 2 equal volumes, one of
which was pelleted and resuspended in 40 µl of methylation buffer and
the other pelleted and resuspended in SDS-PAGE sample buffer. GST-Skb1
was expressed in the E. coli host strain BL21 from the
plasmid pRP259 (a gift from E. Chang), a derivative of pGEX-1 (Amersham
Pharmacia Biotech). GST-Skb1 was purified from bacterial cell lysates
using glutathione-agarose beads following the manufacturer's
recommendations (Amersham Pharmacia Biotech). Beads containing GST-Skb1
were resuspended in methylation buffer. Methylation assays were
performed essentially as described (14). To initiate the methylation
reaction, 5 µl of [3H]adenosyl methionine (specific
activity, 78 Ci/mmol) (PerkinElmer Life Sciences) and 5 µl of myelin
basic protein solution (12 mg/ml in methylation buffer) were added to
each 40-µl volume of immune complex or GST-Skb1 beads in methylation
buffer and incubated 30 min at 30 °C. Reactions were terminated by
placing on ice, adding SDS-PAGE sample buffer, and boiling for 5 min.
Reactions were resolved by SDS-PAGE and exposed to x-ray film for 3-6
days. The remaining portion of each immune complex was boiled for 5 min
and then subjected to SDS-PAGE and subsequent Western blot analysis to
measure the relative amount of Skb1 protein isolated from each GST
precipitation or immunoprecipitation.
Skb1 Is Required for Normal Cell Viability and Polarity in
Hyperosmotic Medium--
Under normal growth conditions, S. pombe skb1 skb1 Skb1 Localizes to Cell Ends, Sites of Septation, and Nuclei in S. pombe Cells--
To obtain additional insights into the role of Skb1
as a morphological regulator, we performed experiments to examine its subcellular localization. To do this, we constructed the plasmid pREPUHASkb1 for expressing Skb1 as a triple hemagglutinin
epitope-tagged protein (HA-Skb1) from the thiamine-repressible
nmt1 promoter (17). S. pombe cells transformed
with pREPUHASkb1 were grown in medium containing thiamine to repress
expression of HA-Skb1, then transferred to medium lacking thiamine, and
grown for 11 h to derepress HA-Skb1 expression prior to
immunofluorescence microscopy. HA-Skb1 protein was detected at either
one or both cell ends in interphase cells and at what appeared to be
the nuclear periphery in both interphase and mitotic cells (Fig.
3, A and B). In a
small percentage of dividing cells, we were able to detect HA-Skb1 at
the septum-forming region (Fig. 3B). The localization of
Skb1 to cell ends is consistent with its role as a regulator of
morphological polarity, because a number of S. pombe
proteins required for proper control of cell polarity has been shown to localize to the cell ends (27, 28).
Hyperosmotic Shock Induces Rapid Delocalization and Enzymatic
Stimulation of Skb1--
To obtain additional molecular evidence of a
role for Skb1 as a mediator of hyperosmotic stress response, we
determined whether its subcellular localization or protein
methyltransferase activity are affected by hyperosmotic shock. S. pombe cells expressing HA-Skb1 were grown in EMM and then shifted
to EMM with 1.5 M KCl. Culture samples were then processed
for immunostaining at various time points after transfer to
hyperosmotic medium. We found that the frequency of cells exhibiting
HA-Skb1 at cell ends and nuclei was markedly reduced within 15 min of
hyperosmotic shock (Fig. 4). After this
initial delocalization, HA-Skb1 protein returned substantially to the
cell ends and nuclei within 1 h of exposure to 1.5 M
KCl.
Immune complexes of both human and budding yeast homologs of Skb1 have
been shown to possess protein methyltransferase activity. We determined
that recombinant Skb1 protein purified from either fission yeast or
bacterial cells likewise possesses protein methyltransferase activity
(Fig. 5A), thereby
demonstrating for the first time that this activity represents an
intrinsic enzymatic function of this class of proteins. To examine
whether Skb1 methyltransferase activity is stimulated by hyperosmotic
shock, cells expressing a c-Myc epitope-tagged Skb1 protein were grown
in EMM and were then shifted to either EMM or EMM + 1.5 M
KCl and incubated for 15 to 60 min prior to lysing the cells and
assaying for Skb1 protein methyltransferase activity. As shown in Fig.
5B, Skb1 methyltransferase activity was stimulated within 15 min of hyperosmotic shock. We conclude from these experiments that
hyperosmotic shock induces both a delocalization of Skb1 protein from
cell ends and nuclei, as well as a concomitant increase in Skb1 protein
methyltransferase activity.
The Human Skb1 Homolog, Skb1Hs, Is Stimulated by Hyperosmotic Shock
in Fission Yeast--
To address whether the methyltransferase
activity of Skb1Hs is stimulated by hyperosmotic shock in fission yeast
cells, we subjected cells expressing Skb1Hs to hyperosmotic shock and
then prepared cell lysates and assayed Skb1Hs protein methyltransferase activity. As shown in Fig. 5C, Skb1Hs methyltransferase
activity, similar to that of fission yeast Skb1, was rapidly stimulated by hyperosmotic shock, suggesting that mediation of hyperosmotic stress
response is likely to represent a conserved function of Skb1-related proteins.
In conclusion, we have demonstrated that a highly conserved protein
methyltransferase, Skb1, is required for normal growth and maintenance
of cell polarity under conditions of hyperosmotic stress in fission
yeast. We have shown that skb1
Importantly, we have shown that the protein methyltransferase activity
of the human Skb1/Hsl7 homolog, Skb1Hs, is induced by hyperosmotic
stress in S. pombe cells. This result provides evidence for
evolutionary conservation of a role for Skb1/Hsl7-related proteins in
mediating cellular response to hyperosmotic stress. Moreover, the fact
that the Skb1Hs enzyme is induced by hyperosmotic stress in S. pombe cells suggests that mechanisms involved in regulating the
function of these highly conserved proteins may have also been
substantially conserved through evolution.
Protein sequence analyses suggest that Skb1/Hsl7-related proteins
belong to the protein arginine methyltransferase family (14, 29). A
variety of eukaryotic proteins of diverse function have been shown to
undergo methylation on arginine or lysine residues (30). Although the
functional significance of this modification remains ill defined in all
but a handful of cases, recent studies have implicated arginine
methylation as being of potential significance in signal transduction
(31), transcriptional regulation (32), and RNA processing (33, 34). The
results presented in this report suggest that protein methylation is
likely to play a role in cellular response to hyperosmotic stress. The
continued characterization of Skb1/Hsl7-related protein
methyltransferases and in particular the identification of Skb1
substrates in the evolutionarily distant fission and budding yeasts
will undoubtedly shed substantial new insights into roles for protein
methylation in eukaryotic organisms.
cells to properly maintain cell
polarity in hyperosmotic conditions results from inefficient
subcellular targeting of F-actin. We show that Skb1 localizes to cell
ends, sites of septation, and nuclei of S. pombe cells.
Hyperosmotic shock results in substantial delocalization of Skb1 from
cell ends and nuclei, as well as stimulation of Skb1 protein
methyltransferase activity. Taken together, our results demonstrate a
new role for Skb1 as a mediator of hyperosmotic stress response in
fission yeast. We show that the protein methyltransferase activity of the human Skb1 homolog, Skb1Hs, is also stimulated by
hyperosmotic stress in fission yeast, providing evidence for evolutionary conservation of a role for Skb1-related proteins as
mediators of hyperosmotic stress response, as well as mechanisms involved in regulating this novel class of protein methyltransferases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
cells (1). These and additional genetic data implicate Skb1 as a
positive modulator of Shk1. However, unlike Shk1, Skb1 is not required
for cell viability, morphological polarity, or mating of S. pombe cells (1). Indeed, the only defect previously attributed to
skb1
mutants under normal growth conditions is that they
divide at a length slightly shorter than that of wild-type S. pombe cells (1, 2). In contrast, overexpression of Skb1 results in
a substantial delay in G2/M progression, suggesting that
Skb1 has a dose-dependent mitotic inhibitory function. The
G2/M delay caused by Skb1 overexpression is dependent on
both Shk1 and the Cdc2 inhibitory kinase Wee1 (2).
5) were several related protein
arginine methyltransferases. These investigators also demonstrated that
immunoprecipitates of Skb1Hs contain protein methyltransferase
activity, suggesting that Skb1Hs either possesses an intrinsic protein
methyltransferase function or associates with a protein
methyltransferase (14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
mutants are only modestly defective in cell growth
and divide at a length just slightly shorter than that of wild-type
S. pombe cells. We demonstrated previously that S. pombe mutants carrying a null mutation in the skb5
gene, which encodes an SH3 domain protein that directly activates Shk1,
are unable to maintain cell polarity under conditions of hyperosmotic
stress (21). Furthermore, PAKs have been implicated in the regulation
of osmotic stress response in budding yeast and mammalian cells
(22-24). We therefore determined whether Skb1 might play a more
prominent role in regulating cell viability or morphology when S. pombe cells are subjected to hyperosmotic stress. Although not
completely inhibited for growth, skb1
cells grew
substantially slower than wild-type S. pombe cells on
minimal medium containing 1.5 M KCl (Fig.
1A). We observed further that skb1
cells exhibited a marked defect in the ability to
maintain cell polarity in hyperosmotic medium, becoming stubby to round in appearance at a high frequency when compared with wild-type S. pombe cells, which retained a primarily rod-like appearance under
the same conditions (Fig. 1B). These observations
demonstrate that Skb1, although largely dispensable for growth and
maintenance of cell morphology under normal growth conditions, plays a
more significant role in regulating cell growth and polarity under conditions of hyperosmotic stress.
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Fig. 1.
Skb1 is required for normal growth and
control of morphological polarity in hyperosmotic medium.
A, wild-type and skb1 S. pombe
cells were streaked onto either EMM agar (top) or EMM agar
containing 1.5 M KCl (EMM+KCl;
bottom) and incubated at 30 °C (4 days for EMM plates and
7 days for EMM+KCl plates). B, wild-type and
skb1
cells were grown in EMM or EMM+KCl liquid for 3 days
prior to photomicroscopy.
Mutants Are Defective in Restoring Localization of F-actin
Patches to Cell Ends After Hyperosmotic Shock--
During interphase,
cortical F-actin patches are concentrated at the growing ends of
S. pombe cells (25). When S. pombe cells are
subjected to hyperosmotic shock, F-actin patches become transiently delocalized from the cell ends and randomly distributed but eventually redistribute to the cell ends after continued incubation in
hyperosmotic medium (26). To determine whether the inability of
skb1
mutants to properly maintain cell polarity in
hyperosmotic medium might correlate with a defect in regulating the
polarization of F-actin patches, we examined the appearance of F-actin
in wild-type and skb1
S. pombe cells after
subjecting them to hyperosmotic stress. Consistent with previously
reported observations (26), we found that F-actin patches became
delocalized from cell ends and randomly distributed in both wild-type
and skb1
cells within 30 min of exposure to EMM
containing 1 M KCl (Fig. 2,
EMM+KCl). After 2.5 h of incubation in EMM + 1 M KCl, F-actin patches became substantially redistributed
to the cell ends in wild-type S. pombe cultures but remained
depolarized in skb1
cells (Fig. 2). After 3.5 h of
incubation in 1 M KCl, cortical F-actin was redistributed
to the ends of skb1
cells but at a lower frequency than
in cultures of wild-type cells. These results suggest that the failure
of skb1
mutants to properly maintain morphological
polarity in hyperosmotic medium is likely to be due, at least in part,
to a defect in effectively repolarizing the localization of F-actin
patches to the cell ends, a process viewed as essential for
establishing and maintaining morphological polarity in S. pombe cells (25).
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Fig. 2.
Skb1 is required for effectively
redistributing F-actin to cell ends after hyperosmotic shock.
A and B, wild-type and skb1
S. pombe cells were grown in EMM and then were transferred
to EMM containing 1 M KCl and incubated at 30 °C. Cells
were fixed and stained with rhodamine-phalloidin at the indicated times
for visualization of F-actin by fluorescence microscopy. A,
fluorescence photomicrographs of F-actin in wild-type (left)
and skb1
(right) cells at 0, 60, and 150 min
after exposure to hyperosmotic medium. B, percentages of
wild-type (black bars) and skb1
(gray
bars) cells with F-actin patches at the cell ends at indicated
times after exposure to hyperosmotic medium. See text for description
of results.
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Fig. 3.
Skb1 localizes to cell ends, sites of
septation, and nuclei of S. pombe cells.
A, indirect immunofluorescence photomicrograph of HA-Skb1 in
wild-type S. pombe cells. Inset, cells
transformed with HA control plasmid. B, HA-Skb1-expressing
cells were immunostained for detection of HA-Skb1 and counterstained
with 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei.
I, interphase; M, mitosis; S,
septation.
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Fig. 4.
Hyperosmotic shock triggers a rapid
delocalization of Skb1 from cell ends and nuclei. HA-Skb1
expressing S. pombe cells were grown in EMM and then were
shifted to EMM containing 1.5 M KCl. Culture samples were
fixed at the indicated times for detection of HA-Skb1 protein by
indirect immunofluorescence microscopy. The percentage of cells
exhibiting HA-Skb1 localization at cell ends (black bars)
and nuclei (gray bars) is indicated in the graph.
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Fig. 5.
The protein methyltransferase activities of
S. pombe and human Skb1 proteins are rapidly
stimulated by hyperosmotic shock of S. pombe
cells. A, fission yeast Skb1 possesses intrinsic
protein methyltransferase activity. The panel at the
far left shows MBP methylation (top) and
immunoblot analysis (bottom) of GST and GST-Skb1 isolated
from S. pombe cells. The adjacent panel shows
results of the same analyses for GST and GST-Skb1 isolated from
E. coli cells. The far right panels show MBP
methylation (top) and immunoblot analysis of c-Myc
epitope-tagged S. pombe (CM-Skb1) and human
(CM-Skb1Hs) proteins expressed and purified from S. pombe cells. B, S. pombe cells expressing
CM-Skb1 were grown in EMM and were then shifted to EMM containing 1.5 M KCl and incubated for the indicated times before assaying
for CM-Skb1 methyltransferase activity. C, S. pombe cells expressing CM-Skb1Hs were subjected to hyperosmotic
shock and assayed for protein methyltransferase activity as described
in B.
mutants are defective in
redistributing F-actin patches to cell ends after hyperosmotic
stress-induced F-actin depolarization. It is likely that the failure of
skb1
cells to properly maintain cell polarity in
hyperosmotic medium is caused, at least in part, by this defect. Correlating with these findings, we found that in response to hyperosmotic stress, the Skb1 protein delocalizes from cell ends and
nuclei and becomes markedly stimulated with respect to its protein
methyltransferase activity. Cumulatively, these data establish a role
for Skb1 as a mediator of hyperosmotic stress response in fission
yeast. Previous genetic studies suggested that Skb1 functions as a
positive modulator of the fission yeast PAK, Shk1. Whereas Shk1 plays
an essential role in regulating the localization of F-actin patches and
cell polarity under normal growth conditions, we consider it likely
that it also shares with Skb1 a role in mediating hyperosmotic stress
response because of the following. (i) Its kinase activity is rapidly
stimulated by hyperosmotic stress. (ii) Similar to Skb1, it becomes
delocalized from cell ends in response to hyperosmotic stress, and
(iii) it is required for proper subcellular targeting of
Skb1.2
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grant R01GM53239 (to S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work and are listed alphabetically.
To whom correspondence should be addressed. Tel.:
713-745-2032; Fax: 713-794-4394; E-mail: smarcus@mdacc.tmc.edu.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.C100096200
2 Y. Qyang, R. Pimental, and S. Marcus, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PAK, p21-activated kinase; HA, hemagglutinin; EMM, essential minimal medium; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; MBP, myelin basic protein.
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