Department of Biology, Graduate School of Science, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan
* Author for correspondence (e-mail: shimoda{at}sci.osaka-cu.ac.jp )
Accepted 16 February 2002
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Summary |
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Key words: C2 domain, HECT domain, G2 arrest, Ubiquitin ligase
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Introduction |
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The ubiquitination of proteins is catalyzed by three enzymes, E1, E2 and
E3. The ubiquitin-activating enzyme (E1) binds to and activates free ubiquitin
(Ub) molecules using ATP; the activated Ub is subsequently transferred to a
ubiquitin-conjugating enzyme (E2). In many cases, ubiquitin-protein ligases
(E3) are necessary for substrate-specific multi-ubiquitination (for reviews,
see Varshavsky, 1997;
Hershko and Ciechanover,
1998
). Ubiquitin-protein ligases are classified as either a RING
finger or a HECT type (for a review, see
Jackson et al., 2000
). Typical
RING-finger-type ubiquitin ligases include the anaphase promoting complex
(APC) and the SCF (Skp1/Cullin/F-box) complex, both of which are involved in
controlling cell cycle progression. HECT-type ubiquitin ligases contain a HECT
(homologous to the E6-AP carboxyl
terminus) domain, which is the ubiquitin-protein ligase catalytic
domain (Huibregtse et al.,
1995
). HECT-type ubiquitin ligases form a thioester intermediate
with Ub via a conserved cysteine (Cys) residue located in the HECT domain
(Scheffner et al., 1995
).
Mammalian Nedd4p and S. cerevisiae Rsp5p constitute a subfamily of
HECT-type ubiquitin ligases. Nedd4/Rsp5 proteins are characterized by a C2
domain and several WW domains, in addition to the catalytic HECT domain (for a
review, see Harvey and Kumar,
1999
). The C2 domain acts as a Ca2+-dependent
phospholipid-binding site. Indeed, the N-terminal C2 domain of mammalian
Nedd4p is required for its localization to the plasma membrane
(Plant et al., 1997
). The WW
domain binds to several proline-rich motifs such as the PY, PPLP and PGM
motifs (Bedford, 2000) (for a review, see
Kay et al., 2000
). Compared
with APC and SCF, little is known about the biological functions of HECT-type
ubiquitin ligases; however, mammalian Nedd4p and budding yeast Rsp5p have been
analyzed extensively and their in vivo substrates have been identified. Human
Nedd4p downregulates epithelial Na+ channel proteins by
ubiquitin-mediated proteolysis (Staub et
al., 1996
), whereas Rsp5p, an essential HECT-type ubiquitin ligase
in Saccharomyces cerevisiae, ubiquitinates the large subunit of RNA
polymerase II (Rpb1p) (Huibregtse et al.,
1997
), uracil permease (Fur4p)
(Galan et al., 1996
), a general
amino-acid permease (Gap1p) (Hein et al.,
1995
) and a receptor protein for
-mating pheromone (Ste2p)
(Hicke et al., 1998
). Rsp5p
also promotes the endocytosis of plasma-membrane-integrated proteins, such as
Ste2p and Fur4p, by ubiquitination (for a review, see
Rotin et al., 2000
).
In the fission yeast Schizosaccharomyces pombe, Pub1p (E6-AP-like
protein ubiquitin ligase), which is encoded by the
pub1+ gene, has been reported to be a HECT-type ubiquitin
ligase (Nefsky and Beach,
1996). Pub1p has been proposed to regulate G2/M transition via
ubiquitination of Cdc25p, which dephosphorylates the phosphotyrosine of Cdc2p
(Russell and Nurse, 1986
;
Gould and Nurse, 1989
;
Dunphy and Kumagai, 1991
;
Gautier et al., 1991
;
Lundgren et al., 1991
;
Millar et al., 1992
).
Disruption of the pub1+ gene markedly reduces the level of
ubiquitinated Cdc25p. In addition to cell cycle regulation, Pub1p is involved
both in cell viability in low pH medium and in leucine transport
(Saleki et al., 1997
;
Karagiannis et al., 1999
).
Pub1p ubiquitin ligase belongs to the Nedd4/Rsp5 subfamily, because it is
composed of a highly conserved HECT domain as well as a single C2 domain and
three WW domains. We have identified two more genes encoding ubiquitin ligases
of this subfamily in the S. pombe genome sequence database (The
Sanger Centre, UK), which we designated pub2+ and
pub3+. In this article, we report that the
pub2+ gene product has ubiquitin-protein ligase activity
in vivo and shares a partially overlapping function with Pub1p.
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Materials and Methods |
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Synchronous culture
Mitotic cell cycles were synchronized using the cdc25-22
temperature-sensitive mutation. cdc25-22 cells were grown in EMM2
until mid-log phase at 25°C and shifted to 36.5°C to arrest the cell
cycle in late G2 phase. After a 4 hour incubation at 36.5°C, the culture
was shifted to 25°C to restart the cell cycle. Synchrony was monitored by
scoring septated cells (Alfa et al.,
1993).
Flow cytometric analysis
Cells were fixed with cold 70% ethanol, resuspended in 50 mM
Na+-citrate buffer and treated with 0.1 mg/ml RNase A for 2 hours
at 37°C. After washing with Na+-citrate buffer, cells were
stained with propidium iodide (PI) at a final concentration of 10 µg/ml.
Stained cells were analyzed by a flow cytometer (FACScaliber, Becton
Dickinson).
Isolation of pub2+ and
pub3+
A 13 kb genomic DNA fragment containing pub2+ was
fortuitously cloned from an S. pombe genomic DNA library
(Ikemoto et al., 2000) during
a previous screening for other genes. A 4.0 kb ClaI/PstI
fragment, containing the pub2+ ORF, was subcloned into a
pBluescript II-KS(+) plasmid (Stratagene, La Jolla, CA). We also isolated the
pub2+ cDNA clone from an S. pombe cDNA library
pTN-RC5 (Nakamura et al.,
2001
). The nucleotide sequences of the genomic and cDNA clones
were determined. The pub2+ gene is split by three introns,
and the exonic sequence potentially encodes a Pub2 protein comprising 671
amino acids. pub2+ proved to be identical to SPAC1805.15c
(The Sanger Centre Genome Sequence Database).
We also identified another ORF that is homologous to Pub1p in the database. This ORF, SPBC16E9.11c, was designated pub3+. The genomic pub3+ gene was isolated by a PCR-based method (see below).
Gene disruption of pub1+ and
pub2+
The pub2+ gene was disrupted by one-step gene
replacement (Rothstein, 1983).
The 1.2 kb HindIII fragment carrying about 60% of the Pub2 ORF was
replaced by a 1.8 kb HindIII fragment containing
ura4+ (Grimm et al.,
1988
). After digestion with EcoRI/PstI, the
linearized DNA fragment harboring the pub2::ura4+ allele
was introduced into the diploid strain C525 using the lithium acetate method
(Okazaki et al., 1990
). Stable
Ura+ transformants were subjected to genomic Southern blotting to
confirm that they carried one copy of pub2::ura4+ and one
copy of pub2+ (data not shown).
The pub1+ gene was disrupted as follows. The genomic
DNA fragment carrying pub1+ in pBluescript II was digested
with NspV and blunt-ended with T4 DNA polymerase. It was further
digested with BglII and ligated to a BamHI/HincII
fragment carrying a ura4+ cassette. The
pub1::ura4+ fragment was amplified with primers designed
to amplify pub1+ genomic DNA and introduced into C525.
Verification of pub1 was conducted by genomic Southern
blotting. Genomic DNA was digested with ClaI. The
XhoI/BglII fragment cut out from pub1+
in pBluescript II was used as the probe for Southern blotting.
Plasmid construction
The plasmid pREP2-Myc was constructed by inserting three copies of Myc into
the NotI site of pREP2. The plasmid pREP81-GFP was constructed by
inserting the mutant version of GFP (GFPS65T) into the
NotI site of pREP81 (Nakamura et
al., 2001). To overexpress the pub2+ gene,
plasmid pREP1-pub2+ was constructed.
pub2+ cDNA was amplified from the cDNA library, pTN-RC5
(Nakamura et al., 2001
), by
PCR with the following primers:
5'-CCAGATCTCATATG(NdeI)GAAAATATTCGCTTG-3' and
5'-CCGCGGCCGC(NotI)-CCTCCGTACCAAATCC-3'. The
amplified DNA was digested with NdeI and NotI and ligated
into pREP1 to generate pREP1-pub2+. The same
NdeI/NotI fragment was inserted into the plasmid pREP81-GFP.
In vitro mutagenesis for the substitution of Pub2 Cys639 to Ala (Pub2CA) was
done by using the above primers and the following primers:
5'-CATACTGCCTTCAATCG-3' and
5'-AAACGATTGAAGGCAGTATG-3'. To amplify the Pub2 HECT domain, the
following primers were used: 5'-CCCCATATGCAGCTAAAGGTTAGCAGAG-3',
5'-CCGCGGCCGCCCTCCGTACCAAATCC-3'. The Pub1-HECTPub2
chimeric protein-encoding gene was constructed with the primers:
5'-ATCCCCGTGAATACTTCTATATTTTGTCTCATGC-3',
5'-GCATGAGACAAAATATAGAAGTATTCACGGGA-3'.
The ubiquitin-encoding gene was amplified in the same way but with the following primers: 5'-GGGCGGCCGC(NotI)ATGCAGATTTTTGTC-3', 5'-GGAGATCT(BglII)TACTTAAGCTTCTTCTTAGG-3'. The amplified DNA fragment was inserted into pREP1-GST after NotI/BglII digestion. For amplification of genomic DNA containing pub1+, the following primers were used: 5'-GTGGATAGCAAATAGTCTTTATGACCAGCC-3', 5'-AGGATTGTTTTACAAGGCTATTGTGGTTGG-3'. The amplified DNA fragment was blunt-ended with T4 DNA polymerase and ligated into the EcoRV site of pBluescript II KS(+). pub1+ cDNA was amplified with the primers 5'-GGCATATG(NdeI)TCAAACTCAGCTCAATCTCG-3' and 5'-CCGCGGCCGC(NotI)CCTCCTGACCAAAACCAATCG-3'. The amplified fragment was inserted into pREP41 after digestion with NdeI/NotI.
Southern and northern blotting
Genomic DNA was prepared according to Hereford et al.
(Hereford et al., 1979). DNA
was restricted, fractionated on 1% agarose gels and then transferred onto
nylon membranes (Biodyne B, Pall Co.). Nonradioactive probes were prepared by
using the DIG-DNA Labeling Kit (Boehringer Mannheim, Mannheim). Hybridization
was carried out according to the manufacturer's instructions using the
DIG-Hybridization Kit (Boehringer Mannheim, Mannheim).
Northern blotting was carried out as follows. Total RNA was prepared from
S. pombe cultures by the method of Jensen et al.
(Jensen et al., 1983). 10
µg of total RNA was fractionated on 1.5% agarose gels and then transferred
onto nylon membranes (Biodyne B, Pall Co.)
(Thomas, 1980
).
pub1+ and pub2+ cDNAs were labeled
with [
-32P]ATP by the random primer method (Feinberg et al.,
1983) and used as hybridization probes.
Immunological detection of Pub2-ubiquitin conjugation
Both pREP1-GST:Ub and pREP2-HECTpub2:Myc plasmids were
introduced into the haploid strain MM72-1D (h-leul-32
ura4-D18). The transformants were grown to mid-log phase in EMM2
containing 20 µM thiamine to repress the nmt1 promoter. After
washing twice with sterilized H2O, the cells were transferred into
EMM2 without thiamine to switch on the nmt1 promoter and incubated
for 20 hours. Cells were disintegrated by glass beads in ice-cold lysis buffer
(20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS) containing 50 mM N-ethylmaleimide and
protease inhibitors; 5 µg/ml aprotinin, 3 µg/ml leupeptin and 1 mM PMSF.
The protein concentration of total cell lysates was determined by the Lowry
method. Whole-cell extracts containing 5 µg of protein were incubated with
20 µl of Glutathione-Sepharose 4B (Pharmacia Biotech) with rotation at
4°C for 1 hour. Glutathione beads were washed at least four times with
RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS) containing protease inhibitors used in lysis buffer.
After washing, the samples were divided into two portions. One was incubated
for 30 minutes at 60°C in SDS-PAGE sample buffer (50 mM Tris-HCl pH 6.8,
2% SDS, 10% glycerol) containing 100 mM dithiothreitol (DTT). The other was
treated in the same way except that 4 M urea was added instead of 100 mM DTT
(Scheffner, 1995). After SDS-PAGE on 10% gels, proteins were transferred onto
polyvinylidene difluoride (PVDF) membranes (Immobilon, Millipore Co.). Western
blotting was performed with a 9E10 anti-c-Myc antibody (1:1000 dilution)
(Sigma) and HRP-conjugated antimouse IgG (1:1000) (Promega).
Localization of Pub1-GFP and Pub2-GFP
Plasmids pREP81-pub1+-GFP,
pREP81-pub2+-GFP and pREP81-GFP were
introduced into MM72-11C (h- leul-32) cells. The
transformed cells were grown to mid-log phase in EMM2 containing 20 µM
thiamine and then transferred to EMM2 without thiamine. Cells were observed
without fixation under a fluorescence microscope (Model BX50, Olympus
Co.).
Subcelluar fractionation by differential centrifugation
Preparation of cell lysates and their fractionation by differential
centrifugation were done as described by Dunn and Hicke
(Dunn and Hicke, 2001).
Spheroplasts were prepared by treating cells by Zymolyase 100T (SEIKAGAKU Co.,
Tokyo) in YE medium containing 1.2 M sorbitol and then mechanically broken in
lysis buffer (20 mM MES pH6.5, 0.1 M NaCl, 5 mM MgCl2, and protease
inhibitors). The lysates were centrifuged first at 13,000 g for 30
minutes to separate precipitate (P13) and supernatant. The latter fraction was
subjected to a second centrifugation at 100,000 g for 30 minutes,
resulting in pellet (P100) and supernatant (S100) fractions. P13 mainly
contains large membrane compartments such as plasma membranes and vacuoles.
The lighter membrane components such as endoplasmic reticulum, Golgi apparatus
and endosomes were recovered in P100. S100 is a cytosolic fraction
(Dunn and Hicke, 2001
). The
quantity of Pub2-HA in each fraction was analyzed by western blotting with
anti-HA antibody (3F10).
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Results |
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The pub2+ gene encodes a 77 kDa protein (Pub2p)
comprising 671 amino acids and containing a single WW domain and a HECT domain
but no C2 domain (Fig. 1A). The
pub3+ gene product (Pub3p) consists of 786 amino acids (89
kDa) and has a C2 domain in its N-terminus, three WW domains in its central
region and a HECT domain in its C-terminus
(Fig. 1A). Pub3p resembles
Pub1p in size and primary structure, with an amino acid identity of 66% (74%
in the HECT domain) (Fig. 1B).
It therefore seems likely that pub1+ and
pub3+ are redundant genes. In support of this, a
pub1 pub3
double disruptant was non-viable
(K.K.T. and C.S., unpublished data).
By contrast, the amino-acid sequence similarity between Pub2p and Pub1p is relatively low (39% identity), and the domain structure is different. Compared with other members of the Nedd4/Rsp5 family, the most prominent feature of Pub2p is its lack of C2 domain. Despite the overall differences between Pub2p and Pub1p/Pub3p, their HECT domains are highly conserved (Fig. 1B,C).
Transcription of pub2+
Although Pub1p has been proposed to negatively regulate the G2/M
transition, the fluctuation of transcript level of pub1+
is not known. We examined transcription of pub1+ and
pub2+ in the cell cycle. Synchronous cell division was
attained by the cdc25-22 temperature-sensitive mutation. Cell cycle
progression of cdc25-22 cells was blocked in late G2 phase at
36.5°C for 4 hours, and the cell cycle was restarted by a shift to
permissive temperature, 24°C. RNA samples were taken every 20 minutes for
5 hours and then subjected to northern analysis with pub1- or
pub2-specific probes. As shown in
Fig. 2A, the mRNA level of
pub2+ was low relative to that of
pub1+. The abundance of pub2+ mRNA did
not fluctuate throughout the cell cycle.
|
We next examined whether the transcription of pub2+ mRNA was affected by stress, such as nutrient starvation, heat shock or osmotic stress. We measured the abundance of pub1+ and pub2+ mRNA after nitrogen starvation (Fig. 2B). Homothallic (h90) cells were grown in nitrogen-rich medium until mid-log phase and then transferred to nitrogen-free medium. Notably, the pub2+ mRNA level was significantly enhanced 2 hours after nitrogen starvation, and this elevated expression level persisted (Fig. 2B). By contrast, the expression of pub1+ was not affected by nutritional state. Upregulation of pub2+ mRNA was also observed in a heterothallic haploid strain (data not shown), suggesting that pub2+ transcription is stimulated by the stress of nitrogen starvation, independent of the mating reaction.
As Ste11p is a key transcription factor for many genes whose expression is
upregulated by starvation (Sugimoto et
al., 1991), we investigated whether the increase in
pub2+ mRNA is regulated by Ste11p. We performed northern
blot analysis using h90 ste11
cells. As shown in
Fig. 2B, the expression of
pub2+ was repressed in ste11
cells;
however, there was still an increase in mRNA levels after starvation. The
Ste11-binding site, called the `TR box', was not present in the putative
promoter region of pub2+. Together, these results suggest
that the pub2+ transcription is affected by the
ste11
mutation but that the Ste11 transcription factor might
have only an indirect effect on transcription of
pub2+.
Phenotypes of pub2
To investigate functions of Pub2p, we disrupted the chromosomal
pub2+ gene by replacing a substantial part of the
pub2+ ORF with the ura4+ cassette.
Tetrad analysis of the heterozygous pub2 diploid cells
revealed that the pub2+ gene is not essential for
vegetative growth. The size and shape of pub2
cells were not
distinguishable from those of isogenic wild-type cells (data not shown). As
pub2+ transcription was stimulated by nitrogen depletion,
we thought that pub2
might be defective in mating and/or
sporulation. h90 pub2
cells were incubated in
liquid sporulation medium (EMM2-N) to induce mating and sporulation. The
frequency of zygotes and asci was scored after 20 and 40 hours in EMM2-N. The
efficiency of conjugation and sporulation of pub2
cells was
indistinguishable from that of wild-type cells (data not shown). Finally, we
found that the germination efficiency of pub2
spores was also
normal (data not shown). Thus, we conclude that pub2+ is
not required for sexual development.
We also examined the sensitivity of the pub2 mutant to
other environmental stresses. The pub2
cells were exposed to
high and low incubation temperature (18-37°C), high osmolarity (1.2 M
sorbitol) and high concentrations of salt solutions (1.2 M NaCl and 0.5 M
CaCl2). The growth of pub2
cells under these
conditions was not different from that of wild-type cells. Saleki et al.
(Saleki et al., 1997
) have
shown that the pub1
mutant is sensitive to low pH. Some
auxotrophic strains are not able to grow at pH6.8, and pub1
can tolerate high pH (Karagiannis et al.,
1999
). We therefore examined whether pub2
cells
showed similar traits. As shown in Fig.
3A, pub2
formed colonies on EMM2 minimal medium at
pH 3.5, but not at pH 6.8, as do wild-type cells.
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Overexpression of pub2+
To gain insight into the function of Pub2p in vegetative cells, we
overexpressed the epitope-tagged Pub2 (Pub2-HA) in wild-type cells.
Overexpression was controlled by a strong nmt1 promoter, which is
thiamine repressible. After 16 hours of thiamine removal, Pub2-HA had
accumulated as revealed by western blotting using an anti-HA monoclonal
antibody 12CA5 (Fig. 4A, lane
1, 3). Cells overexpressing pub2+ become elongated
(Fig. 4Ba,c), and cell
proliferation was repressed (Fig.
4C). Flow cytometric analysis revealed that most of the
pub2+-overexpressing haploid cells contained 2C DNA
(Fig. 4D). Seemingly,
substantial proportions of the culture showed DNA content higher than 2C. This
might occur because of the remarkable elongation of
pub2+-overexpressing cells. Immunostaining with an
anti-tubulin antibody (TAT-1) showed that the cells contained only cytoplasmic
microtubules, indicating that they were in interphase (data not shown).
Together, these results strongly suggest that the
pub2+-overexpressing cells arrested in the G2 phase of the
cell cycle.
|
Nefsky and Beach (Nefsky and Beach,
1996) have demonstrated that Pub1p is involved in ubiquitination
of Cdc25p, which dephosphorylates the phospho-Tyr15 of Cdc2p. It is
possible that Pub2p may also ubiquitinate Cdc25p, as this would explain the
Pub2-induced delay of G2 progression. If this is the case, levels of Cdc25p
should be reduced in pub2+-overexpressing cells. We
incubated strain OM1715 harboring pREP1-pub2+, in which
Cdc25-6HA is integrated at the chromosomal cdc25+ locus,
in medium lacking thiamine. During the incubation, protein samples were
removed and analyzed for Cdc25-6HA by an anti-HA monoclonal antibody (12CA5).
Unexpectedly, levels of Cdc25-6HA increased when Pub2p was overproduced
(Fig. 4E). Therefore, Pub2p is
not likely to be involved in the degradation of Cdc25p. The accumulation of
Cdc25p in pub2+-overexpressing cells might be due to a
secondary effect of cell cycle arrest in G2 phase. Accumulation of Cdc25p in
G2 arrest cells has been reported (Moreno
et al., 1990
).
To address the mechanism of G2 arrest caused by Pub2 overproduction, we overexpressed the pub2+ gene in wee1-50 or cdc25-22 temperature-sensitive mutants. Wee1p is a kinase that inhibits G2/M transition by phosphorylating Cdc2p. Therefore, the wee1 mutation accelerates the commitment to M phase, resulting in a small cell size. Pub2p was overproduced in the wee1-50 cells. At both restrictive and permissive temperatures, wee1-50 cells failed to grow when pub2+ was overexpressed (data not shown). Cdc25p regulates the onset of M phase by dephosphorylating Cdc2p. If Pub2-induced G2 arrest is mediated by Cdc25p, the cdc25-22 temperature-sensitive mutation might strengthen the G2 arrest phenotype. However, growth inhibition and cell elongation were not observed in cdc25-22 cells at 26 or 30°C unless pub2+ on a multicopy expression vector, pREP1, was overexpressed (data not shown). These results suggest that the G2 arrest phenotype arising from pub2+ overexpression is caused by a mechanism that is not related to Wee1 kinase and Cdc25 protein phosphatase.
Another possibility is that Pub2p overproduction activates checkpoint
machinery, leading to cell cycle arrest at G2 phase. If so, the G2 arrest
phenotype caused by Pub2 overproduction might depend on the checkpoint genes.
To inspect this possibility, we overexpressed pub2+ in
rad1, rad3
, cds1
and
chk1
. The cell elongation phenotype was observed in either
disruptant (data not shown), indicating that pub2+
overexpression activates neither DNA replication checkpoint nor DNA damage
checkpoint. These observations imply that pub2+
overexpression affects G2/M transition by an unknown mechanism.
Genetic interaction between pub1+ and
pub2+
The pub2-null mutant exhibited no apparent defects in vegetative
growth. The expression level of pub1+ was higher than that
of pub2+ in vegetative cells
(Fig. 2A). We therefore
speculated that Pub1p and Pub2p might share an overlapping function. To
examine this possible redundancy between Pub1p and Pub2p, we investigated the
growth properties of pub1 pub2
cells. We found that
this double disruptant was viable at 30°C
(Fig. 3B), and its morphology
was not different from wild-type cells. Nefsky and Beach
(Nefsky and Beach, 1996
) have
reported that cells bearing pub1
divide at a noticeably
smaller cell size but form colonies even at 37°C. We constructed a
pub1-null mutant, pub1::ura4+, in which virtually
the whole sequence of the pub1 ORF was deleted, and examined the
temperature sensitivity of this null mutant. This pub1
mutant
was unable to form colonies on complete medium at 37°C
(Fig. 3B). We next carefully
examined the effect of incubation temperature on the growth of single and
double disruptant strains. As shown in Fig.
3B, the pub1
pub2
double mutant did not
form colonies, even at 35.5°C, whereas both pub1
and
pub2
single mutants grew well. This observation indicates
that, at least in vegetative cells, Pub1p and Pub2p carry out a similar
function.
To study further the genetic relationship between pub1+
and pub2+, we tested whether overexpression of
pub2+ can override the temperature sensitivity of
pub1 cells. As mentioned above, strong overexpression of Pub2p
is toxic to cells, the pub2+ gene was moderately expressed
by a weaker nmt41 promoter. Neither cell elongation nor growth arrest
was detected when Pub2p was expressed from nmt41 promoter in a
wild-type background. The pub1
cells, which were transformed
with pREP41-pub2+, were incubated at 37°C for 3 days.
As shown in Fig. 7B, cells
moderately overexpressing pub2+ grew, in contrast to
control pub1
cells bearing the empty vector pREP41. This
result confirms that Pub2p must share some overlapping functions with
Pub1p.
|
Localization of Pub2p
It has been shown that Nedd4p is localized to the plasma membrane via its
C2 domain (Plant et al., 1997;
Plant et al., 2000
). However,
a C2-domain-truncated variant of Rsp5p still associates with the membrane
fraction (Dunn and Hicke,
2001
); therefore, the exact role of the C2 domain in the
Nedd4/Rsp5 family of ubiquitin ligases remains controversial. Notably, Pub2p
is unique among the other family members in that it lacks a C2 domain.
To define the cellular localization of Pub2p, a Pub2-GFP fusion protein was
expressed by the weakest version of the nmt promoter, nmt81.
A multicopy plasmid containing the
nmt81-pub2+-GFP fusion gene was introduced into
wild-type strain MM72-11C. As a negative control, pREP81-GFP was also
introduced into the same strain. As the localization of Pub1p has not been
documented so far, we thus also observed the fluorescent signals in the cells
expressing the nmt81-pub1+-GFP fusion gene. Like
the authentic Pub2 protein, overexpression of Pub2-GFP induced growth arrest
and cell elongation (data not shown). The
nmt41-pub1+-GFP fusion gene complemented the
temperature sensitivity of pub1 (data not shown). These
results indicated that both Pub1-GFP and Pub2-GFP were functional. As
Fig. 5A shows, Pub1-GFP
localized to the plasma membrane and to unidentified cytoplasmic bodies. The
latter structures might be endosomes, because a similar staining image was
seen for GFP-fused membrane-bound proteins targeted to vacuoles
(Katzmann et al., 2001
;
Morishita et al., 2002
).
Basically the same staining pattern was observed, when Pub1-GFP was expressed
by the authentic promoter. On the other hand, Pub2-GFP was present both in the
nuclei and in the cytoplasm. Furthermore, we noted that Pub2-GFP localized to
the cell surface in the polar regions (Fig.
5Ac). This observation suggests that Pub2p localizes to the plasma
membrane independently of a C2 domain. It is plausible that the cell-surface
localization of other members of the Nedd4/Rsp5 family may be attained in a
manner both dependent on, and independent of, the C2 domain.
|
Localization of Pub2-GFP was observed with cells expressing the fusion gene by the nmt81 promoter because the expression by the native pub2 promoter was too low to visualize any fluorescent signals. Therefore, the subcellular distribution of Pub2p was explored by subcellular fractionation using a Pub2-HA integrant strain (KKT87). As shown in Fig. 5B, Pub2-HA was detected not only in the soluble fraction (S100) but also in the low (P13) and high speed (P100) pellet fractions. Treatment of lysates with a high concentration of salts (e.g., 0.5 M NaCl) increased the proportion of Pub2-HA in S100, although treatment with 1% Triton X-100 showed no such consequence (data not shown). These results strongly suggested that a portion of Pub2-HA was associated with cellular structures, supporting the microscopic observation that Pub2-GFP was present at the plasma membrane in the polar regions.
Pub2 is thiol-ubiquitinated in vivo
HECT-type ubiquitin ligases directly bind to a ubiquitin molecule through
the conserved cysteine residue located in the HECT domain
(Scheffner et al., 1995). In
Pub2p, this residue corresponds to cysteine 639
(Fig. 1B). To confirm the
significance of this residue in the function of Pub2p, we generated an alanine
mutation at Cys639 (Pub2CA). To test the function of Pub2CA, we noted the fact
that the high overexpression of pub2+ is toxic. We
overexpressed Pub2CA in h- wild-type cells under the
control of the strong nmt1 promoter. The accumulation of Pub2CA was
confirmed by western blotting (Fig.
4A, lane 2, 4). Overexpression of Pub2CA had little influence on
cell elongation (Fig. 4Bb,d)
and cell proliferation (data not shown). Consistent with this observation, no
accumulation of Cdc25-6HA was found in the pub2CA-overexpressing
cells (Fig. 4E). These
observations strongly suggest that Pub2p requires the conserved Cys639 residue
for correct function and so acts as a HECT-type ubiquitin-protein ligase.
Next, we examined whether Pub2p forms a thioester linkage with ubiquitin via Cys639. As we predicted that putative Pub2p-Ub intermediates would be highly unstable, we fused only the HECT domain, which lacks a putative substrate-binding site, to an Myc epitope (HECTPub2-Myc) instead of generating a full-length Pub2-Myc fusion protein. The HECTPub2-Myc fusion protein was co-expressed with a GST-Ub fusion protein in wild-type cells. GST-Ub was pulled down from cell lysates by glutathione beads, and the precipitate was treated with 100 mM DTT to cleave any thioester bond that might have been formed between HECTPub2-Myc and GST-Ub. The presence of HECTPub2-Myc protein was detected by western analysis with an anti c-Myc antibody (9E10). When HECTPub2-Myc and GST-Ub were co-expressed, detection of the HECTPub2-Myc signal was dependent on addition of DTT (Fig. 6, lane 2, 3). However, when HECTPub2-Myc and GST alone were co-expressed, the HECTPub2-Myc signal was not detected at all (Fig. 6, lane 1). Next, we tested whether the alanine mutant HECT tagged with Myc (HECT-CAPub2-Myc) binds to GST-Ub. As shown in Fig. 6 (lane 4, 5), the alanine mutant HECT did not associate with ubiquitin proteins. Together, these results support the notion that Cys639 of Pub2p forms a thioester bond with ubiquitin.
|
HECTPub2 might serve as a catalytic domain
Finally, we addressed the question of whether or not Pub2p has
ubiquitin-protein ligase activity. It has been established that Pub1p is a
HECT-type ubiquitin-protein ligase. We reasoned that if the HECT domain of
Pub2p (HECTPub2) has catalytic activity, then a protein chimera of
Pub1p, whose HECT domain is replaced with HECTPub2, would be
functional. This chimeric protein (Pub1-HECTPub2;
Fig. 7A) was overexpressed from
the attenuated nmt41 promoter in temperature-sensitive
pub1 cells. As shown in
Fig. 7B,
Pub1-HECTPub2 suppressed the temperature sensitivity of
pub1
cells. By contrast, Pub1-HECT-CAPub2 did not
complement the temperature sensitivity. These experiments suggest that the
wild-type HECT domain of Pub2p can act as the catalytic domain of Pub1
ubiquitin ligase and that the Cys639 residue is essential for this catalytic
activity.
![]() |
Discussion |
---|
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---|
We have presented evidence to show that Pub2p is thiol-ubiquitinated
(Fig. 6). In terms of domain
structure, Pub2p is unique among the Nedd4/Rsp5 protein family, as it lacks a
C2 domain and contains only one WW domain
(Fig. 1A). We found that a
single knock-out of any of the pub genes does not render the cells
non-viable; however, a pub1 pub3
double
knock-out mutant is non-viable, indicating that Pub1p and Pub3p have redundant
functions (K.K.T. and C.S., unpublished). By contrast, our results suggested
that Pub2p shares only partially overlapping function with Pub1p. The
pub1+ gene is constitutively transcribed in vegetative
cells at relatively high levels, whereas the pub2+ gene is
transcribed at low levels. Interestingly, the transcription of
pub2+ is greatly enhanced by nitrogen starvation
(Fig. 2). The abundance of
pub3+ mRNA is extremely low in vegetative cells and is not
increased by nitrogen starvation (K.K.T. and C.S., unpublished). Such
different control of gene expression suggests that the Nedd4/Rsp5-related
proteins of S. pombe have different cellular functions during
vegetative growth under various environmental conditions. Moderate
overexpression of the pub2+ gene suppressed the
temperature sensitivity of pub1
cells
(Fig. 7B). Conversely,
disruption of pub2+ intensified the
temperature-sensitivity phenotype of pub1
cells
(Fig. 3B). These findings
suggest that pub1 and pub2 may have partly overlapping
functions.
On the basis of the following observations, Pub1p has been assumed to have
a role in controlling the cell cycle. Pub1 cells prematurely
traverse the G2/M boundary. A pub1
wee1-50ts double
mutant is non-viable at the restrictive temperature, probably owing to mitotic
catastrophe. Pub1 ubiquitin ligase ubiquitinates Cdc25 phosphatase, a crucial
inducer of M phase, and the level of Cdc25p is elevated in
pub1
cells (Nefsky and
Beach, 1996
). Together, these facts support the idea that Pub1p is
a negative regulator of G2/M transition. We therefore addressed whether Pub2p
is also involved in cell cycle control. Although pub2 disruptants
were normal in their progression through the cell cycle, high overexpression
of pub2+ caused a marked cell elongation and the
repression of cell multiplication (Fig.
4A-D). Therefore, Pub2p, in conjunction with Pub1p, possibly
regulates the progression of G2 or the start of M-phase. However, as
overexpression of pub2+ did not reduce levels of Cdc25p
(Fig. 4E), it is unlikely that
Pub2p performs precisely the same functions as Pub1p in cell cycle
regulation.
It should be noted that the phenotype of pub1 mutants is
pleiotropic. They are sensitive to acidic pH (pH 3.5) and have reduced
repression of leucine uptake in the presence of ammonium ions
(Saleki et al., 1997;
Karagiannis et al., 1999
).
These phenotypes imply that Pub1p is involved in ubiquitination and
degradation of leucine-specific permease in the plasma membrane
(Karagiannis et al., 1999
).
This notion is strongly supported by our observation in this study that
Pub1-GFP is localized to the cell membrane
(Fig. 5Ab). In budding yeast,
Rsp5p is also necessary for adaptation to nutrient limitation. When exogenous
amino acids are limited, S. cerevisiae cells repress the pathway that
regulates the uptake of specific amino acids. Under starvation conditions,
several amino-acid-specific permeases such as tryptophan permease (Tat2p) and
histidine permease (Hip1p) are subjected to Rsp5-mediated ubiquitination,
targeted to vacuoles and degraded therein. Conversely, the broad-range
amino-acid permease Gap1p is targeted to the plasma membrane and stabilized
(Hein et al., 1995
;
Beck et al., 1999
). For fission
yeast, there are no data concerning the regulation of amino-acid permeases in
nitrogen-limited medium. It is possible that Pub2p is responsible for
regulating the ubiquitination of membrane proteins such as amino acid
permeases. Our present results showing that Pub2p appears to be located in the
plasma membrane and that pub2+ is preferentially expressed
under starvation conditions favor such a possibility. Future studies should
examine whether Pub2p is implicated in the ubiquitination of membrane
proteins, especially under starvation conditions.
![]() |
Acknowledgments |
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