From the Instituto de Microbiología-Bioquímica/Centro de Investigación del Cáncer, Departamento de Microbiología y Genética, Edificio Departamental, Campus Miguel de Unamuno, CSIC/Universidad de Salamanca, 37007 Salamanca, Spain
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ABSTRACT |
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The Saccharomyces cerevisiae Cdc6
protein is necessary for the formation of pre-replicative complexes
that are required for firing DNA replication at origins at the
beginning of S phase. Cdc6p protein levels oscillate during the cell
cycle. In a normal cell cycle the presence of this protein is
restricted to G1, partly because the CDC6 gene
is transcribed only during G1 and partly because the Cdc6p
protein is rapidly degraded at late G1/early S phase. We
report here that the Cdc6p protein is degraded in a
Cdc4-dependent manner, suggesting that phosphorylated Cdc6
is specifically recognized by the ubiquitin-mediated proteolysis machinery. Indeed, we have found that Cdc6 is ubiquitinated in vivo and degraded by a Cdc4-dependent mechanism. Our
data, together with previous observations regarding Cdc6 stability,
suggest that under physiological conditions budding yeast cells degrade
ubiquitinated Cdc6 every cell cycle at the beginning of S phase.
In eukaryotes, duplication and segregation of the genome are
strictly regulated processes that cells must alternate for successful cell division. Studies of yeast have suggested that this coordination is achieved by means of the Ser/Thr kinase activity of
p34cdc2/cdc28, given that activation of this kinase is required
first for initiating genome replication and second for cells to enter
mitosis (1). Nevertheless, initiation of DNA synthesis in replicating
cells requires chromatin to be competent for the
p34cdc2/cdc28-mediated activation. At the molecular level,
competency means that a multisubunit protein complex, called
pre-replicative complex (pre-RC),1 must be formed at
origins of replication (ARSs) previous to fire initiation of DNA
replication. Association with chromatin is a hierarchical process in
which ARSs first recruit the origin recognition complex, then Cdc6p,
and finally the Mcm complex, consisting of Mcm2-7 proteins (2, 3). In
budding yeast Saccharomyces cerevisiae, formation of
pre-replicative complexes is dependent upon Cdc6, because association
of the Mcm proteins relies on the presence of a functional
CDC6 gene product (3, 4). Furthermore, direct genetic
evidence indicates that CDC6 is rate-limiting for the initiation of DNA replication by interacting with origin recognition complex at origins (5).
In S. cerevisiae the ubiquitin-mediated proteolysis may play
a role in cell cycle (6). Proteolysis may trigger the initiation of S
phase and the transition from metaphase to anaphase by two distinct
ubiquitin-conjugation pathways, the former requiring CDC34
and the latter involving the anaphase-promoting complex (reviewed in
Refs. 7 and 8). In G1 for example, Cln/Cdc28-phosphorylated Cln2p is targeted for degradation in a Cdc53p-dependent
manner (9). Comparably, proteolysis of Sic1p relies on Cdc4p at the G1 to S phase transition (10). In fact, Sic1p must be
Cdk-phosphorylated prior to its in vitro
Cdc34-dependent ubiquitination (11). It is well known that
the Cdc4p protein acts in concert with Cdc34p and Cdc53p to
ubiquitinate proteins (12). Given the importance of some of these
substrates, it is accepted that the Cdc4p-Cdc34p-Cdc53 complex controls
the G1 to S phase transition (reviewed in Ref. 7). All
these data support the notion that this complex may act to specifically
target degradation of proteins implicated in cell cycle control in a
CDC28-dependent phosphorylation manner (13-15).
Cdc34p is an E2-type ubiquitin-conjugating enzyme that together with
Cdc53p and Skp1p form the core complex of ubiquitination for
degradation of G1 cyclins Cln1p and Cln2p and the Cdk
inhibitor Sic1p. These ubiquitination processes require different F box proteins, whereas Grr1p participates in targeting G1
cyclins (15, 16), Cdc4p may interact with the Cdk inhibitor once it has
been phosphorylated (10, 11). It has been argued that Cdc4p may physically interact both with phosphorylated substrates and Skp1p to
form the ubiquitinating complex (reviewed in Ref. 12).
This paper addresses the mechanism degrading the key DNA replication
initiator Cdc6p protein at the G1 to S phase boundary. Cdc6p is accumulated in ubiquitin-mediated proteolysis mutants, cdc4-1 and cdc34-2. In accordance with recently
published data (17), our results indicate that Cdc4p and Cdc34p
participate in Cdc6p proteolysis, suggesting that the initiator protein
is phosphorylated for degradation through the proteasome. Consistent with this hypothesis, we present data showing that the Cdc6p protein is
ubiquitinated in vivo in budding yeast before it is degraded.
Strains, Plasmids, Culture Conditions, and General
Techniques--
All S. cerevisiae strains were derived from
15Dau unless indicated. 15Dau is a MATa derivative of BF264-15D (18).
Temperature-sensitive and deletion mutants used were as follows:
cdc4-1 leu2 ura3; cdc4-1 GAL1:CDC6(URA3) leu2 ura3; cdc4-1
sic1::URA3 leu2 ura3; cdc34-2 ura3 his3; cdc34-2
sic1::URA3 leu2 ura3; and cdc6::kanMX4
GAL1:CDC6 (URA3).
Cells were grown in YEPD (1% yeast extract, 2% Bactopeptone, 2%
glucose), YEPR (1% yeast extract, 2% Bactopeptone, 2% raffinose), or
YEPGal (1% yeast extract, 2% Bactopeptone, 2.5% galactose), except
when selecting for plasmids. In this case, cultures were grown in
minimal media with supplemented amino acids. The strain deleted for
CDC6 conditionally grows on galactose media thanks to the
GAL1:CDC6 allele inserted at the ura3 locus, as
checked by Southern blotting. Repressing with glucose results in cell cycle block due to the lack of any functional Cdc6 product, as described previously (19). Induction of genes from the CUP1 promoter was accomplished by growing cells in YEPD suplemented with 25 µM CuSO4 according to Ecker et al.
(20). General molecular techniques were performed as described (21,
22).
The DNA content of individual cells was measured using a Becton
Dickinson FACScan. Cells were prepared for flow cytometry by staining
them with propidium iodide following the method of Hutter and Eipel
(23).
Preparation of Cdc6 Antibodies--
An
NdeI-BamHI CDC6 containing fragment
was cloned into bacterial expression vector pT7-7. Escherichia
coli BL21-DE3 cells transformed with this plasmid were induced to
express the fusion protein by
isopropyl-1-thio-
The antibodies were affinity purified on nitrocellulose filter prefixed
with Cdc6Ha fusion protein purified to homogeneity. This antiserum was
incubated with the filter, washed with phosphate-buffered saline
solution, and eluted by 100 mM glycine HCl, pH 2.5. The Cdc6 affinity purified antibody was also characterized by immunoblot.
Mouse polyclonal antibodies were obtained as described for rabbit
antibodies with some modifications as follows: 12 µg of purified and
refolded Cdc6264 protein per dosage were intraperitoneally
injected into mouse with Freund's complete adjuvant. 10 days after the
third injection, ascites fluid was collected, centrifuged to discard
cell debris, and the antibody-containing supernatant titrated and
characterized by immunoblot (Fig. 3) and used in the indicated dilutions.
Protein Extract Preparation--
Soluble protein extracts were
prepared as described previously (24). Cells were collected, washed,
and broken in 30 µl of HB buffer by glass beads. HB buffer contains
60 mM Western Blotting--
Protein extracts and immunoprecipitates
were electrophoresed using 10 or 8-16% gradient SDS-polyacrylamide
gels (25). For Western blots, 40 µg of total protein extracts from
each sample were blotted to nitrocellulose, and proteins were detected
using Detection of CDC6 Gene Product--
The CDC6 gene
encodes a polypeptide of predicted molecular mass of 56 kDa. In order
to study the Cdc6 protein in budding yeast,
Affinity purified rabbit antibodies detected two bands by Western blot,
not detected by preimmune serum, of approximately 57 and 65 kDa in
cdc4-1 cells blocked in G1 (Fig.
1). The fastest species was absent from
protein extracts of strains deleted for CDC6, suggesting
that could be Cdc6. Moreover, cells overexpressing CDC6
accumulated the same molecular weight band (Fig. 1), so we concluded
that this antibody detected specifically endogenous p57CDC6.
Affinity purified mouse polyclonal Cdc6 Protein Becomes Stabilized on Ubiquitin-mediated
Proteolysis-deficient Mutant Yeast Strains--
Cdc6p and Sic1p
proteins become unstable late in G1. Given that Sic1p is
degraded through the ubiquitin-mediated proteolysis pathway (10, 11),
we were interested in studying whether stabilization of Cdc6p is
regulated in a similar way. For this reason, we monitored the Cdc6
protein content by Western blot with antiserum to Cdc6 in
cdc4-1 and cdc34-2 mutants incubated at the
restrictive temperature. Samples were taken every hour as shown in Fig.
2. Microscopic and flow cytometry
analysis confirmed that the cdc4-1 and cdc34-2 mutant strains arrested with the characteristic multibudded phenotype and that cells had a 1C DNA content. In both mutant backgrounds p57CDC6 accumulated as a single band (Fig. 2a
for cdc4-1 and data not shown for cdc34-2).
Accumulation of a given protein could be an indirect consequence
of the cell cycle defect of the mutant under analysis. A first approach
to understand whether or not Cdc4 affects Cdc6 stability was to examine
the rate of Cdc6 disappearance in promoter shut-off experiments in a
cdc4-1 GAL1-10:CDC6 strain. Expression
of CDC6 was induced in nocodazole-arrested cells by incubating them on galactose for 60 min at 37 °C. Transcription was
repressed by addition of glucose and Cdc6 stability estimated by
Western blotting either at 25 °C (permissive temperature) or 37 °C (restrictive temperature) (Fig. 2c). While Cdc6
became unstable at the permissive temperature, its levels remained
constant up to 50 min at the restrictive temperature. Therefore, cells
blocked in G2/M require Cdc4 to degrade ectopically
produced Cdc6.
These results together with data recently shown by others (17) suggest
that Cdc6p degradation requires CDC4, CDC34, and CDC53 gene products, because Cdc6p is stabilized in
cdc4/34/53 temperature-sensitive mutants. Nevertheless, it
has been described that cdc4 and cdc34 mutants
accumulate p40SIC1, a potent Cdk inhibitor (10). Cdc6p contains
eight potential Cdk consensus phosphorylation sites, and it is an
in vitro substrate for this kinase as well as an in
vivo phosphoprotein (26); furthermore, Cdc6p accumulates in
cdc28 temperature-sensitive mutants.2 Then, it
is formally possible that Cdc6p stabilizes in cdc4 mutants as a direct consequence of the drop in CDC28-associated
kinase activity due to the accumulation of Sic1p. Alternatively, this accumulation might be the consequence of a defect in CDC4
function related to Cdc6p degradation. To distinguish between these two possibilities we studied Cdc6p accumulation in two different mutant backgrounds, cdc4-1 single and cdc4-1
Cdc6 Is Ubiquitinated in Vivo--
The Cdc4p protein then plays an
unknown but essential role in the pathway of degradation of the Cdc6p
protein (Ref. 17 and this work). It has been argued that Cdc4p may
physically interact both with phosphorylated substrates and Skp1p to
form the ubiquitinating complex (12). If this is true it should be
possible to block cdc4-1 mutant cells, accumulating Cdc6p in
a phosphorylated but non-ubiquitinated form, release them from the
block, and detect ubiquitinated-Cdc6p protein prior to degradation.
With the purpose of examining the kinetic of accumulation and
degradation of ubiquitinated forms of the Cdc6p protein, an indirect
strategy was designed. A cdc4-1 yeast strain was transformed
with a plasmid carrying the ubiquitin gene tagged with the Ha epitope
(Ub-Ha) under the control of the CUP1 promoter (see
"Experimental Procedures"). Upon Cu2+ induction at
25 °C, permissive conditions for the cdc4 allele, Ub-Ha
efficiently labeled many polypeptides, as shown by Western blot with
Cdc6 Protein Is Ubiquitinated by a Cdc4-dependent
Mechanism--
To investigate if the Cdc6 protein was
polyubiquitinated in a Cdc4-dependent manner as suggested
by experiments described above, we studied Cdc6 ubiquitination in block
and release experiments in cdc4-1 and cdc4-1
Different Degree of Accumulation of Cdc6p in cdc4-1 Mutants S. cerevisiae 15Dau or W303 Strains--
Genetic interaction of mutant
alleles of two different genes may reflect in vivo direct
protein association or an association-independent but functional
interaction between wild type gene products. This genetic approach is
called synthetic lethality analysis. It has been shown that Cdc4p and
Cdc6p proteins interact through the amino terminus of Cdc6p (17), then
it is expected that cdc6ts mutants may interact with
cdc4ts mutant yeast strains. Indeed, in our strain
background, GAL1:CDC6 exhibited at 25 °C synthetic lethal
interactions with the cdc4-1 mutation (Fig.
5a); furthermore, Cdc6p
overproduction rendered multiple and elongated budded cells, phenocopying the cdc4-1 terminal arrest phenotype at the
restrictive temperature.
These data are partially in contrast with the recently published
results of Drury et al. (17). They have published that overproduction of Cdc6p in cdc4-1 mutants does not block
cell growth, although overproduction causes dramatic changes in cell morphology at the permissive temperature in a W303 S. cerevisiae strain (17).2 This phenotype is very
similar to the cdc4-1 arrest phenotype and is
indistinguishable from the terminal phenotype of the cdc4-1 GAL1-10:CDC6 described above. Since Cdc6p
overproduction affects distinctly S. cerevisiae 15Dau and
W303 strains (17, 26, 27), we wondered if these contradictory results
may be explained by differences in Cdc6p accumulation in these strain
backgrounds. We monitored p57CDC6 content by Western analysis
with rabbit antiserum to Cdc6p in parallel experiments with
cdc4-1 mutants of the two strain backgrounds, 15Dau and
W303. Exponentially growing cultures were shifted to the restrictive
temperature. Samples were taken at 1-h intervals for FACS analysis and
protein detection. Flow cytometry and microscopic analysis demonstrated
that cells were arrested in G1. Western analysis of cell
extracts revealed that Cdc6p accumulated more rapidly and to a greater
extent in S. cerevisiae 15Dau cells (Fig. 5b),
suggesting a more dramatic phenotype of the cdc4-1 mutation in this strain background. In this context, the overproduction of Cdc6p
in a cdc4-1 15Dau mutant strain is likely to allow cells to
accumulate the initiator protein to higher levels than
cdc4-1 W303. This circumstance might diminish further Cdc4-1
protein function causing the stabilization of Sic1p that would mimic
the arrest phenotype of the cdc4-1 allele at the restrictive temperature.
A distinguishing feature of the Cdc6/Cdc18 class of proteins is
their oscillatory pattern of appearance each cell cycle during G1 due in part to its periodic destruction as cells enter S
phase (17, 19). Recently, it has been reported that the Cdc6p protein interacts with Cdc4p and that this interaction is required for its
degradation (17). To address details regarding the mechanism and the
timing of Cdc6p degradation further, we have combined genetic analysis
and yeast biochemistry in studying these questions. The major
conclusion of this report is that Cdc6p is ubiquitinated each cell
cycle in a Cdc4p-dependent manner.
Several important details regarding the Cdc4-Cdc34-mediated degradation
of Cdc6 are reported in this paper. First, although Drury et
al. (17) reported that cdc4ts, cdc34ts, and
cdc53ts mutants do not degrade Cdc6p, their experiments were
made ectopically expressing CDC6 from the
GAL1-10 promoter. Corroborating their results, we
report here that Cdc6p accumulates at the block point of
cdc4ts and cdc34ts mutants (Fig. 2). Second, the
Cdc4-Cdc34-Cdc53 complex controls the G1 to S phase
transition ubiquitinating for degradation the potent Cdk inhibitor Sic1
(10, 11) (reviewed in Refs. 7 and 12). As previously discussed (Fig.
2), our results regarding the accumulation of Cdc6p in
cdc4-1 The closest homologue of CDC6 in fission yeast,
cdc18+ is also required for DNA replication
(28). Cdc18 protein levels oscillate during the cell cycle, peaking at
G1 as Cdc6 does (19, 29). Although at present it is unknown
if the Cdc18 protein is degraded at the same time that DNA replication
is initiated, given that the overexpression of
cdc18+ induces continuous DNA synthesis (29,
30), it has been suggested that proteolysis of this protein might play
a key role in limiting DNA replication to once per cell cycle. On the
other hand, it has been shown that Cdc18 interacts in vivo
with Pop1 and Sud1, Cdc4-related proteins in fission yeast, and that
Cdc18 protein accumulates in pop1 and sud1
mutants as well as in proteasome-deficient mutants of the fission
yeast, in the latter mutants possibly forming ubiquitin conjugates (31,
32). These data and the results presented here show that degradation of
Cdc6/Cdc18 class of initiator proteins is conserved at least in simple
eukaryotes such as yeast. Despite these similarities, the degree of
competence of this degradation mechanism in controlling genome ploidy
appears to be slightly different in fission and budding yeast. In
Schizosaccharomyces pombe, deletion of either
pop1+ or sud1+ results in
cells becoming polyploids, suggesting that Cdk-regulated proteolysis
may be critical to ensure a single round of DNA replication (31-33).
Ploidy control in budding yeast appears to be a Cdk-controlled redundant mechanism in which some of the pre-RCs components may be
degraded, as is the case for Cdc6 (Refs. 17, 19, and this work),
whereas association and/or localization of others may be regulated by
Cdk phosphorylation (34-37). Consistent with this notion,
cdc4ts mutants do not polyploidize under restrictive
conditions (10), or cells expressing a stable form of Cdc6 do not
re-replicate (17). In any case, budding yeast Cdc4-regulated Cdc6p
proteolysis is part of the mechanism restricting genome duplication to
S phase, because in the absence of Cdc6p no pre-RCs are formed (4). Multicellular eukaryotes apparently have developed a slightly different
mechanism in which nuclear non-phosphorylated Cdc6 associates with
chromatin; meanwhile, Cdk-phosphorylated Cdc6 is probably membrane-bound or cytosolic and therefore unable to interact with chromatin. This has been suggested for Xenopus and
Homo sapiens Cdc6 protein homologues (38-39). Whether or
not degradation of nuclear Cdc6 plays a role in maintenance of genome
ploidy in higher eukaryotes remains to be elucidated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside. Cdc6 protein was
purified to homogeneity from inclusion bodies by solubilization in 6 mM NaH2PO4, 4 mM
Na2HPO4, 2% SDS, pH 6.8 buffer. Solubilized protein was loaded into a 11% SDS-polyacrylamide electrophoresis gel
and purified by electroelution. SDS was removed and fusion protein
partially refolded dialyzing against 25 mM Tris, 192 mM glycine, pH 8.3 buffer. Protein concentration was
estimated by comparing with bovine serum albumin standards. Rabbits
were immunized with 25 µg of full-length Cdc6. After the third
injection, antibody-containing serum was collected and titrated by immunoblot.
-glycerophosphate, 15 mM P-nitrophenyl
phosphate, 25 mM MOPS, pH 7.2, 15 mM
MgCl2, 15 mM EGTA, 1 mM
dithiothreitol, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 20 µg/ml leupeptin
and aprotinin. Glass beads were washed with 500 µl of HB buffer, and the supernatant was recovered. Protein concentration was measured using
the BCA assay kit (Pierce).
-Cdc6 affinity purified polyclonal mouse antibody (1:3000), or rabbit (1:100), or the anti-Cdc28 polyclonal antibody (9) (1:5000). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies and the ECL kit (Amersham Pharmacia Biotech) were used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Cdc6 polyclonal
antisera were produced by injecting rabbits with the full-length
protein or mouse with a truncated Cdc6 polypeptide (named
Cdc6264) both made in E. coli (see
"Experimental Procedures"). This 30.6-kDa polypeptide lacks the
carboxyl-terminal 249 amino acids downstream of the region interacting
with Cdc28 and Cdc4 (17, 26). Cdc6 protein was only detected in
immunoblots of protein extracts of cdc28ts,2
cdc4-1, or cdc34-2 mutants blocked at the
restrictive temperature. Neither with rabbit
-Cdc6 nor with mouse
-Cdc6264 antibodies did we detect Cdc6 protein in
immunoblots of extracts from asynchronous cultures of wild type cells,
indicating that it is not an abundant protein.
-Cdc6264 antibodies
identified three bands of approximately 53, 57, and 70 kDa present in
lysates from cdc4-1 cells blocked in G1. The p57
species was absent in cell lysates of a strain deleted for CDC6 and
prominent in extracts from cells overexpressing that gene (Fig. 3),
indicating that this single band is the endogenous Cdc6.
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Fig. 1.
Immunoblot detection of the Cdc6
protein. Upper panel, protein extracts prepared from
cells deleted for CDC6 (left lane) or
cdc4-1 mutant cells incubated at 37 °C for 30 or 60 min
(middle lanes), or cells overexpressing CDC6
(right lane) were analyzed by immunoblotting with affinity
purified rabbit antiserum to Cdc6 or Cdc28. Lower panel,
induction of Ub-Ha in cdc4-1 mutant cells. Immunoblot assay
of cell extracts from cdc4-1 ubiquitin-Ha-expressing strain
after 0, 1, and 2 h of induction of the copper metallothionein
(CUP1) promoter. Western blots were probed with Ha
monoclonal antibody. The band in lane 0H is a yeast protein
that cross-reacts with the Ha antibody as described previously
(40).
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Fig. 2.
Wild type p57CDC6 becomes stable
either in G1 or in G2 in a
Cdc4-dependent manner. a, immunoblot analysis of
cell extracts from cdc4-1 single mutant or cdc4-1
sic1::URA3 double mutant strains arrested at 37 °C.
Cells were grown on YEPD media until they reached mid-log phase and
then were blocked at 37 °C. Samples were taken at 1-h intervals, and
split in two for FACS and Western analysis. Cell lysates were resolved
by SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose membranes, and probed with rabbit -Cdc6 or
-Cdc28
antibodies, the latter for loading control. b, DNA content
analysis of cdc4-1 and cdc4-1
sic1::URA3 strains. Flow cytometry analysis and
microscopic examination on samples taken at indicated intervals showed
that after 3 h at the restrictive temperature cells were uniformly
blocked in G1 (for cdc4-1) or G2
(for cdc4-1 sic1::URA3), and by 4 h all cells
had the multibudded phenotype associated to the cdc4-1
mutation. c, immunoblot analysis of cell extracts from
cdc4-1 GAL1-10:CDC6. This double
mutant strain was blocked with nocodazole at 25 °C, and galactose
was added for CDC6 induction for 60 min at 37 °C.
Afterward, GAL1-10:CDC6 expression was repressed
by addition of glucose (time 0). Culture was split in two
and further incubated at 25 or 37 °C. Samples were taken at
indicated intervals, processed for Western blotting, and probed with
affinity purified
-Cdc6 or
-Cdc28 antibodies.
sic1 double mutants. The latter strain, although
incapable of performing the Cdc4 function at the restrictive
temperature (37 °C) and in contrast with the single mutant, arrests
with a 2C DNA content (Fig. 2b) consistent with a
G2-like arrest as previously shown by others (10). We therefore analyzed by Western blot the content of Cdc6 protein in a
cdc4-1 sic1::URA3 double mutant
arrested at 37 °C by taking samples at regular intervals. As shown
in Fig. 2, Cdc6 also accumulated rapidly under these conditions, and
the kinetics of stabilization was comparable to the cdc4-1
single mutant. Similar results were obtained for cdc34-2 and
cdc34-2 sic1::URA3 strains.
We conclude that Cdc6p is degraded by the
CDC4-CDC34 ubiquitin-mediated pathway because
these data indicate that it is due to a genuine defect on
CDC4-CDC34 function.
-Ha monoclonal antibody (Fig. 1, lower panel). By using this efficient labeling, we wished to see if Cdc6p was one of the
Ub-Ha-tagged proteins. A block and release experiment was carried out
in which samples were taken at regular intervals for immunoprecipitation and DNA content analysis. Either cdc4-1
or cdc4-1 CUP1:UBHA strains were
incubated at 25 °C in the presence of Cu2+ for 2 h
before shifting to 37 °C. Cells were incubated for an additional
2 h at this temperature to allow them to accumulate Cdc6p and then
were released at 25 °C. After releasing, samples were taken at 0, 5, 20, 35, and 65 min; protein extracts were quantitated,
immunoprecipitated with
-Ha monoclonal antibody, and immunoblotted
using mouse
-Cdc6 polyclonal antisera. This affinity purified
antibody recognizes a single Cdc6-related band in cells overexpressing
CDC6 or in cdc4-1-blocked cultures (Fig. 3a, left panel) but
reacts with multiple high molecular weight bands as well as low
molecular weight bands in cdc4-1
CUP1:UBHA (Fig. 3a, right panel);
these bands were absent in the cdc4-1 control strain grown
under identical conditions (Fig. 3a, right panel),
suggesting that they could correspond to Cdc6p ubiquitin conjugates. To
investigate this possibility a second block and release experiment was
done in which samples were immunoprecipitated with crude antisera to
Cdc6 and immunoblotted with
-Ha antibody obtaining comparable
results (Fig. 4a, left panel).
Given that after releasing cultures from the block the proteolytic
machinery is re-activated, we interpret that low molecular weight bands are Cdc6p degradation products. These analyses revealed that Cdc6p protein was ubiquitinated in vivo. Ubiquitin conjugates of
Cdc6p reached a maximum at 5 and 20 min after releasing cells at
25 °C and then disappeared, that is to say coincident with S phase initiation as analyzed by flow cytometry (Fig. 3b).
Ubiquitinated-Cdc6p forms were not further detected. Our results,
together with previous observations demonstrating that the Cdc6 protein
is degraded early in S phase (17, 19), suggest that p57CDC6 is
polyubiquitinated and degraded as soon as budding yeast cells initiate
genome replication.
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Fig. 3.
Cdc6 is ubiquitinated in
vivo. a, left panel, a mouse antibody that
specifically detects the Cdc6 protein in yeast cell extracts.
Immunoblot analysis with affinity purified mouse -Cdc6 antibody of
protein extracts from strains deleted for CDC6 (left
lane), overexpressing CDC6 (middle lane),
and cdc4-1 incubated 1 h at 37 °C (right
lane). As for Fig. 1, the CDC6 deletion control sample
was obtained by repressing on glucose CDC6 expression in the
strain cdc6::URA3 GAL1:CDC6; nevertheless it was
able to grow on galactose-based media. Right panel,
immunodetection of ubiquitinated forms of Cdc6 in S. cerevisiae cells. cdc4-1 or cdc4-1 Ub-Ha
mutant cells were blocked for 2 h at 37 °C and then released at
25 °C (permissive temperature). Ubiquitin-Ha (Ub-Ha) was expressed
from the copper metallothionein (CUP1) promoter by
previously incubating cells in YEPD with 25 µM
Cu2+ for 2 h and then shifting them to 37 °C in the
same media to start the experiment as described above. Samples were
taken at indicated intervals for immunoprecipitation (Ip).
Protein extracts were immunoprecipitated with Ha monoclonal antibody,
blotted, and probed with affinity purified mouse
-Cdc6 antibody.
b, flow cytometry analysis of DNA content of
cdc4-1 and cdc4-1 Ub-Ha cells. Aliquots of
samples described in a (right panel) were
analyzed by FACS. Note that 65 min after releasing at the permissive
temperature (25 °C) most cells have synchronously completed S
phase.
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Fig. 4.
p57CDC6 is ubiquitinated in
G1 or G2 by a Cdc4-mediated mechanism.
a, immunodetection of ubiquitinated Cdc6 in
cdc4-1 CUP1:UBHA or cdc4-1
sic1 CUP1:UBHA S. cerevisiae cells.
Ub-Ha-induced cells were blocked 2 h at 37 °C and then released
at 25 °C. Samples were taken as indicated for immunoprecipitation
with crude mouse
-Cdc6 antibody. Immunoprecipitates were blotted and
then probed with
-Ha monoclonal antibody. The 52-kDa band present in
all samples corresponds to mouse Ig. b, immunoblot analysis
of cdc4-1 GAL1-10:CDC6
CUP1:UBHA. As in Fig. 2c, cells incubated in
the presence of Cu2+ (to induce
CUP1:UBHA) were blocked in nocodazole at
25 °C. CDC6 was expressed for 60 min at 37 °C by
addition of galactose; afterward its expression was repressed with
glucose (time 0). Culture was split in two and incubated at
25 °C or 37 °C. Samples were taken at regular intervals. Protein
samples were immunoprecipitated with
-Ha monoclonal antibody,
blotted, and probed with affinity purified mouse
-Cdc6
antibody.
sic1 mutant strains transformed with a plasmid containing
the CUP1:UBHA allele. As discussed earlier even
though both strains are defective for Cdc4 function at 37 °C, the
latter double mutant mimics a G2 block due to the lack of
sic1 function (10). Cells expressing Ub-Ha were blocked at
37 °C to accumulate Cdc6. After releasing at 25 °C samples were
immunoprecipitated with crude antisera to Cdc6 and immunoblotted with
-Ha monoclonal antibody. High molecular weight bands, Cdc6-ubiquitin
conjugates, were detected in both mutant backgrounds (Fig.
4a); these bands were absent in control strains lacking the
CUP1:UBHA plasmid (data not shown). These results
suggest that formation of ubiquitin conjugates of Cdc6 requires the
Cdc4 function. Nevertheless, a second strategy was used to show a Cdc4
dependence of Cdc6 ubiquitination. Similar to the experiment described
in Fig. 2c, cdc4-1 GAL1-10:CDC6 CUP1:UBHA cells expressing Ub-Ha were blocked in
nocodazole for 2.5 h at 25 °C. Flow cytometry and microscopic
analysis demonstrated that cells were arrested in G2/M
(data not shown). Expression of CDC6 was induced at 37 °C
in these nocodazole-arrested cells by addition of galactose. After 60 min, transcription was repressed by adding glucose. Appearance of
ubiquitin conjugates of Cdc6 was tested either at 25 (permissive
temperature) or 37 °C (restrictive temperature) (Fig.
4b). In this experiment samples were immunoprecipitated with
-Ha monoclonal antibody and immunoblotted with affinity purified
-Cdc6. Consistent with a Cdc4-dependent ubiquitination, multiple high and low molecular weight bands were detected at the
permissive temperature for the cdc4-1 allele. These bands were absent at the restrictive temperature (Fig. 4b, left
panel). Under these conditions ubiquitinated Cdc6 was
immunoprecipitated even 45 min after repressing CDC6
expression; we interpret that this fact is a consequence of the massive
expression of the CDC6 gene from the
GAL1-10 promoter. Together these results indicate that a Cdc4-dependent mechanism ubiquitinates Cdc6 for
degradation in budding yeast.
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Fig. 5.
Synthetic lethality among CDC4
and CDC6 mutants in S. cerevisiae
15Dau strain background. a, overexpression of
CDC6 is lethal in a 15Dau cdc4-1 strain
background. Upon galactose induction the double mutant arrests as
multibudded cells with a single nuclei. Dilutions of equal numbers of
cells of cdc4-1 single mutant and cdc4-1
GAL1:CDC6 double mutant strains were incubated at 25 °C
on glucose (upper panel) or galactose (lower
panel) plates for 48 h and photographed. The single mutant
control is shown for comparison. b, different degree of
accumulation of wild type Cdc6 protein in S. cerevisiae
15Dau or W303 strain backgrounds. Western blots on cdc4-1
15Dau and cdc4-1 W303 mutants are shown. Protein samples
were obtained at indicated intervals, electrophoresed, blotted, and
probed with rabbit antiserum to Cdc6 or Cdc28, the latter as a loading
control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sic1 double mutants indicate that the
initiator protein stabilizes due to a genuine defect in the
ubiquitination pathway rather than a drop in kinase activity. Third,
the requirement of Cdc4 and Cdc34 suggests that Cdc6 degradation may
involve ubiquitination of the initiator protein; however, it does not
prove it. Nevertheless, we show here cdc4-1 block and
release experiments in which Cdc6p is detected forming ubiquitin
conjugates. Our data, together with previous observations regarding
Cdc6 stability (17, 19), show that ubiquitination and proteolysis are
consecutive steps in Cdc6p degradation that occur soon after the
transition of G1 to S phase is initiated. Moreover, by
arresting cells in different points of the cell cycle, we have shown
that Cdc6 is ubiquitinated in a Cdc4-dependent manner (Fig.
4). Although these data suggest that proteolysis of Cdc6 might be
catalyzed by the proteasome, at present we do not know if this is the
case. Further experiments need to be done in order to show that
proteasome is indeed involved in Cdc6 proteolysis even though it is
likely to be the mechanism.
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ACKNOWLEDGEMENTS |
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We thank Francisco Antequera, Jaime Correa, Karim Labib, Sergio Moreno, and the ASP laboratory for many helpful discussions. We thank Karim Labib for critically reading this manuscript.
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FOOTNOTES |
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* This research was supported by CICYT and Fundación Ramón Areces grants (to A. B.).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.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 34 923121589; Fax: 34 923224876; E-mail: abn{at}gugu.usal.es.
2 M. Sánchez, A. Calzada, and A. Bueno, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: pre-RC, pre-replicative complex; MOPS, 4-morpholinepropanesulfonic acid; Cdk, cyclin-dependent kinase; Ha, hemagglutinin; Ub, ubiquitin; FACS, fluorescence-activated cell sorter; ARS, autonomously replicating sequence.
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REFERENCES |
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