From the Division of Biological Science, Graduate School of Science, Nagoya University and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Furo-cho, Chikusa, Nagoya 464-8602, Japan
Received for publication, August 30, 2002, and in revised form, October 9, 2002
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ABSTRACT |
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Physical interactions among clock-related
proteins KaiA, KaiB, KaiC, and SasA are proposed to be important for
circadian function in the cyanobacterium Synechococcus
elongatus PCC 7942. Here we show that the Kai proteins and SasA
form heteromultimeric protein complexes dynamically in a circadian
fashion. KaiC forms protein complexes of ~350 and 400-600 kDa during
the subjective day and night, respectively, and serves as a core of the
circadian protein complexes. This change in the size of the
KaiC-containing complex is accompanied by nighttime-specific
interaction of KaiA and KaiB with KaiC. In various arrhythmic mutants
that lack each functional Kai protein or SasA, circadian rhythms in
formation of the clock protein complex are abolished, and the size of
the protein complexes is dramatically affected. Thus,
circadian-regulated formation of the clock protein complexes is
probably a critical process in the generation of circadian rhythm in cyanobacteria.
Circadian rhythms, biological oscillations with a period of ~24
h, are observed in many physiological events of a wide variety of
organisms. At the molecular level, several clock-related genes have
been identified and intensively analyzed in cyanobacteria, Neurospora, Arabidopsis, Drosophila,
and mammals (1, 2).
Cyanobacteria are the simplest organisms known to have the circadian
clock and provide both genetic and physiological model systems (3, 4).
Previously we have monitored circadian gene expression in the
cyanobacterium Synechococcus elongatus PCC 7942 using a
bioluminescence (luciferase) reporter gene set (5), isolated various
mutants that impaired normal circadian phenotypes (6), and identified
the clock gene cluster kaiABC (7). Molecular genetic studies
suggested functions of KaiA and KaiC as positive and negative elements
for kaiBC expression, respectively, to form a negative
feedback loop (7). This regulatory loop has been proposed as an
important step to drive circadian oscillation in kaiBC
expression (7).
To elucidate the molecular mechanism of the cyanobacterial circadian
clock, it is essential to discover the biochemical properties of the
Kai proteins. KaiB and KaiC accumulate in a circadian fashion in
cyanobacterial cells, peaking at circadian time
(CT)1 15-16 in continuous
light conditions. (CT indicates the subjective time of a day (h) in
constant environmental conditions; CT 0 corresponds to the subjective
dawn. In contrast, Zeitgeber time (ZT) is used to indicate time in a
light-day cycle; ZT 0 indicates dawn time in a light-day cycle.) This
accumulation follows kaiBC mRNA rhythms by ~8 h (8).
KaiC binds to ATP in vitro through nucleotide-binding domains (P-loops), and mutations of these motifs severely alter the
circadian function (4, 9). Moreover, KaiC undergoes autophosphorylation
in vitro (9). Another important characteristic of the Kai
proteins is their physical association in various combinations in yeast
cells, in vitro, and in Synechococcus cells (10).
A long period mutation of KaiA affects the strength of the KaiA-KaiC interaction, suggesting an important role of such protein interactions in circadian time-keeping (10). We have also identified another KaiC-interacting partner, SasA, which is a sensory histidine kinase (11). Although SasA is not an essential oscillator component, SasA is
important in enhancing kaiBC expression and likely forms a
secondary loop with KaiC to stabilize the Kai-based primary molecular
circuit. SasA is also involved in metabolic growth control under
day/night cycle conditions (11). However, biochemical functions and
molecular behaviors of the Kai proteins and SasA remain largely unknown
in Synechococcus cells.
In this study, we biochemically examined temporal profiles of the clock
protein interactions in Synechococcus. Gel filtration chromatographic analysis demonstrated that the Kai proteins and SasA
form large protein complexes and suggested that the complexes change
their stoichiometry during the circadian cycle. Immunoprecipitation studies confirmed that the Kai proteins and SasA circadianly associate in large KaiC-containing protein complexes. Dramatic changes in the
size of Kai/SasA-containing complexes in kaiA-,
kaiB-, kaiC-, and sasA-inactivated
strains further support that circadian formation of the clock protein
complexes has a function in the circadian regulatory mechanism.
Bacterial Strains and Cultures--
NUC39 and NUC38 (10) were
used as the wild-type and kaiABC-depleted
Synechococcus strains, respectively. We also used
kaiA-, kaiB-, and kaiC-inactivated
strains (7), a sasA-disrupted strain (11), and the
KaiC(K52H) mutant strain (9). Synechococcus cells
were grown at 30 °C in BG-11 liquid medium under continuous light
(LL) conditions of 46 µmol Immunoblotting Analysis--
Synechococcus cells were
grown in BG-11 liquid medium to an optical density of 0.2 at 730 nm (OD730). The cells were exposed to two cycles of 12-h
light/12-h dark (LD) alternation and then placed in LL conditions and
harvested in LD and LL. The cell pellets were collected from the
culture and then immediately resuspended in 200 µl of binding buffer,
BB5 (50 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2, 0.2% glycerol, 0.1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 3 µg/ml
leupeptin, 3 µg/ml pepstatin, 0.1 mM NaF, 0.1 mM Na3VO4, 0.1 mg/ml DNase I, pH
8.0), and disrupted by sonication using the Ultrasonic Liquid Processor
(Misonix) or the BioRupter (Cosmo) at 4 °C. After centrifugation,
the resulting supernatant was used for immunoblotting,
immunoprecipitation, or gel filtration chromatographic analyses.
Immunoblot analyses, shown in Figs. 1 and 4B, were performed
with 5 µg of total cell extracts that were subjected to SDS-PAGE on
10 or 15% gels and blotted onto nitrocellulose membranes. The KaiA,
KaiB, KaiC, and SasA proteins were analyzed with specific antisera (10,
11) at a 1:500, 1:2000, 1:2000, and 1:2000 dilution, respectively, and
detected with the enhanced chemiluminescence (ECL) method
(Amersham Biosciences) according to the manufacturer's protocol.
The specificities of these antibodies have been confirmed and
described previously (10, 11).
Immunoprecipitation--
Immunoprecipitation was performed as
described previously (11) with some modifications. Briefly, protein
extracts in 360 µl of BB5 (total, 500 µg of protein) were prepared
as described above. Then the extracts were supplemented with 40 µl of
5% bovine serum albumin in 0.1 M KCl and then incubated
with a 10-µl bed volume of prewashed AffiGel-Hz beads (Bio-Rad)
coupled to purified rabbit anti-KaiC IgG at 4 °C for 2 h. After
six washes with 1 ml of BB5 without DNase I, the beads were resuspended
in 80 µl of SDS sample buffer without reducing agents. Proteins were
eluted by vortexing gently for 10 min at room temperature. After
centrifugation, the supernatant was collected, supplemented with
0.1% 2-mercaptoethanol and 0.1% bromphenol blue, and subjected
to SDS-PAGE and immunoblotting.
Gel Filtration Chromatography--
All protein
chromatographic steps were performed with an Amersham Biosciences FPLC
system. Aliquots (100 µl) of cell extracts in BB5 as prepared above
were size-fractionated by gel filtration chromatography with Superdex
200 (Amersham Biosciences) at 4 °C. Protein standards used for
calibration of the gel filtration column included thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactose dehydrogenase
(140 kDa), bovine serum albumin (67 kDa), carbonic anhydrase (30 kDa),
and KaiA, KaiB, and SasA Interact with KaiC in a Circadian
Manner--
To analyze temporal dynamics of clock protein
interactions, we first evaluated the temporal accumulation profiles of
the Kai proteins and SasA in Synechococcus cells. Soluble
protein extracts were prepared from cells that had been collected at
4-h intervals either during one LD cycle or during LL conditions.
Immunoblotting analysis confirmed that the amounts of the KaiB and KaiC
proteins oscillate, peaking at subjective dusk, and the amount of KaiA protein oscillates with a low amplitude rhythm under an LD cycle or LL
conditions (Fig. 1A) as
reported previously (8).
KaiC is a master circadian regulator, proposed to be a negative factor
for kaiBC expression (7). Recent analysis suggested that
KaiC is also responsible for activation of the kaiBC
promoter (11). To examine whether KaiC interacts with other
clock-related proteins, such as positive factors KaiA and SasA, in a
phase-dependent manner, we performed co-immunoprecipitation
analysis using anti-KaiC antiserum. The resulting immunoprecipitated
materials were blotted and probed with anti-KaiA, anti-KaiB, anti-KaiC,
or anti-SasA antiserum. The results demonstrated that KaiC interacts
rhythmically with KaiA, KaiB, and SasA (Fig. 1B). KaiA,
KaiB, and SasA bound to KaiC more abundantly during subjective night in
LL. The level of SasA interacting with KaiC was abundant at 12-16 in
LL; KaiC also accumulated abundantly at this time. On the other
hand, the levels of KaiA and KaiB interacting with KaiC followed the
KaiC accumulation rhythms with a 4-h delay, peaking at hours 20-24 in
LL (see "Discussion").
KaiA, KaiB, KaiC, and SasA Form Large Protein Complexes in a
Circadian Fashion--
The Kai proteins and SasA interact in a
circadian manner, but the in vivo dynamics of their complex
formation is still largely unknown. To estimate the size of clock
protein-containing complexes in cells, soluble protein extracts were
prepared from Synechococcus cells at hours 4, 10, 16, and 22 in LD cycles, size-fractionated by filtration chromatography, and then
subjected to immunoblotting analyses.
As shown in Fig. 2A, KaiA
showed a dramatic change in its size throughout an LD cycle. KaiA
occurred as a complex of ~60 kDa, which is close to the size of a
KaiA dimer at ZT 10, whereas it occurred as both a ~500-kDa complex
and as the ~60-kDa complex at ZT 16. At ZT 22 and ZT 4, KaiA was
fractionated more broadly, and the ~60-kDa complex and a fraction
tail of smaller size were more abundant at ZT 4. These results indicate
that KaiA forms larger protein complexes in the night and is present in
smaller forms in the day.
KaiB was fractionated mainly as two portions: a broad tail of the
larger fractions (200-600 kDa) and a smaller ~20-kDa fraction, which
is close to the size of a KaiB dimer. At ZT 10, KaiB was mainly found
in the ~20-kDa fraction. By contrast, at ZT 22 most of KaiB molecules
were present in larger complex fractions, and only faint signals were
detected from the ~20-kDa portion.
KaiC forms protein complexes of ~350 kDa at ZT 4 and 10, whereas it
was present in larger complexes of 500-670 kDa at ZT 16 and 22. Less
KaiC was detected at ZT 10 and 16 in a fraction of the ~60-kDa
portion, which might corresponded to its monomeric form.
SasA was found during the daytime (ZT 4 and 10) in mainly two portions,
peaking at 400-600 and 200 kDa, respectively. In the nighttime (ZT 16 and 22), the 200-kDa signal disappeared, and SasA was present as large
protein complexes of 400-600 kDa. As summarized in Fig. 2B,
these data show that during the night all four clock-related proteins
were mainly co-fractionated in ~400-600-kDa portions, whereas they
separated into different fractions during the daytime, especially at ZT 10.
Next we examined the size of the clock-related proteins in cells
cultured under LL conditions after two LD cycles (Fig.
3A). The results were
essentially similar to those under LD cycles. Clock-related protein
molecules were more abundant during subjective night, occurring as
400-600-kDa protein complexes, and they separated into different
fractions during subjective day (Fig. 3B). All these results
support the hypothesis that the clock-related proteins interact with
each other and form large protein complexes during (subjective) night
and tend to dissociate from each other during (subjective) day.
Effects of Disruption of Clock Genes on the Size of the Clock
Protein Complexes--
To characterize the nighttime-specific
large complexes, we performed gel filtration chromatography on the Kai
and SasA proteins from various clock gene-inactivated strains (Fig.
4A). Inactivation of the
kaiA, kaiB, or kaiC gene each
abolishes circadian rhythms (7), and disruption of the sasA
gene reduces kaiBC expression with lowered amplitude and a
shortened period length in a light intensity-dependent manner (11).
In both kaiB- and kaiC-null mutants, KaiA failed
to form the 400-500-kDa complex and was present predominantly as the
~60-kDa form throughout the day (Fig. 4A; data not shown).
These results indicate that both KaiB and KaiC are required for the
400-600-kDa KaiA-containing complex during the (subjective) night.
KaiB also failed to form the large complex in both kaiA- and
kaiC-null mutants and was exclusively present as a 20-kDa
form. Thus, both KaiA and KaiC are necessary for the large
KaiB-containing protein complex.
In both kaiA- and kaiB-null strains, KaiC and
SasA were co-present as 440-670-kDa forms, which are significantly
larger than the major form of the night-specific KaiC complex. The size
of these complexes is, however, very close to that of the larger SasA
complex or larger tail of the KaiC complex in the wild-type strain in
LL 22 (Fig. 4A). The 440-670-kDa SasA-containing complex in
the mutant strains probably contains KaiC because this form is
dramatically reduced in the kaiC-null mutant (Fig.
4A). These results support the hypothesis that SasA
serves as a main partner for KaiC in the absence of KaiA or KaiB (see
"Discussion").
We also characterized the size of Kai protein complexes in the
sasA-deficient strain under LL conditions (50 µmol
m
To better understand the relationship between a biochemical property of
KaiC and the protein complex formation, we analyzed in vivo
clock protein behaviors in a KaiC(K52H) strain in which an
ATP-binding domain of KaiC (P-loop1) was mutated (9). This mutation
dramatically reduces the ATP binding activity of KaiC in
vitro, and the mutant cyanobacterial strain nullifies the
circadian clock (9). We first examined the level of the Kai proteins and SasA in the mutant. Accumulation of all four proteins did not
exhibit any detectable circadian fluctuations (Fig. 4B).
Compared with wild-type strains, KaiA and KaiB were present
constitutively at relatively high levels. In contrast, the accumulation
level of KaiC was reduced dramatically in this strain (Fig.
4B). Because the kaiBC promoter activity is not
evidently down-regulated in the KaiC(K52H) strain (9), it is
plausible that the ATP binding property is important for stability of
the KaiC protein. The level of SasA in the mutant strain was similar to
that in the wild-type cells. Immunoprecipitation analysis using
anti-KaiC antiserum failed to detect KaiC-interacting KaiA or KaiB
protein, but faint signals were observed for SasA bound to KaiC (Fig.
4C). Gel filtration analysis followed by immunoblotting
confirmed that, in the KaiC(K52H) mutant, KaiA and KaiB did not
form the protein complexes of >400 kDa and instead were found only in
distinct fractions of ~60 and ~14 kDa, respectively (Fig
4A). SasA was dominantly found as a complex of ~140 kDa,
while lesser amounts of SasA were also detected broadly in fractions of
~400-600 kDa (data not shown). These profiles were essentially the
same as in the kaiC-deficient mutant (Fig. 4A)
and were the same as the day phase patterns in the wild-type strain
(Fig. 3).
KaiC Forms Large Protein Complexes with KaiA, KaiB, and
SasA--
All the results mentioned above support KaiC as a scaffold
protein for the heteromultimeric clock protein complexes. Thus, an
immunoprecipitation assay with anti-KaiC antiserum was performed using
KaiC-containing fractions after the gel filtration assay (fractions
19-24, 28, and 29), and results were examined to determine whether
KaiA, KaiB, and SasA were co-precipitated with KaiC. As shown in Fig.
5, A and B, KaiA
and KaiB interacted with KaiC solidly in the 400-600-kDa fractions,
peaking at ~500 kDa during the subjective night. The SasA protein
interacted with KaiC in the 400-670-kDa complexes, especially in the
500-670-kDa region. The level of SasA bound to KaiC also exhibited a
circadian rhythm, while the amplitude was lower compared with the
rhythms of KaiA and KaiB interacting with KaiC. These results further
confirmed that the Kai proteins and SasA interact to form 400-670-kDa
protein complexes in a circadian fashion.
We have previously demonstrated that KaiC enhances KaiA-KaiB
interaction in vitro and in the yeast two-hybrid system and
predicted formation of a heteromultimeric complex that contains the
three Kai proteins (10). We also reported limited information on
KaiC-SasA and KaiB-SasA interactions in cyanobacteria by
immunoprecipitation analysis at two points of the day (11). In
contrast, this report provides more comprehensive details on in
vivo dynamics of the clock protein complex formation, which must
be a basis for understanding molecular actions of these circadian clock
elements in the cell.
Our results showed that the Kai proteins and SasA are present in
various complexes during the circadian cycle. In the subjective day,
KaiC forms complexes ranging from 350 to 440 kDa. The small KaiC-containing complex (350 kDa; fraction 23 in Figs. 2 and
3) corresponds to the size of a KaiC hexamer (calculated mass of 354 kDa). This could be possible because the amino acid sequence of KaiC is
similar to those of RecA/DnaB superfamily proteins, which generally
form hexamers (12). In the subjective night, KaiC additionally forms
larger protein complexes, most commonly around 500 kDa (Figs. 2 and 3).
Importantly, in kaiC-null mutant strains, KaiA, KaiB, and
SasA did not form the large complexes, and all four clock proteins were
dissociated from each other (Fig. 4A). Therefore, we propose
a role for KaiC (or the KaiC hexamer) as a scaffold protein to form the
large heteromultimeric clock protein complexes.
In addition to the complexes of >300 kDa, we observed that KaiA and
KaiC from wild-type cells were also co-fractionated in ~60-kDa
fractions (Fig. 3). Two observations suggest that KaiA and KaiC are not
associating with each other in a 60-kDa complex. First, these small
KaiA and KaiC signals were not affected in kaiC- and
kaiA-inactivated mutants, respectively (Fig. 4A).
Second, these ~60-kDa forms of KaiA and KaiC did not
co-immunoprecipitate (Fig. 5). Thus, these signals might simply
represent a KaiA dimer (calculated mass of 65.2 kDa) and a KaiC monomer
(58.0 kDa).
Our results also revealed the importance of SasA for formation of
heteromultimeric clock protein complexes. Because the KaiC-interacting sensory domain of SasA is similar to the full-length KaiB protein, KaiB
and SasA have been proposed to compete with each other for binding to
KaiC (11). Consistently in the kaiB-inactivated strain we
found abnormal dominance of 500-670-kDa forms of KaiC, which co-fractionated with SasA. Dominance of these forms of KaiC was also
observed in kaiA-null mutants, in which the levels of KaiB and KaiC were reduced, although the level of SasA was not affected (Fig. 4A). Thus, molecular excess of SasA compared with KaiB
and the absence of KaiA-KaiC interaction both could enhance KaiC-SasA interaction and reduce KaiB-KaiC association in the
kaiA-inactivated strain.
SasA is dominantly present as a 140-200-kDa form at CT 4 in the
wild-type strain and throughout the circadian cycle in the kaiC-null and kaiABC-null mutants. Thus, SasA is
capable of forming a multimeric protein complex even without the Kai
proteins. Because histidine kinases often dimerize, a SasA dimer may be
interacting solidly with a still unknown cognate response regulator to
form the 140-200-kDa complex. Note that homotypic interaction of SasA has been demonstrated in vitro previously (11). The
night-specific 500-670-kDa KaiC-containing complex might be formed by
the binding of a 140-200-kDa SasA complex to a KaiC hexamer, which
conjugates with KaiA and/or KaiB according to subjective time. So far,
however, we do not have any evidence that SasA binds indirectly to KaiA or KaiB through association with a KaiA/KaiB-interacting KaiC complex.
This lack of evidence is because our anti-KaiA, anti-KaiB, and
anti-SasA antisera are not sufficient for immunoprecipitation studies.
Nevertheless, it is plausible that there are several co-existing
populations of multimeric complexes even during the late subjective
night. It is necessary to find detailed stoichiometry of the complexes
to gain a better understanding of the characteristics of the clock proteins.
How do the clock protein complexes function for circadian timing in the
cell? Considering a temporal profile of the clock protein complexes and
a circadian kaiBC expression pattern, we speculate a certain
day/night-specific Kai/SasA complex could be either a positive
or negative regulator at certain times of the day for kaiBC
gene expression. When the kaiBC expression level is low at
CT 0 the Kai/SasA proteins form large complexes. By contrast, when the
kaiBC mRNA level is high at CT 8-10 (7, 11), the Kai
proteins and SasA dissociate from each other. Fig. 6 illustrates one possibility for a
kaiBC regulatory loop by these protein complexes. In the
early subjective day, a smaller (~60-kDa) KaiA-containing protein
complex, possibly a KaiA dimer or a KaiB monomer or dimer, and a
~350-kDa KaiC-containing protein complex (a hexamer?) accumulate in
the Synechococcus cells. Thus, the small KaiA complex and/or
the 350-kDa (smaller) KaiC complex may function as positive elements.
There has been a speculation that the KaiC protein has some positive
function (11, 13). As a result, KaiB and KaiC accumulate, peaking at CT
16 at which time KaiA, KaiB, and SasA interact with the KaiC hexamer
and kaiBC expression becomes reduced. At around CT 22, most
KaiA, KaiB, and SasA molecules are complexed with KaiC, and the
kaiBC mRNA reaches its trough level. We suggest the
large KaiC complexes containing KaiA, KaiB, and SasA as negative
regulators. When these complexes start dissociating and
the KaiB and KaiC protein levels decrease in the next subjective early
morning, the kaiBC transcription begins increasing by
derepression. Interestingly, the peak of the Kai protein association is
delayed several hours compared with that of KaiB and KaiC accumulation
(Figs. 1 and 2). This may be an important step for producing a delay in
the negative action to achieve proper 24-h molecular periodicity.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 s
1 from white
fluorescent lamps.
-lactalbumin (14.4 kDa). The protein samples fractionated
by gel filtration chromatography were precipitated with
trichloroacetate, and each was resolved in 50 µl of the SDS sample
buffer. Five microliters of each sample were subjected to SDS-PAGE and
immunoblotting. Two or three independent experiments obtained similar
results at each time point.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Circadian rhythms of the KaiC protein in its
accumulation and association with other clock proteins.
A, Western blot analysis. Accumulation levels of KaiA, KaiB,
KaiC, and SasA were examined. Synechococcus cells were
collected at the indicated timing in LD and LL conditions. Total
proteins (5 µg) were fractionated by SDS-PAGE on 15% gels and then
analyzed by immunoblotting using antibodies of each of the anti-Kai or
anti-SasA antisera. The white, hatched, and
black bars indicate subjective day, subjective night, and
darkness, respectively. B, co-immunoprecipitation assay. The
protein extracts were used for immunoprecipitation with anti-KaiC IgG
as a primary antiserum. Immune complexes were resolved and analyzed by
Western blotting with specific antisera.
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Fig. 2.
Nighttime-specific formation of clock protein
complexes under LD cycle conditions. A,
Synechococcus cells were collected at hours 4, 10, 16, and
22 in an LD cycle (noted as ZT 4, 10, 16 and 22; ZT 0 indicates dawn
time in an LD cycle), and cell extracts were size-fractionated by gel
filtration chromatography. Each fraction was analyzed by immunoblotting
with specific antisera. Asterisks indicate the peaks of
standard proteins and their native molecular size. Two or three
independent experiments obtained essentially the same patterns:
representative data are shown here. B, densitometric
data of panel A are shown to emphasize that clock-related
proteins were detected in different fractions from each other in a
daytime, whereas they were co-fractionated in a nighttime. The maximal
level for each protein was set as 1.
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Fig. 3.
Circadian rhythm in clock protein complex
formation under LL conditions. A,
Synechococcus cells were collected at hours 4, 10, 16, and
22 in LL, and cell extracts were size-fractionated by gel filtration
chromatography followed by immunoblotting as described in Fig.
2A. Similar data were obtained from two or three independent
experiments for each time point: representative data are shown here.
B, densitometric data of panel A are shown. Note
that in LL conditions, the clock-related proteins were detected in
different fractions from each other during subjective daytime, whereas
they were co-fractionated during subjective nighttime. The maximal
level for each protein was set as 1.
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Fig. 4.
Effects of inactivation of kai
genes and sasA on formation of clock protein
complexes. A, gel filtration assay followed by
immunoblotting analysis. Cell extracts were collected from
kaiA-inactivated (kaiA ),
kaiB-inactivated (kaiB
),
kaiC-disrupted (
kaiC), kaiABC
cluster-depleted (
kaiABC), and sasA-disrupted
(
sasA) strains and an arrhythmic KaiC P-loop mutant
(K52H) strain. Gel filtration and immunoblotting assays were performed
as described in Fig. 2A. Profiles of subjective
night-specific large clock protein complex formations in wild-type
strains are also shown as a control (WT). The Kai proteins
and SasA did not form large protein complexes in the
kaiC-disrupted and K52H strains. kaiA- and
kaiB-null mutants also showed abnormalities in clock protein
complex formation (see text). Note that in these arrhythmic mutants,
each clock protein showed no circadian change in its size by the gel
filtration assay (data not shown). B, expression of KaiABC
and SasA proteins in the KaiC(K52H) mutant strain examined by
immunoblotting. Note that accumulation of KaiC was dramatically lowered
in the mutant throughout a day. C, immunoprecipitation
analysis using anti-KaiC antibody for the KaiC(K52H) strain. The
cell extracts were analyzed as described in Fig. 1B. The
asterisks indicate cross-reacting signals due to anti-IgG.
IP, immunoprecipitation.
2 s
1). Under these conditions, the mutant
strain is arrhythmic with severely reduced levels of KaiB and KaiC
(11). KaiA was fractionated as ~60-kDa proteins in the
sasA-null mutant and was not present in the 400-600-kDa
fractions as was the case in the kaiC-deficient strain (Fig.
4A). We were unable to obtain reliable data on the size of
these proteins by gel filtration because of their low concentrations.
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Fig. 5.
KaiA, KaiB, and SasA co-exist in the large
KaiC-containing protein complexes. A,
co-immunoprecipitation assay following the gel filtration
chromatography analysis. Cell extracts from the wild-type
Synechococcus strain were prepared at the indicated times in
LL and were size-fractionated by gel filtration chromatography as
described in Fig. 3A. The KaiC-containing fractions (19-24,
28, and 29 in Fig. 3A) were then subjected to
immunoprecipitation assay with anti-KaiC IgG followed by immunoblotting
as described in Fig. 1B. Five micrograms each of total cell
extracts were used as control input samples (Ctr).
B, densitometric data of panel A were used to
compare the strength of interactions between KaiC and KaiA, KaiB, or
SasA in each fraction, dependent on circadian time. The maximal level
of each protein in a circadian cycle was set to 1. IP,
immunoprecipitation.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Fig. 6.
A possible model for circadian formation and
functions of clock protein complexes. The details of this model
are described in the text (see "Discussion"). B and
C in white and gray circles indicate
KaiB and KaiC proteins respectively. × represents transcriptional
inactivation of KaiBC mRNA.
We do not yet know how these protein complexes activate or inhibit the kaiBC promoter. It is possible that these protein complexes contain as yet unknown transcriptional activators or inhibitors. Alternatively, the circadianly changing KaiC-containing complexes might affect the topology or superhelicity of the chromosomal DNA, thereby affecting gene expression, as proposed (4) with respect to a similarity of the primary structure of KaiC to those of RecA (recombinase) and DnaB (DNA helicase). Moreover, KaiC was recently found to be phosphorylated in a circadian fashion (13), and protein-protein interaction would affect this KaiC phosphorylation to modulate activities of the complexes.
Finally, we found the accumulation level of KaiC was severely reduced
in the KaiC(K52H) strain (Fig. 4B). As the
kaiBC promoter activity is not evidently down-regulated in
this mutant (9), an ATP binding activity is probably important for the
stability of the KaiC protein. Dissociation between the KaiA and KaiB
proteins and their failure to form large protein complexes in the
P-loop mutant seem primarily due to a dramatic loss of KaiC
accumulation (Fig. 4B). The KaiC level was too low in this
mutant to obtain reliable information on the size of the mutant KaiC
complex by the gel filtration assay under our experimental conditions.
Another discovery related to the stability of the Kai proteins is the relatively enhanced accumulation levels of KaiB and KaiC in ZT 12-24
compared with those in CT (LL) 12-24 in the wild-type strains (Fig.
1). Although we do not know whether this is due to a
dark/light-dependent control on transcription, translation,
or degradation, this phenomenon might be related to entrainment of the
clock by photic stimuli.
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ACKNOWLEDGEMENTS |
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We thank Takahiro Nakamura (Nagoya University), Takashi Kageyama (Kyoto University), and Toshio Iwasaki (Nippon Medical School) for technical suggestions, Jun Tomita (Nagoya University) for helpful discussion on the Kai protein accumulation profiles, Taeko Nishiwaki (Nagoya University) for the Synechococcus KaiC(K52H) strain, and our laboratory members for valuable comments.
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FOOTNOTES |
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* This research was supported in part by grants-in-aid from the Japanese Society for Promotion of Science (13680778 to H. I.), the Japanese Ministry of Education, Culture, Sports, Science, and Technology (11233203 to H. I. and T. K. and COE 13CE2005 to T. K.), and the Kurata Memorial Hitachi Science and Technology Foundation (to H. I.) and by the Inoue Research Award for Young Scientists from the Inoue Foundation (to H. I.).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.
To whom correspondence should be addressed: Division of Biological
Science, Graduate School of Science, Nagoya University, Furo-cho,
Chikusa, Nagoya 464-8602, Japan. Tel.: 81-52-789-2507; Fax:
81-52-789-2963; E-mail: iwasaki@bio.nagoya-u.ac.jp.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M208899200
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ABBREVIATIONS |
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The abbreviations used are: CT, circadian time; ZT, Zeitgeber time; LL, continuous light; LD, 12-h light/12-h dark.
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