Circadian Formation of Clock Protein Complexes by KaiA, KaiB, KaiC, and SasA in Cyanobacteria
2003; Elsevier BV; Volume: 278; Issue: 4 Linguagem: Inglês
10.1074/jbc.m208899200
ISSN1083-351X
AutoresHakuto Kageyama, Takao Kondo, Hideo Iwasaki,
Tópico(s)Light effects on plants
ResumoPhysical 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. 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 time Zeitgeber time continuous light 12-h light/12-h dark 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 (1Dunlap J.C. Cell. 1999; 96: 271-290Abstract Full Text Full Text PDF PubMed Scopus (2391) Google Scholar, 2Young M.W. Kay S.A. Nat. Rev. Genet. 2001; 2: 702-715Crossref PubMed Scopus (932) Google Scholar). Cyanobacteria are the simplest organisms known to have the circadian clock and provide both genetic and physiological model systems (3Iwasaki H. Kondo T. Plant Cell Physiol. 2000; 41: 1013-1020Crossref PubMed Scopus (43) Google Scholar, 4Mori T. Johnson C.H. Semin. Cell Dev. Biol. 2001; 12: 271-278Crossref PubMed Scopus (84) Google Scholar). Previously we have monitored circadian gene expression in the cyanobacterium Synechococcus elongatus PCC 7942 using a bioluminescence (luciferase) reporter gene set (5Kondo T. Strayer C.A. Kulkarni R.D. Taylor W. Ishiura M. Golden S.S. Johnson C.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5672-5676Crossref PubMed Scopus (387) Google Scholar), isolated various mutants that impaired normal circadian phenotypes (6Kondo T. Tsinoremas N.F. Golden S.S. Johnson C.H. Kutsuna S. Ishiura M. Science. 1994; 266: 1233-1236Crossref PubMed Scopus (255) Google Scholar), and identified the clock gene cluster kaiABC (7Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar). Molecular genetic studies suggested functions of KaiA and KaiC as positive and negative elements for kaiBC expression, respectively, to form a negative feedback loop (7Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar). This regulatory loop has been proposed as an important step to drive circadian oscillation in kaiBCexpression (7Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar). 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 (8Xu Y. Mori T. Johnson C.H. EMBO J. 2000; 19: 3349-3357Crossref PubMed Scopus (108) Google Scholar). KaiC binds to ATP in vitro through nucleotide-binding domains (P-loops), and mutations of these motifs severely alter the circadian function (4Mori T. Johnson C.H. Semin. Cell Dev. Biol. 2001; 12: 271-278Crossref PubMed Scopus (84) Google Scholar, 9Nishiwaki T. Iwasaki H. Ishiura M. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 495-499Crossref PubMed Scopus (161) Google Scholar). Moreover, KaiC undergoes autophosphorylationin vitro (9Nishiwaki T. Iwasaki H. Ishiura M. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 495-499Crossref PubMed Scopus (161) Google Scholar). Another important characteristic of the Kai proteins is their physical association in various combinations in yeast cells, in vitro, and in Synechococcus cells (10Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (146) Google Scholar). 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 (10Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (146) Google Scholar). We have also identified another KaiC-interacting partner, SasA, which is a sensory histidine kinase (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). 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 (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). 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. NUC39 and NUC38 (10Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (146) Google Scholar) were used as the wild-type and kaiABC-depletedSynechococcus strains, respectively. We also usedkaiA-, kaiB-, and kaiC-inactivated strains (7Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar), a sasA-disrupted strain (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), and the KaiC(K52H) mutant strain (9Nishiwaki T. Iwasaki H. Ishiura M. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 495-499Crossref PubMed Scopus (161) Google Scholar). Synechococcus cells were grown at 30 °C in BG-11 liquid medium under continuous light (LL) conditions of 46 μmol−2 s−1 from white fluorescent lamps. 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 mmEDTA, 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 4 B, 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 (10Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (146) Google Scholar,11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) 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 (10Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (146) Google Scholar, 11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar).Figure 4Effects of inactivation of kaigenes and sasA on formation of clock protein complexes. A, gel filtration assay followed by immunoblotting analysis. Cell extracts were collected fromkaiA-inactivated (kaiA −),kaiB-inactivated (kaiB −),kaiC-disrupted (ΔkaiC), kaiABCcluster-depleted (ΔkaiABC), and sasA-disrupted (ΔsasA) strains and an arrhythmic KaiC P-loop mutant (K52H) strain. Gel filtration and immunoblotting assays were performed as described in Fig. 2 A. 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 thekaiC-disrupted and K52H strains. kaiA- andkaiB-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. 1 B. Theasterisks indicate cross-reacting signals due to anti-IgG.IP, immunoprecipitation.View Large Image Figure ViewerDownload (PPT) Immunoprecipitation was performed as described previously (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) 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. 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 α-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. 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. 1 A) as reported previously (8Xu Y. Mori T. Johnson C.H. EMBO J. 2000; 19: 3349-3357Crossref PubMed Scopus (108) Google Scholar). KaiC is a master circadian regulator, proposed to be a negative factor for kaiBC expression (7Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar). Recent analysis suggested that KaiC is also responsible for activation of the kaiBCpromoter (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). 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. 1 B). 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"). 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. 2 A, 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. 2 B, 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. 3 A). 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. 3 B). 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. 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. 4 A). Inactivation of thekaiA, kaiB, or kaiC gene each abolishes circadian rhythms (7Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar), and disruption of the sasAgene reduces kaiBC expression with lowered amplitude and a shortened period length in a light intensity-dependent manner (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). 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. 4 A; 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- andkaiC-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. 4 A). 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. 4 A). 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 thesasA-deficient strain under LL conditions (50 μmol m−2 s−1). Under these conditions, the mutant strain is arrhythmic with severely reduced levels of KaiB and KaiC (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). KaiA was fractionated as ∼60-kDa proteins in thesasA-null mutant and was not present in the 400–600-kDa fractions as was the case in the kaiC-deficient strain (Fig. 4 A). We were unable to obtain reliable data on the size of these proteins by gel filtration because of their low concentrations. To better understand the relationship between a biochemical property of KaiC and the protein complex formation, we analyzed in vivoclock protein behaviors in a KaiC(K52H) strain in which an ATP-binding domain of KaiC (P-loop1) was mutated (9Nishiwaki T. Iwasaki H. Ishiura M. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 495-499Crossref PubMed Scopus (161) Google Scholar). This mutation dramatically reduces the ATP binding activity of KaiC in vitro, and the mutant cyanobacterial strain nullifies the circadian clock (9Nishiwaki T. Iwasaki H. Ishiura M. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 495-499Crossref PubMed Scopus (161) Google Scholar). 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. 4 B). 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. 4 B). Because the kaiBC promoter activity is not evidently down-regulated in the KaiC(K52H) strain (9Nishiwaki T. Iwasaki H. Ishiura M. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 495-499Crossref PubMed Scopus (161) Google Scholar), 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. 4 C). 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 (Fig4 A). 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. 4 A) and were the same as the day phase patterns in the wild-type strain (Fig. 3). 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 (10Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (146) Google Scholar). We also reported limited information on KaiC-SasA and KaiB-SasA interactions in cyanobacteria by immunoprecipitation analysis at two points of the day (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). 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 and3) 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 (12Leipe D.D. Aravind L. Grishin N.V. Koonin E.V. Genome Res. 2000; 10: 5-16PubMed Google Scholar). 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. 4 A). 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- andkaiA-inactivated mutants, respectively (Fig. 4 A). 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 (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). 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. 4 A). 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 thekaiA-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 thekaiC-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 (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). 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 kaiBCgene expression. When the kaiBC expression level is low at CT 0 the Kai/SasA proteins form large complexes. By contrast, when thekaiBC mRNA level is high at CT 8–10 (7Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar, 11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), the Kai proteins and SasA dissociate from each other. Fig. 6 illustrates one possibility for akaiBC 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 (11Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 13Iwasaki H. Nishiwaki T. Kitayama Y. Nakajima M. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15788-15793Crossref PubMed Scopus (245) Google Scholar). 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 thekaiBC 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. We do not yet know how these protein complexes activate or inhibit thekaiBC 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 (4Mori T. Johnson C.H. Semin. Cell Dev. Biol. 2001; 12: 271-278Crossref PubMed Scopus (84) Google Scholar) 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 (13Iwasaki H. Nishiwaki T. Kitayama Y. Nakajima M. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15788-15793Crossref PubMed Scopus (245) Google Scholar), 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. 4 B). As thekaiBC promoter activity is not evidently down-regulated in this mutant (9Nishiwaki T. Iwasaki H. Ishiura M. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 495-499Crossref PubMed Scopus (161) Google Scholar), 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. 4 B). 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. 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 theSynechococcus KaiC(K52H) strain, and our laboratory members for valuable comments.
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