Reiko Murakami

Characterization of an Arabidopsis thaliana mutant with impaired psbO, one of two genes encoding extrinsic 33-kDa proteins in photosystem II

A 33‐kDa protein component of the oxygen‐evolving complex in photosystem II is essential for photosynthesis, and it has been believed that mutants with deletion of this 33‐kDa protein are not found in higher plants. We report here the first isolation of an Arabidopsis thaliana mutant with a defect in one of the genes for the 33‐kDa proteins, psbO, and an intact gene (psbO2). This mutant showed considerable growth retardation, suggesting that there is a functional difference between psbO and psbO2.

Functional dissection of two Arabidopsis PsbO proteins: PsbO1 and PsbO2.

PsbO protein is an extrinsic subunit of photosystem II (PSII) and has been proposed to play a central role in stabilization of the catalytic manganese cluster. Arabidopsis thaliana has two psbO genes that express two PsbO proteins; PsbO1 and PsbO2. We reported previously that a mutant plant that lacked PsbO1 (psbo1) showed considerable growth retardation despite the presence of PsbO2 [Murakami, R., Ifuku, K., Takabayashi, A., Shikanai, T., Endo, T., and Sato, F. (2002) FEBS Lett523, 138–142]. In the present study, we characterized the functional differences between PsbO1 and PsbO2. We found that PsbO1 is the major isoform in the wild‐type, and the amount of PsbO2 in psbo1 was significantly less than the total amount of PsbO in the wild‐type. The amount of PsbO as well as the efficiency of PSII in psbo1 increased as the plants grew; howeVER, it neVER reached the total PsbO level observed in the wild‐type, suggesting that the poor activity of PSII in psbo1 was caused by a shortage of PsbO. In addition, an in vitro reconstitution experiment using recombinant PsbOs and urea‐washed PSII particles showed that oxygen evolution was better recoVERed by PsbO1 than by PsbO2. Further analysis using chimeric and mutated PsbOs suggested that the amino acid changes Val186→Ser, Leu246→Ile, and Val204→Ile could explain the functional difference between the two PsbOs. Therefore we concluded that both the lower expression level and the inferior functionality of PsbO2 are responsible for the phenotype observed in psbo1.

ATPase activity and its temperature compensation of the cyanobacterial clock protein KaiC

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria. KaiC is a homohexamer; its subunit contains duplicated halves, each with a set of ATPase motifs. Here, using highly purified KaiC preparations of the thermophilic cyanobacterium Thermosynechococcus elongatus BP‐1 produced in Escherichia coli, we found that the N‐ and C‐terminal domains of KaiC had extremely weak ATPase activity. ATPase activity showed temperature compensation in wild‐type KaiC, but not in KaiCS431A/T432A, a mutant that lacks two phosphorylation sites. We concluded that KaiC phosphorylation is involved in the ATPase temperature‐compensation mechanism—which is probably critical to the stability of the circadian clock in cyanobacteria—and we hypothesized the following temperature‐compensation mechanism: (i) The C‐terminal phosphorylation sites of a KaiC hexamer subunit are phosphorylated by the C‐terminal domain of an adjacent KaiC subunit; (ii) the phosphorylation suppresses the ATPase activity of the C‐terminal domain; and (iii) the phosphorylated KaiC spontaneously dephosphorylates, resulting in the recover of ATPase activity.

The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria

Background: The function of KaiB remains to be solved. Results: Dimeric KaiB1–94 generated circadian oscillation in vitro, but it did not in cells. Conclusion: KaiB tetramer-dimer transformation is responsible for the regulation of the SasA-mediated clock output pathway. Significance: We demonstrated the role of KaiB in the regulation of the SasA-KaiC interaction, involved in the transmission of time-information from KaiABC-machinery to transcription apparatus. The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C-terminal deletion (KaiB1–94), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB1–94 interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB1–94 also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C-terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB1–94 showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB1–94.

Phase-dependent generation and transmission of time information by the KaiABC circadian clock oscillator through SasA-KaiC interaction in cyanobacteria.

Circadian clocks allow organisms to predict environmental changes of the day/night cycle. In the cyanobacterial circadian clock machinery, the phosphorylation level and ATPase activity of the clock protein KaiC oscillate with a period of approximately 24 h. The time information is transmitted from KaiC to the histidine kinase SasA through the SasA autophosphorylation‐enhancing activity of KaiC, ultimately resulting in genome‐wide transcription cycles. Here, we showed that SasA derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP‐1 has the domain structure of an orthodox histidine kinase and that its C‐terminal domain, which contains a phosphorylation site at His160, is responsible for the autophosphorylation activity and the temperature‐ and phosphorylation state‐dependent trimerization / hexamerization activity of SasA. SasA and KaiC associate through their N‐terminal domains with an affinity that depends on their phosphorylation states. Furthermore, the SasA autophosphorylation‐enhancing activity of KaiC requires the C‐terminal ATPase catalytic site and depends on its phosphorylation state. We show that the phosphotransfer activity of SasA is essential for the generation of normal circadian gene expression in cyanobacterial cells. Numerical simulations suggest that circadian time information (free phosphorylated SasA) is released mainly by unphosphorylated KaiC during the late subjective night.

Direct interaction between KaiA and KaiB revealed by a site-directed spin labeling electron spin resonance analysis.

In cyanobacteria, three clock proteins, KaiA, KaiB and KaiC, play essential roles in generating circadian oscillations. The interactions of these proteins change during the circadian cycle. Here, we demonstrated direct interaction between KaiA and KaiB using electron spin resonance spectroscopy. We prepared cystein (Cys)‐substituted mutants of Thermosynechococcus elongatus KaiB, labeled specifically their Cys residues with spin labels and measured the ESR spectra of the labeled KaiB. We found that KaiB labeled at the 64th residue showed spectral changes in the presence of KaiA, but not in the presence of KaiC or bovine serum albumin as a negative control. KaiB labeled at the 101st residue showed no such spectral changes even in the presence of KaiA. The results suggest that KaiB interacts with KaiA in the vicinity of the 64th residue of KaiB. Further analysis demonstrated that the C‐terminal clock‐oscillator domain of KaiA is responsible for this interaction.

Allocation of Absorbed Light Energy in PSII to Thermal Dissipations in the Presence or Absence of PsbS Subunits of Rice

The thermal dissipation (TD) of absorbed light energy in PSII is considered to be an important photoprotection process in photosynthesis. A major portion of TD has been visualized through the analysis of Chl fluorescence as energy quenching (qE) which depends on the presence of the PsbS subunit. Although the physiological importance of qE-associated TD (qE-TD) has been widely accepted, it is not yet clear how much of the absorbed light energy is dissipated through a qE-associated mechanism. In this study, the fates of absorbed light energy in PSII with regard to different TD processes, including qE-TD, were quantitatively estimated by the typical energy allocation models using transgenic rice in which psbS genes were silenced by RNA interference (RNAi). The silencing of psbS genes resulted in a decrease in the light-inducible portion of TD, whereas the allocation of energy to electron transport did not change over a wide range of light intensities. The allocation models indicate that the energy allocated to qE-TD under saturating light is 30-50%. We also showed that a large portion of absorbed light energy is thermally dissipated in manners that are independent of qE. The nature of such dissipations is discussed.

Functionally important structural elements of the cyanobacterial clock-related protein Pex

Pex, a clock‐related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged‐helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25‐bp sequence that contained an imperfectly inverted repeat sequence as the Pex‐binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pexs DNA‐binding activity and examined the effects of various Ala‐substitutions in the α3 helix and wing region of Pex on in vitro DNA‐binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA‐binding activity, whereas those carrying a substitution in the α3 helix did display specific binding activity. But the latter were less thermostable than wild‐type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its α3 helix is probably important to its stability.

Structural characterization of the circadian clock protein complex composed of KaiB and KaiC by inverse contrast-matching small-angle neutron scattering

The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC. Through interactions among the three Kai proteins, the phosphorylation states of KaiC generate circadian oscillations in vitro in the presence of ATP. Here, we characterized the complex formation between KaiB and KaiC using a phospho-mimicking mutant of KaiC, which had an aspartate substitution at the Ser431 phosphorylation site and exhibited optimal binding to KaiB. Mass-spectrometric titration data showed that the proteins formed a complex exclusively in a 6:6 stoichiometry, indicating that KaiB bound to the KaiC hexamer with strong positive cooperativity. The inverse contrast-matching technique of small-angle neutron scattering enabled selective observation of KaiB in complex with the KaiC mutant with partial deuteration. It revealed a disk-shaped arrangement of the KaiB subunits on the outer surface of the KaiC C1 ring, which also serves as the interaction site for SasA, a histidine kinase that operates as a clock-output protein in the regulation of circadian transcription. These data suggest that cooperatively binding KaiB competes with SasA with respect to interaction with KaiC, thereby promoting the synergistic release of this clock-output protein from the circadian oscillator complex.

Circadian oscillations of KaiA-KaiC and KaiB-KaiC complex formations in an in vitro reconstituted KaiABC clock oscillator.

The circadian clock is an endogenous biological mechanism that generates autonomous daily cycles in physiological activities. The phosphorylation levels of KaiC oscillated with a period of 24 h in an ATP‐dependent clock oscillator reconstituted in vitro from KaiA, KaiB and KaiC. We examined the complex formations of KaiA and KaiB with KaiC in the KaiABC clock oscillator by fluorescence correlation spectrometry (FCS) analysis. The formation of KaiB‐containing protein complex(es) oscillated in a circadian manner, with a single peak at 12 h and single trough at 24 h in the circadian cycle, whereas that of KaiA‐containing protein complex(es) oscillated with two peaks at 12 and 24 h. FCS and surface plasmon resonance analyses showed that the binding affinity of KaiA for a mutant KaiC with Ala substitutions at the two phosphorylation sites considered to mimic the nonphosphorylated form of KaiC (np‐KaiC) was higher than that for a mutant KaiC with Asp substitutions at the two phosphorylation sites considered to mimic the completely phosphorylated form of KaiC (cp‐KaiC). The results from the study suggest that a KaiA‐KaiB‐cp‐KaiC ternary complex and a KaiA‐np‐KaiC complex were formed at 12 and 24 h, respectively.