Yohko Kitayama

KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria

Cyanobacterial clock proteins KaiA and KaiC are proposed as positive and negative regulators in the autoregulatory circadian kaiBC expression, respectively. Here, we show that activation of kaiBC expression by kaiA requires KaiC, suggesting a positive feedback control in the cyanobacterial clockwork. We found that robust circadian phosphorylation of KaiC. KaiA was essential for in vivo KaiC phosphorylation and activated in vitro KaiC autophosphorylation. These effects of KaiA were attenuated by the kaiA2 long period mutation. Both the long period phenotype and the abnormal KaiC phosphorylation in this mutant were suppressed by a previously undocumented kaiC mutation. We propose that KaiA-stimulated circadian KaiC phosphorylation is important for circadian timing.

KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB and KaiC proteins are essential for generation of circadian rhythms. We quantitatively analyzed the intracellular dynamics of these proteins and found a circadian rhythm in the membrane/cytosolic localization of KaiB, such that KaiB interacts with a KaiA–KaiC complex during the late subjective night. KaiB–KaiC binding is accompanied by a dramatic reduction in KaiC phosphorylation and followed by dissociation of the clock protein complex(es). KaiB attenuated KaiA‐enhanced phosphorylation both in vitro and in vivo. Based on these results, we propose a novel role for KaiB in a regulatory link among subcellular localization, protein–protein interactions and post‐translational modification of Kai proteins in the cyanobacterial clock system.

A KaiC-Interacting Sensory Histidine Kinase, SasA, Necessary to Sustain Robust Circadian Oscillation in Cyanobacteria

Both regulated expression of the clock genes kaiA, kaiB, and kaiC and interactions among the Kai proteins are proposed to be important for circadian function in the cyanobacterium Synechococcus sp. strain PCC 7942. We have identified the histidine kinase SasA as a KaiC-interacting protein. SasA contains a KaiB-like sensory domain, which appears sufficient for interaction with KaiC. Disruption of the sasA gene lowered kaiBC expression and dramatically reduced amplitude of the kai expression rhythms while shortening the period. Accordingly, sasA disruption attenuated circadian expression patterns of all tested genes, some of which became arrhythmic. Continuous sasA overexpression eliminated circadian rhythms, whereas temporal overexpression changed the phase of kaiBC expression rhythm. Thus, SasA is a close associate of the cyanobacterial clock that is necessary to sustain robust circadian rhythms.

A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria

The circadian phosphorylation cycle of the cyanobacterial clock protein KaiC has been reconstituted in vitro. The phosphorylation profiles of two phosphorylation sites in KaiC, serine 431 (S431) and threonine 432 (T432), revealed that the phosphorylation cycle contained four steps: (i) T432 phosphorylation; (ii) S431 phosphorylation to generate the double‐phosphorylated form of KaiC; (iii) T432 dephosphorylation; and (iv) S431 dephosphorylation. We then examined the effects of mutations introduced at one KaiC phosphorylation site on the intact phosphorylation site. We found that the product of each step in the phosphorylation cycle regulated the reaction in the next step, and that double phosphorylation converted KaiC from an autokinase to an autophosphatase, whereas complete dephosphorylation had the opposite effect. These mechanisms serve as the basis for cyanobacterial circadian rhythm generation. We also found that associations among KaiA, KaiB, and KaiC result from S431 phosphorylation, and these interactions would maintain the amplitude of the rhythm.

ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria.

Self-sustainable oscillation of KaiC phosphorylation has been reconstituted in vitro, demonstrating that this cycle is the basic time generator of the circadian clock of cyanobacteria. Here we show that the ATPase activity of KaiC satisfies the characteristics of the circadian oscillation, the period length, and the temperature compensation. KaiC possesses extremely weak but stable ATPase activity (15 molecules of ATP per day), and the addition of KaiA and KaiB makes the activity oscillate with a circadian period in vitro. The ATPase activity of KaiC is inherently temperature-invariant, suggesting that temperature compensation of the circadian period could be driven by this simple biochemical reaction. Moreover, the activities of wild-type KaiC and five period-mutant proteins are directly proportional to their in vivo circadian frequencies, indicating that the ATPase activity defines the circadian period. Thus, we propose that KaiC ATPase activity constitutes the most fundamental reaction underlying circadian periodicity in cyanobacteria.

Dual KaiC-based oscillations constitute the circadian system of cyanobacteria

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB, and KaiC proteins are essential for the generation of circadian rhythms. Both in vivo and in vitro, phosphorylation of KaiC is regulated positively by KaiA and negatively by KaiB and shows circadian rhythmicity. The autonomous circadian cycling of KaiC phosphorylation is thought to be the basic pacemaker of the circadian clock and to control genome-wide gene expression in cyanobacteria. In this study, we found that temperature-compensated circadian oscillations of gene expression persisted even when KaiC was arrested in the phosphorylated state due to kaiA overexpression. Moreover, two phosphorylation mutants showed transcriptional oscillation with a long period. In kaiA-overexpressing and phosphorylation-deficient strains, KaiC oscillated and transient overexpression of phosphorylation-deficient kaiC reset the phase of the rhythm. These results suggest that transcription- and translation-based oscillations in KaiC abundance are also important for circadian rhythm generation in cyanobacteria. Furthermore, at low temperature, cyanobacteria can show circadian rhythms only when both the KaiC phosphorylation cycle and the transcription and translation cycle are intact. Our findings indicate that multiple coupled oscillatory systems based on the biochemical properties of KaiC are important to maintain robust and precise circadian rhythms in cyanobacteria.

Proteins Found in a CikA Interaction Assay Link the Circadian Clock, Metabolism, and Cell Division in Synechococcus elongatus

Diverse organisms time their cellular activities to occur at distinct phases of Earths solar day, not through the direct regulation of these processes by light and darkness but rather through the use of an internal biological (circadian) clock that is synchronized with the external cycle. Input pathways serve as mechanisms to transduce external cues to a circadian oscillator to maintain synchrony between this internal oscillation and the environment. The circadian input pathway in the cyanobacterium Synechococcus elongatus PCC 7942 requires the kinase CikA. A cikA null mutant exhibits a short circadian period, the inability to reset its clock in response to pulses of darkness, and a defect in cell division. Although CikA is copurified with the Kai proteins that constitute the circadian central oscillator, no direct interaction between CikA and either KaiA, KaiB, or KaiC has been demonstrated. Here, we identify four proteins that may help connect CikA with the oscillator. Phenotypic analyses of null and overexpression alleles demonstrate that these proteins are involved in at least one of the functions--circadian period regulation, phase resetting, and cell division--attributed to CikA. Predictions based on sequence similarity suggest that these proteins function through protein phosphorylation, iron-sulfur cluster biosynthesis, and redox regulation. Collectively, these results suggest a model for circadian input that incorporates proteins that link the circadian clock, metabolism, and cell division.

KaiC intersubunit communication facilitates robustness of circadian rhythms in cyanobacteria

The cyanobacterial circadian clock is the only model clock to have been reconstituted in vitro. KaiC, the central clock component, is a homohexameric ATPase with autokinase and autophosphatase activities. Changes in phosphorylation state have been proposed to switch KaiC’s activity between autokinase and autophosphatase. Here we analyse the molecular mechanism underlying the regulation of KaiC’s activity, in the context of its hexameric structure. We reconstitute KaiC hexamers containing different variant protomers, and measure their autophosphatase and autokinase activities. We identify two types of regulatory mechanisms with distinct functions. First, local interactions between adjacent phosphorylation sites regulate KaiC’s activities, coupling the ATPase and nucleotide-binding states at subunit interfaces of the CII domain. Second, the phosphorylation states of the protomers affect the overall activity of KaiC hexamers via intersubunit communication. Our findings indicate that intra-hexameric interactions play an important role in sustaining robust circadian rhythmicity.

Exchange of ADP with ATP in the CII ATPase domain promotes autophosphorylation of cyanobacterial clock protein KaiC.

Significance The circadian clock protein KaiC cyclically phosphorylates and dephosphorylates itself with a periodicity of ∼24 h. Unlike the reactions mediated by conventional protein phosphatases, dephosphorylation of KaiC occurs via reversal of the phosphorylation reaction. This unusual mechanism suggests that the KaiC phosphorylation rhythm is sustained by periodic shifts in the equilibrium of the reversible autophosphorylation reaction, the molecular basis of which has never been elucidated. In this study, we found that KaiA promoted the forward reaction by stimulating exchange of KaiC-bound ADP for exogenous ATP. KaiB inhibited KaiA, promoting retention of KaiC-bound ADP, and thereby facilitating the reverse reaction. We propose that KaiA and KaiB sustain the circadian oscillation by regulating this reversible reaction at the level of substrate availability. The cyanobacterial circadian oscillator can be reconstituted in vitro. In the presence of KaiA and KaiB, the phosphorylation state of KaiC oscillates with a periodicity of ∼24 h. KaiC is a hexameric P-loop ATPase with autophosphorylation and autodephosphorylation activities. Recently, we found that dephosphorylation of KaiC occurs via reversal of the phosphorylation reaction: a phosphate group attached to Ser431/Thr432 is transferred to KaiC-bound ADP to generate ATP, which is subsequently hydrolyzed. This unusual reaction mechanism suggests that the KaiC phosphorylation rhythm is sustained by periodic shifts in the equilibrium of the reversible autophosphorylation reaction, the molecular basis of which has never been elucidated. Because KaiC-bound ATP and ADP serve as substrates for the forward and reverse reactions, respectively, we investigated the regulation of the nucleotide-bound state of KaiC. In the absence of KaiA, the condition in which the reverse reaction proceeds, KaiC favored the ADP-bound state. KaiA increased the ratio of ATP to total KaiC-bound nucleotides by facilitating the release of bound ADP and the incorporation of exogenous ATP, allowing the forward reaction to proceed. When both KaiA and KaiB were present, the ratio of ATP to total bound nucleotides exhibited a circadian rhythm, whose phase was advanced by several hours relative to that of the phosphorylation rhythm. Based on these findings, we propose that the direction of the reversible autophosphorylation reaction is regulated by KaiA and KaiB at the level of substrate availability and that this regulation sustains the oscillation of the phosphorylation state of KaiC.

Elucidation of the Role of Clp Protease Components in Circadian Rhythm by Genetic Deletion and Overexpression in Cyanobacteria

In the cyanobacterium Synechococcus elongatus PCC7942, KaiA, KaiB, and KaiC are essential elements of the circadian clock, and Kai-based oscillation is thought to be the basic circadian timing mechanism. The Kai-based oscillator coupled with transcription/translation feedback and other intercellular factors maintains the stability of the 24-hour period in vivo. In this study, we showed that disruption of the Clp protease family genes clpP1, clpP2, and clpX and the overexpression of clpP3 cause long-period phenotypes. There were no significant changes in the levels of the clock proteins in these mutants. The overexpression of clpX led to a decrease in kaiBC promoter activity, the disruption of the circadian rhythm, and eventually cell death. However, after the transient overexpression of clpX, the kaiBC gene expression rhythm recovered after a few days. The rhythm phase after recovery was almost the same as the phase before clpX overexpression. These results suggest that the core Kai-based oscillation was not affected by clpX overexpression. Moreover, we showed that the overexpression of clpX sequentially upregulated ribosomal protein subunit mRNA levels, followed by upregulation of other genes, including the clock genes. Additionally, we found that the disruption of clpX decreased the expression of the ribosomal protein subunits. Finally, we showed that the circadian period was prolonged following the addition of a translation inhibitor at a low concentration. These results suggest that translational efficiency affects the circadian period and that clpX participates in the control of translation efficiency by regulating the transcription of ribosomal protein genes.