Stoichiometric interactions between cyanobacterial clock proteins KaiA and KaiC.
We determined the stoichiometry of KaiA-KaiC interactions. Using immunoblotting and two-dimensional Native- and SDS-PAGE (2DNS-PAGE) analysis, we demonstrated that the reaction products of KaiA-KaiC interactions in the presence of ATP consisted of only phosphorylated KaiC whereas in the presence of the unhydrolyzable analogue 5'-adenylylimidodiphosphate (AMPPNP) they consisted of KaiA and KaiC. In the presence of ATP, the KE (molar ratio of KaiA dimer to KaiC hexamer giving half saturation in the enhancement of KaiC phosphorylation) was 0.25, and IAsys affinity biosensor analysis demonstrated that 1 molecule of KaiA dimer interacted with 1 molecule of KaiC hexamer. In the presence of AMPPNP, the ratio of KaiA dimer to KaiC hexamer in KaiA-KaiC complexes was determined to be 2 by 2DNS-PAGE, Native-PAGE/Scatchard plot, and IAsys analyses. These results suggest that 2 molecules of KaiA dimer can interact with 1 molecule of KaiC hexamer, and that interactions of at least 1 molecule of KaiA dimer with 1 molecule of KaiC hexamer are enough to enhance the phosphorylation of KaiC by KaiA at an almost saturated level.
Hayashi
Fumio
F
Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan.
Ito
Hiroki
H
Fujita
Masayasu
M
Iwase
Ryo
R
Uzumaki
Tatsuya
T
Ishiura
Masahiro
M
D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D015374:Biosensing Techniques; D056925:Circadian Rhythm Signaling Peptides and Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D007700:Kinetics; D046911:Macromolecular Substances; D010766:Phosphorylation
D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C114269:KaiA protein, cyanobacteria; C114271:KaiC protein, cyanobacteria; D046911:Macromolecular Substances; D000266:Adenylyl Imidodiphosphate