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Atomic force microscopy analysis of SasA‐KaiC complex formation involved in information transfer from the Kai ABC clock machinery to the output pathway in cyanobacteria
Author(s) -
Murakami Reiko,
Hokonohara Hitomi,
Che DockChil,
Kawai Tomoji,
Matsumoto Takuya,
Ishiura Masahiro
Publication year - 2018
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/gtc.12574
Subject(s) - sasa , random hexamer , biology , biophysics , trimer , atg8 , mutant , biochemistry , crystallography , chemistry , botany , apoptosis , dimer , organic chemistry , autophagy , gene
The cyanobacterial clock oscillator is composed of three clock proteins: KaiA, KaiB and KaiC. SasA, a KaiC‐binding EnvZ‐like orthodox histidine kinase involved in the main clock output pathway, exists mainly as a trimer (SasA 3mer ) and occasionally as a hexamer (SasA 6mer ) in vitro. Previously, the molecular mass of the SasA‐Kai C DD complex, where Kai C DD is a mutant KaiC with two Asp substitutions at the two phosphorylation sites, has been estimated by gel‐filtration chromatography to be larger than 670 kDa. This value disagrees with the theoretical estimation of 480 kDa for a SasA 3mer ‐KaiC hexamer (KaiC 6mer ) complex with a 1:1 molecular ratio. To clarify the structure of the SasA‐KaiC complex, we analyzed Kai C DD with 0.1 mmol/L ATP and 5 mmol/L MgCl 2 (Mg‐ ATP ), SasA and a mixture containing SasA and Kai C DD 6mer with Mg‐ ATP by atomic force microscopy ( AFM ). Kai C DD images were classified into two types with height distribution corresponding to Kai C DD monomer (Kai C DD 1mer ) and Kai C DD 6mer , respectively. SasA images were classified into two types with height corresponding to SasA 3mer and SasA 6mer , respectively. The AFM images of the SasA‐Kai C DD mixture indicated not only Kai C DD 1mer , Kai C DD 6mer , SasA 3mer and SasA 6mer , but also wider area “islands,” suggesting the presence of a polymerized form of the SasA‐Kai C DD complex.