The phototroph-specific β-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria

Kumiko Kondo,Masayuki Izumi,Kosuke Inabe,Keisuke Yoshida,Mari Imashimizu,Toshiharu Suzuki,Toru Hisabori

doi:10.1016/j.jbc.2021.101027

Kumiko Kondo, Masayuki Izumi + Show 5 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101027

Copy DOI

Abstract

The FoF1 synthase produces ATP from ADP and inorganic phosphate. The γ subunit of FoF1 ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole FoF1 complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire FoF1 ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of FoF1 ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of FoF1 ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.

Highlights

Photosynthetic organisms utilize FoF1 ATP synthase (FoF1) for a solar-to-chemical energy conversion system to produce ATP, the universal energy currency for cells
Catalytic sites that are necessary for the ATP synthesis/hydrolysis reactions are present in the β subunit
Our results demonstrated that the phototroph-specific β-hairpin structure of the γ subunit of FoF1 critically contributes to its ATP synthesis activity, in addition to suppressing ATP hydrolysis

Summary

Results

To investigate the role of the β-hairpin on ATP synthesis function, we purified FoF1 preparations from S. 6803, as follows. The resulting FoF1 preparation was subjected to SDS-PAGE (Fig. 3A, lane WT). The validity of the bands was confirmed by peptide mass fingerprinting after trypsin digestion, N-terminal sequencing, or immunoblot analyses (Table 1 and Fig. 3B). The complete segregation and replacement of endogenous sll1327, which encodes the γ subunit, with the mutated gene were validated by PCR analysis (Fig. 2D) and DNA sequence analysis. The two amino acids located at the tip of the turn region or the entire β-hairpin structure were not present in the two mutants (Fig. 2B). No significant differences were observed in subunit stoichiometry (Fig. 3, A and B) or expression level, which was assessed based on immunoblotting using β subunit–specific antibodies (Fig. 2, E and F), between the WT and the mutant strains. Subunits of the FoF1 ATP synthase of S. 6803 identified by peptide mass fingerprint analysis and N-terminal sequencing analyses

39 TLTVRVIT c

Discussion

Experimental procedures