Figure 9.

Mapping the critical residues of CF₁β required for the interaction with BFA3. A, Diagram of CF₁β and various deletions (I, II, III, II-1, II-2, and II-3) of CF₁β. The positions of the point mutation (mβ1-mβ12) are indicated by yellow dots. B, Yeast two-hybrid analysis of the interaction between BFA3 and various deletions of AthCF₁β. C, Amino acid sequence alignment of the interacting region (AthCF₁βII-2) corresponding to amino acid residues 166 to 275 in Arabidopsis CF₁β. The positions of the point mutation (mβ5-mβ9) are indicated. The residues for nucleotide binding in CF₁β are indicated by closed triangles. Ath, Arabidopsis thaliana; Gma, Glycine max; Osa, Oryza sativa; Zma, Zea mays; Smo, Selaginella moellendorffii; Ppa, Physcomitrella patens; Cre, Chlamydomonas reinhardtii; Syn, Synechocystis sp. PCC 6803; Nos, Nostoc sp. PCC 7120; The, Thermosynechococcus elongatus BP-1; Rin, Richelia intracellularis. D, Yeast two-hybrid analysis of the interaction between BFA3 and SynCF₁β variants. In these variants, the residues in SynCF₁β were point mutated to the corresponding residues in AthCF₁β and designated as mSynCF₁β1- mSynCF₁β12. E, Quantitative α-galactosidase assay. The strength of interaction was quantified by assaying α-galactosidase activity in yeast colonies using p-nitrophenyl α-d-galactopyranoside as substrate. Mean and SD of α-Gal activity were obtained from three independent colonies. F, Yeast two-hybrid analysis of the interaction between BFA3 and the AthCF₁β589 variant. In the AthCF₁β589 variant, the residues in AthCF₁β at the positions of mβ5, mβ8, and mβ9 (A and C) were point mutated to the corresponding residues in SynCF₁β. G, Quantitative α-galactosidase assay performed as in E.