FIGURE 4 Overview of homo and hetero-dimeric interactions at different interfaces. (A) S1:S1’, (B) L1:L1’, (C) L1’:S1 and L1:S1’, (D) L1:S1 and L1’:S1’. Heterodimeric interface interactions noticed between two loop regions of catalytic domains are highlighted in dotted squares. OsAGPase subunits are displayed in colored cartoons; interacting residues are displayed in colored ball-stick models and annotated with black and red fonts. Intra/intermolecular H-bonds are showed in black dotted lines.
Due to the lack of available structural data on the native forms (heterotetramers) of AGPases in higher plants, several previous studies have relied on homology models derived from the X-ray crystal structure of the potato AGPase SS homotetramer.29 Based on the proposed side-by-side and up-side-down model for the heterotetrameric form of potato tuber AGPase,11,12 similar modeling approaches have been employed for other plant AGPases as well. This approach has been applied to generate models for various plant AGPases based on the structural characteristics observed in the potato tuber AGPase.47
To investigate the differences in the structural configuration among the models, a yeast-two-hybrid experiment was carried out (Figure 5). Reciprocal interactions were observed between OsL1 and OsS1 and between OsS1 and OsL1 of rice AGPase, while no homotypic interactions were observed between OsS1 and OsS1 or between OsL1 and OsL1. Similar interaction profiles were also observed for the potato AGPase StLS:StSS. We also observed that OsS2a, another rice small subunit of rice AGPase, did not interact with either itself or OsS2b (data not shown). Likewise, the cytosolic rice AGPase subunits (OsL2:OsS2b) exhibited reciprocal interactions. While no homotypic interactions were observed for OsS2b, a positive homotypic interaction was observed between OsL2. These findings suggest that the cytosolic rice AGPase possesses a distinct structural configuration from the plastidial rice and potato AGPases. In a previous study,48 unprocessed sequences of OsL1 and OsS1 were used, whereas in our study, we excluded the chloroplast-transit peptides from the sequences to utilize the mature forms of the subunits instead. Nonetheless, the protein interaction profiles for both rice AGPases were consistent with those previously reported,48suggesting the transit peptides in the sequence does not interfere with protein interaction in the yeast-two-hybrid system.