FIGURE 5 Yeast-two-hybrid analysis of protein-protein interaction. The AGPase subunits were fused to GAL4-binding and GAL4-activation domains, which were then utilized as bait and prey, respectively. The bait and prey constructs were tested in synthetic growth media: SD/-2 (SD medium lacking L-leucine and L-tryptophan) and SD/-4 (SD medium lacking L-leucine, L-tryptophan, adenine, and L-histidine).
Tang et al.48 conducted more analysis using truncated forms of the large and small subunits: catalytic and β-helix domains of AGPases OsL1:OsS1 and OsL2:OsS2b. The study revealed that the β-helix domain alone of OsL1 can interact with intact OsS1, and vice versa for OsS1 with OsL1. However, an intriguing observation emerged as the catalytic domain of OsL1 showed interaction with intact OsS1, whereas the catalytic domain of OsS1 did not exhibit any interaction with intact OsL1. This finding implies that the catalytic domains of OsL1 and OsS1 possess distinct configurations. Similar interaction profiles were observed for OsL2 and OsS2b.
Previously Baris et al.49 proposed that the LS:SS dimer of potato AGPase showed greater stability when the two subunits interacted through their β-helix domains compared to their catalytic domains. Thus, it is evident that the primary interactions between the large and small subunits are mediated by their β-helix domains, while conditional interactions occur between the catalytic domains of the two subunits. Collectively, the proposed models and protein interaction analyses suggest that AGPase OsL1:OsS1 possess a distinct structural configuration in comparison to AGPase OsL2:OsS2b, but potentially have a similar configuration to StLS:StSS. The reason why the small subunits of rice and potato AGPases do not interact with each other in the yeast-two-hybrid system, despite the ability of potato small subunits to form a functional homotetramer,50 remains to be resolved.
In our previous study, we proposed the direct association OsL2:OsS2b based on a combination of experimental and computational methods, where we demonstrated that the heterotetrametric association of cytosolic AGPases OsL2:OsS2b,27 which exhibits structural and configurational similarities to that observed in potato tuber AGPase.11,12 Nevertheless, we discovered a distinct type of heterotetrametric association of OsL1:OsS1. Unlike the previously observed patterns (up-down and side-by-side heterodimers), the OsL1:OsS1 subunits form up-down homodimers and side-by-side heterodimers, presenting a unique arrangement (Figure 6).