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).