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.