Identification and Characterization of the Interaction between ZmSBEIIb and ZmPAD1 in Maize

The starch branching enzymes (SBEs) play critical roles in forming branched structures (α-1,6-glycosidic linkages) of starch molecules in maize ( Zea mays L.). Here, we use a yeast two-hybrid system to screen for ZmSBEIIb-binding proteins to identify proteins binding to ZmSBEIIb to facilitate a more thorough characterization of ZmSBEIIb. By screening a cDNA expression library, a physical interactor of ZmSBEIIb was identified, which is highly homologous with the α-subunit of 20S proteasome and thus named as ZmPAD1. In order to obtain more detailed information about this interaction, the ZmPAD1 interacting with ZmSBEIIb and thereby impacting proteolytic processes were further studied. First, we determined the interaction strength between ZmPAD1 and different truncated ZmSBEIIb baits in the yeast two-hybrid system; second, we confirmed this interaction with GST pull-down and co-immunoprecipitation assays in vitro and in vivo; third, we applied MG115 to inhibit the function of the proteasome in maize suspension-cultured cells for changing the ZmSBEIIb protein content. In our opinion, the interaction between ZmPAD1 and ZmSBEIIb may play a role leading to ZmSBEIIb degradation by 20S proteasome. These results provide a valuable ZmSBEIIb-binding protein resource for analyzing ZmSBEIIb function and provide new insights into the potential protein regulatory mechanism that controls the function of ZmSBEIIb.