Biochemical and genetic analyses of physical associations among Zea mays starch biosynthetic enzymes
نویسندگان
چکیده
Mutations affecting specific starch biosynthetic enzymes commonly have pleiotropic effects on other enzymes in the same metabolic pathway. Such genetic evidence indicates functional relationships between components of the starch biosynthetic system including starch synthases (SS), starch branching enzymes (BE), and starch debranching enzymes (DBE), however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/or DBEs associate physically with each other in multisubunit complexes. To test this hypothesis, this study sought to identify stable associations between SSI, SSIIa, SSIII, SBEI, SBEIIa, and SBEIIb from maize amyloplasts. Three separate detection methods, yeast two-hybrid, co-immunoprecipitation, and affinity purification using recombinant proteins as the solid phase ligand were used to identify specific protein-protein interactions. Numerous instances were detected of specific pairs of proteins associating either directly or indirectly in the same multi-subunit complex. Gel permeation chromatography of proteins extracted from maize amyloplasts revealed two high molecular weight complexes of approximately 600kDa (C600) and 300kDa (C300) containing either SSIIa, SSIII, SBEIIa, and SBEIIb, or SSIIa, SBEIIa, and SBEIIb, respectively. To further characterize these interactions, genetic analyses tested the interdependence of specific starch biosynthetic enzymes on each other for assembly into the complexes. Association of SSIIa, SBEIIa, and SBEIIb into C600 was found to require the presence of SSIII, however, loss of SSIII did not affect assembly of the C300 complex. Further purification of the complexes through successive chromatography steps
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