Date of Award
Doctor of Philosophy
Theses & dissertations (Interdisciplinary)
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 demonstrated SSIII, SSIIa, SBEIIa, and SBEIIb co-purified with C600 and the latter three proteins co-purified with C300. These data support the hypothesis that all four enzymes are present in C600 and that SSIII mediates assembly of the other three proteins into that quaternary structure. Additional proteins that co-purified with each complex were identified, specifically pyruvate orthophosphate dikinase (PPDK) and sucrose synthase1. Both of these proteins bound to a specific conserved, non-catalytic fragment of SSIII expressed in E. coli, and co-immunoprecipitated with SSIII. Association of PPDK and starch biosynthetic enzymes suggests a means of global regulation of carbon partitioning between protein and starch in developing seeds.;Finally, the observed, specific interactions among the SSs and SBEs were further examined. Direct protein-protein interactions were reconstituted in a minimal system. Three full-length maize enzymes (SSI, SBEIIa, and SBEIIb) and one conserved domain known to participate in complex formation (SSIIIHD) were expressed in E. coli and purified. Pull-down experiments revealed direct binding between SSIIIHD and SSI, SBEIIa, and SBEIIb. This binding occurred in the absence of any other maize factors. SSIIIHD and SSI were phosphorylated after incubation with soluble extracts of maize amyloplasts. Phosphorylation of SSIIIHD enhanced its ability to bind SSI. These data provide novel information about the specificity of interactions between starch biosynthetic enzymes, and further demonstrate that phosphorylation is likely to play a regulatory role in assembly and activity of the complexes.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Tracie Ann Hennen-Bierwagen
Hennen-Bierwagen, Tracie Ann, "Biochemical and genetic analyses of physical associations among Zea mays starch biosynthetic enzymes" (2008). Retrospective Theses and Dissertations. 15684.