2007 Annual Report 1a.Objectives (from AD-416) Identify domains on sucrose synthase (SuSy) that are essential for binding to membrane and F-actin, and to elucidate the mechanisms that regulate the interactions. 1b.Approach (from AD-416) Proteolytic recombinant fragments of SuSy will be produced and tested for their ability to bind to specific lipids and to liposomes, to identify potential membrane-binding domains, and to F-actin, to identify the actin-binding domain. Fragments that associate will be sequenced by automated Edman degradation or mass spectrometric analysis at Michigan State University. The role of phosphorylation of Ser15 and Ser170 in the association of SuSy with membranes and F-actin in situ will be assessed using phosphorylation-state specific antibodies. The in vivo significance of specific residues and domains will be examined using transient and stable transformation of maize with wildtype and mutant forms of SuSy. 3.Progress Report This report serves to document research conducted under a Reimbursable Agreement between ARS and the U.S. Department of Energy. Additional details of research can be found in the report for the parent project 3611-21000-020-00D, Identifying and Manipulating Determinants of Photosynthate Production and Partitioning. Studies have focused on the enzyme sucrose synthase, which plays an important role in the metabolism of sucrose in seeds and tubers. There are three isoforms of SUS in maize, referred to as SUS1, SUS-SH1, and SUS2. SUS is generally considered to be tetrameric protein but recent evidence suggests that SUS can also occur as a dimeric protein. The formation of tetrameric SUS is regulated by sucrose concentration in vitro and this could also be an important factor in the cellular localization of the protein. We found that high sucrose concentrations, which promote tetramer formation, also inhibit the binding of SUS1 to actin filaments in vitro. Previously, high sucrose concentrations were shown to promote SUS association with the plasma membrane. The specific regions of the SUS molecule involved in oligomerization are not known, but we identified a region of the SUS1 moelcule by bioinformatic analysis that was predicted to form a coiled coil. We demonstrated that this sequence could, in fact, self-associate as predicted for a coiled coil, but truncation analysis with the full-length recombinant protein suggested that it was not responsible for formation of dimers or tetramers. However, the coiled coil may function in binding of other proteins to SUS1. Overall, sugar availability may differentially influence the binding of SUS to cellular structures, and these effects may be mediated by changes in the oligomeric nature of the enzyme.