Investigation of the localization, oligomerization status, and binding activities of maize sucrose synthase

Sucrose synthase (SUS) is an important enzyme in plant metabolism; it is a reversible enzyme that acts physiologically to cleave sucrose into UDP-glucose and fructose. SUS is a cytosolic protein, however it can also associate with the plasma membrane where it is thought to provide UDP-glucose for cellulose synthesis. In maize (Zea mays, L) there are three known isoforms of SUS: SUS1, SUS-SH1, and SUS2.;Little is known about the individual contribution of the SUS isoforms to metabolism. Using isoform-specific antibodies, the three isoforms were evaluated individually. Intracellular localization of the SUS isoforms was studied by cellular fractionation in leaves and kernels and it was found that SUS1 and SUS-SH1 associated with membranes, while SUS2 did not. The lack of membrane associated SUS2 indicated that it might have a unique role in sucrose metabolism. It was also established that SUS2 existed predominantly as a hetero-oligomer with SUS1, while SUS-SH1 was present only as homo-oligomers in kernels. Together these results showed the importance of SUS isoforms in both cytosolic and membrane associated sucrose metabolism, and provided evidence for isoform specific functional roles.;SUS was previously thought to only form a tetramer in vivo, however results reported here indicated that SUS may also exist as monomers and dimers. Two factors that influenced oligomerization were pH and the concentration of sucrose, where high sucrose and slightly acidic pH promoted tetramer formation. The ability of SUS to "sense" sucrose concentrations may be a mechanism to control localization of the enzyme in response to sugar availability. Oligomerization status may play a role in the binding activities of SUS, as high concentrations of sucrose promoted binding to membranes, but inhibited binding to actin.;Recombinant SUS1 C-terminal truncations showed that even the first 50 amino acids of SUS1 could bind to microsomes from kernels as well as to cellulose. The secondary structure of the first 50 amino acids of SUS may be causally involved in the binding activities. Taken together, the results presented in this dissertation gave a better understanding of the dynamic regulation of SUS and documented its specific and vital role in plant metabolism.