Molecular and Biochemical Characterization of Sucrose Synthase from Developing Castor Oil Seeds
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Sucrose synthase (SUS) is the major sucrolytic enzyme of developing fruits, seeds, and tubers responsible for establishing sink strength while allocating imported photosynthate into specific biosynthetic pathways. While SUS has been implicated in directly channeling imported sucrose into starch, xyloglucan, and cellulose biosynthesis, comparatively little is known about its biochemical properties or role in supporting triacylglycerol biosynthesis in developing oil seeds. This thesis examines SUS in the oil-rich endosperm of developing castor oil seeds (Ricinus communis L.). The dominant SUS isozyme from developing endosperm was purified and identified via LC MS/MS as RcSUS1, one of five predicted SUS isozymes encoded by the castor genome. Immunological studies using phosphosite specific antibodies revealed that RcSUS1 is highly phosphorylated at Ser11 during early seed development. The ~10% of RcSUS1 that associates with microsomal membranes was hypophosphorylated relative to its cytosolic counterpart. However, Ser11 phosphorylation affected neither RcSUS1 microsomal association nor its kinetic properties. Instead, decreased Ser11 phosphorylation preceded a marked decline in RcSUS1 activity and polypeptide levels during seed maturation or following sucrose supply removal, suggesting that it triggers its proteolytic turnover. The Ca2+-dependent protein kinase that catalyzes RcSUS1 Ser11 phosphorylation in the developing endosperm was highly purified, thoroughly characterized, and identified as RcCDPK2. Recombinant RcCDPK2 effectively phosphorylated RcSUS1 and its corresponding dephosphopeptide at Ser11. The SUS interactome of developing castor endosperm was assessed via co-immunopurification with anti-RcSUS1-IgG. A 41-kDa polypeptide that co-immunopurified with RcSUS1 was identified as reversibly glycosylated polypeptide 1 (RcRGP1). RcRGP1 was in vivo phosphorylated at Tyr40, Thr165, and Thr338. It is hypothesized that RcSUS1 forms a metabolon with RcRGP1, channeling UDPG directly into hemicellulose and/or glycoprotein/glycolipid biosynthesis. Sucrolytic activity of RcSUS1 was allosterically inhibited by trehalose 6-phosphate (T6P) (I50 ~ 200 μM), the first observation of its kind. In plants, T6P is a key signaling metabolite that acts as both a signal and negative feedback regulator of sucrose levels. Feedback inhibition of RcSUS1 by T6P is hypothesized to help regulate sink strength while playing a role in T6P homeostasis. Collective results of this thesis contribute to our understanding of oil seed SUS while identifying novel mechanisms by which phosphorylation and T6P signaling may control SUS activity.