Submitted to: Plant Physiology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 15, 2003
Publication Date: March 1, 2004
Citation: HARDIN, S.C., HUBER, S.C. 2004. Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves. Plant Physiology and Biochemistry. 42:197-208. Interpretive Summary: Plants produce and feed themselves sucrose. The agricultural productivity of most crops such as maize, wheat and soybean, is dependent upon the ability of leaves to produce sucrose, as an end product of photosynthesis, and the ability of growing parts such as roots, stems and seeds, to utilize the sugar in growth and development. Recent evidence indicates that sucrose is not only a primary nutrient but also an important 'signal compound' that can control gene expression. Consequently, understanding the mechanisms that control the utilization of sucrose in crop plants is very important. The enzyme sucrose synthase is recognized to play an important role in the metabolism of sucrose in seeds and tubers, and the enzymatic activity of this enzyme is often correlated closely with organ growth. Hence, factors that influence the activity of this enzyme may impact plant growth and development. Recent results suggest that degradation of the sucrose synthase protein molecule is tightly controlled and may involve a large multiprotein complex known as the 'proteasome.' Furthermore, phosphorylation of the sucrose synthase protein at a specific serine residue may be the trigger that initiates its degradation via this selective process. Understanding the complex mechanisms that control sucrose metabolism may produce new approaches to regulate crop growth.
Technical Abstract: The serine-170 (S170) calcium-dependent protein kinase phosphorylation site of maize (Zea mays L.) sucrose synthase (SUS) (EC 184.108.40.206) has been implicated in the post-translational regulation of SUS protein stability. To clarify the proteolytic process and the role of phosphorylation, SUS degradation and proteasome activities were studied in the maize leaf elongation zone. Size-exclusion chromatography resolved two peaks of proteasome-like proteolytic activity. The large molecular mass ( 1350 kDa) peak required Mg2+ and ATP for maximal activity and was inhibited by the proteasome inhibitors MG132 and NLVS. Anion-exchange chromatography resolved a similar proteolytic activity that was activated by ATP, characteristics that are consistent with those of a 26S-proteasome. Appropriately, immunoblotting revealed the presence of a 26S-proteasome sub-unit and highly ubiquitinated proteins within the active fractions eluted from both columns. The smaller molecular mass ( 600 kDa) peak represented only 40 % of the total proteasome-like activity and is likely a maize 20S-proteasome as it was activated in vitro by low levels of SDS. S170 phosphorylated SUS (pS170-SUS) was detected as both high molecular mass (HMM) forms and proteolytic fragments that co-eluted with 26S-proteasome activities on both size-exclusion and anion-exchange columns. Conditions that maintained maximal 26S-proteasome activity reduced the amounts of pS170-SUS recovered. In vitro, the 26S-proteasome degraded SUS and proteasome-specific inhibitors reduced SUS proteolysis. HMM-SUS conjugates were produced in vitro and immunoprecipitations suggested that some SUS might be ubiquitinated in vivo. The results suggest that S170 phosphorylation promotes the formation of HMM, ubiquitin-SUS conjugates that are targeted for 26S-proteasome-dependent degradation.