title: Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves.
abstract: The serine-170 (S170) calcium-dependent protein kinase phosphorylation site of maize (Zea mays L.) sucrose synthase (SUS) (EC 2.4.1.13) 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 ( approximately 1350 kDa) peak required Mg(2+) 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 subunit and highly ubiquitinated proteins within the active fractions eluted from both columns. The smaller molecular mass ( approximately 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 sodium dodecyl sulfate (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 can be targeted for 26S-proteasome-dependent degradation.
authors.affiliation: United States Department of Agriculture-Agricultural Research Service, Photosynthesis Research Unit and Departments of Plant Biology and Crop Sciences, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Shane C
authors.initials: SC
authors.lastname: Hardin
authors.forename: Steven C
authors.initials: SC
authors.lastname: Huber
mesh_terms: D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010766:Phosphorylation; D018515:Plant Leaves / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D014452:Ubiquitins / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology*
chemical_list: D009097:Multienzyme Complexes; D014452:Ubiquitins; D005964:Glucosyltransferases; C022699:sucrose synthase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex