Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves

Shane C. Hardin, Steven C. Huber

Research output: Contribution to journalArticlepeer-review

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 (∼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 subunit 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 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.

Original languageEnglish (US)
Pages (from-to)197-208
Number of pages12
JournalPlant Physiology and Biochemistry
Volume42
Issue number3
DOIs
StatePublished - Mar 2004

Keywords

  • AMC
  • Amido-methyl coumarin
  • CDPK
  • CP
  • Calcium-dependent protein kinase
  • Core particle
  • DTT
  • Dithiothreitol
  • E64
  • EDTA
  • Maize
  • N-(trans-epoxysuccinyl)-leucine-4-guanidinobutylamide
  • Phosphorylation
  • Proteasome
  • Sucrose synthase
  • Ubiquitin

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science

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