Journal Article

The Ubiquitin–Proteasome Pathway is Involved in Rapid Degradation of Phosphoenolpyruvate Carboxylase Kinase for C4 Photosynthesis

Masakazu Agetsuma, Tsuyoshi Furumoto, Shuichi Yanagisawa and Katsura Izui

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 3, pages 389-398
Published in print March 2005 | ISSN: 0032-0781
Published online March 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci043
The Ubiquitin–Proteasome Pathway is Involved in Rapid Degradation of Phosphoenolpyruvate Carboxylase Kinase for C4 Photosynthesis

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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In C4 photosynthesis, phosphoenolpyruvate carboxylase (PEPC) is the enzyme responsible for catalyzing the primary fixation of atmospheric CO2. The activity of PEPC is regulated diurnally by reversible phosphorylation. PEPC kinase (PEPCk), a protein kinase involved in this phosphorylation, is highly specific for PEPC and consists of only the core domain of protein kinase. Owing to its extremely low abundance in cells, analysis of its regulatory mechanism at the protein level has been difficult. Here we employed a transient expression system using maize mesophyll protoplasts. The PEPCk protein with a FLAG tag could be expressed correctly and detected with high sensitivity. Rapid degradation of PEPCk protein was confirmed and shown to be blocked by MG132, a 26S proteasome inhibitor. Furthermore, MG132 enhanced accumulation of PEPCk with increased molecular sizes at about 8 kDa intervals. Using anti-ubiquitin antibody, this increase was shown to be due to ubiquitination. This is the first report to show the involvement of the ubiquitin–proteasome pathway in PEPCk turnover. The occurrence of PEPCks with higher molecular sizes, which was noted previously with cell extracts from various plants, was also suggested to be due to ubiquitination of native PEPCk.

Keywords: C4 photosynthesis; Phosphoenolpyruvate carboxylase kinase; 26S Proteasome; Protein degradation; Ubiquitination; CaMV, cauliflower mosaic virus; CHX, cycloheximide; Ctag-PEPCk, PEPCk conjugated with a FLAG tag at the C-terminus; FtPEPCk, Flaveria trinervia PEPCk; Ntag-sGFP, sGFP(S65T) conjugated with a FLAG tag at the N-terminus; Ntag-PEPCk, PEPCk conjugated with a FLAG tag at the N-terminus; PEPC, phosphoenolpyruvate carboxylase; PEPCk, PEPC kinase; PMSF, phenylmethylsulfonyl fluoride; PNP, phosphorylated peptide of the N-terminal region of PEPC; sGFP(S65T), synthetic green-fluorescent protein S65T mutant; ZmPEPC, Zea mays C4-type PEPC

Journal Article.  7301 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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