The ubiquitin-proteasome pathway is involved in rapid degradation of phosphoenolpyruvate carboxylase kinase for C4 photosynthesis.
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.
Agetsuma
Masakazu
M
Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502 Japan.
Furumoto
Tsuyoshi
T
Yanagisawa
Shuichi
S
Izui
Katsura
K
D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D037142:Flaveria / Q000201:enzymology* / Q000235:genetics; D008970:Molecular Weight; D010766:Phosphorylation / Q000187:drug effects; D010788:Photosynthesis / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011523:Protoplasts / Q000187:drug effects / Q000201:enzymology; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D003313:Zea mays / Q000201:enzymology / Q000235:genetics
D004791:Enzyme Inhibitors; D025801:Ubiquitin; C069767:phosphoenolpyruvate carboxylase kinase; D017346:Protein Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex