Determination of a chloroplast degron in the regulatory domain of chlorophyllide a oxygenase.
Chlorophyll b is one of the major photosynthetic pigments of plants. The regulation of chlorophyll b biosynthesis is important for plants in order to acclimate to changing environmental conditions. In the chloroplast, chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), a Rieske-type monooxygenase. The activity of this enzyme is regulated at the level of protein stability via a feedback mechanism through chlorophyll b. The Clp protease and the N-terminal domain (designated the A domain) of CAO are essential for the regulatory mechanism. In this study, we aimed to identify the specific amino acid residue or the sequence within the A domain that is essential for this regulation. To accomplish this goal, we randomly introduced base substitutions into the A domain and searched for potentially important residues by analyzing 1,000 transformants of Arabidopsis thaliana. However, none of the single amino acid substitutions significantly stabilized CAO. Therefore, we generated serial deletions in the A domain and expressed these deletions in the background of CAO-deficient Arabidopsis mutant. We found that the amino acid sequence (97)QDLLTIMILH(106) is essential for the regulation of the protein stability. We furthermore determined that this sequence induces the destabilization of green fluorescent protein. These results suggest that this sequence serves as a degradation signal that is recognized by proteases functioning in the chloroplast.
Institute of Low Temperature Science, Hokkaido University, N19 W8 Kita-Ku, Sapporo 060-0819, Japan. Electronic address: sakuraba@pop.lowtem.hokudai.ac.jp.
Yasuhito
Y
Sakuraba
Institute of Low Temperature Science, Hokkaido University, N19 W8 Kita-Ku, Sapporo 060-0819, Japan.
Ryouichi
R
Tanaka
Research Institute for Biological Sciences Okayama, Okayama 716-1241, Japan.
Akihiro
A
Yamasato
Institute of Low Temperature Science, Hokkaido University, N19 W8 Kita-Ku, Sapporo 060-0819, Japan.
Ayumi
A
Tanaka
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002734:Chlorophyll / Q000235:genetics / Q000378:metabolism; D000077194:Chlorophyll A; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability / Q000502:physiology; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D017384:Sequence Deletion
D029681:Arabidopsis Proteins; D002734:Chlorophyll; C037184:chlorophyll b; D010105:Oxygenases; C115272:chlorophyll a oxygenase; D049071:Endopeptidase Clp; D000077194:Chlorophyll A