A slow maturation of a cysteine protease with a granulin domain in the vacuoles of senescing Arabidopsis leaves.
Arabidopsis RD21 is a cysteine protease of the papain family. Unlike other members of the papain family, RD21 has a C-terminal extension sequence composed of two domains, a 2-kD proline-rich domain and a 10-kD domain homologous to animal epithelin/granulin family proteins. The RD21 protein was accumulated as 38- and 33-kD proteins in Arabidopsis leaves. An immunoblot showed that the 38-kD protein had the granulin domain, whereas the 33-kD protein did not. A pulse-chase experiment with Bright-Yellow 2 transformant cells expressing RD21 showed that RD21 was synthesized as a 57-kD precursor and was then slowly processed to make the 33-kD mature protein via the 38-kD intermediate. After a 12-h chase, the 38-kD intermediate was still detected in the cells. These results indicate that the N-terminal propeptide was first removed from the 57-kD precursor, and the C-terminal granulin domain was then slowly removed to yield the 33-kD mature protein. Subcellular fractionation of the Bright-Yellow 2 transformant showed that the intermediate and mature forms of RD21 were localized in the vacuoles. Under the acidic conditions of the vacuolar interior, the intermediate was found to be easily aggregated. The intermediate and the mature protein were accumulated in association with leaf senescence. Taken together, these results indicate that the intermediate of RD21 was accumulated in the vacuoles as an aggregate, and then slowly matured to make a soluble protease by removing the granulin domain during leaf senescence.
Yamada
K
K
Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
Matsushima
R
R
Nishimura
M
M
Hara-Nishimura
I
I
D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism; D020224:Expressed Sequence Tags; D036341:Intercellular Signaling Peptides and Proteins; D016200:Maturation-Promoting Factor; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D000077153:Progranulins; D014617:Vacuoles / Q000201:enzymology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D004792:Enzyme Precursors; D036341:Intercellular Signaling Peptides and Proteins; D000077153:Progranulins; D014764:Viral Proteins; D016200:Maturation-Promoting Factor; D003546:Cysteine Endopeptidases