A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan |
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Authors: | Masaya Yamamoto |
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Institution: | Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan |
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Abstract: | Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY∗ is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY∗, which carries a mutation homologous to yeast CPY∗, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY∗-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY∗-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY∗-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells. |
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Keywords: | Endoplasmic reticulum (ER) ER-associated degradation (ERAD) Arabidopsis thaliana Serine carboxypeptidase |
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