A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan.
A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan.
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DOI：
10.1016/j.bbrc.2010.02.001
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发表时间：
2010-03
影响因子：
3.1
通讯作者：
Masaya Yamamoto;Mitsuyoshi Kawanabe;Y. Hayashi;T. Endo;S. Nishikawa
中科院分区：
文献类型：
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作者：
Masaya Yamamoto;Mitsuyoshi Kawanabe;Y. Hayashi;T. Endo;S. Nishikawa
内质网（ER）中产生的错误折叠蛋白质通过ER相关降解（ERAD）机制降解。本文报道了植物细胞ERAD分析实验系统的建立。羧肽酶Y（Carboxypeptidase Y，CPY）是一种空泡酶，其突变体CPY β能被酵母中的ERAD降解。由于拟南芥具有AtCPY（酵母CPY的直向同源物），我们构建并表达了AtCPY和GFP以及AtCPY β（其携带与酵母CPY β同源的突变）和A.拟南芥细胞虽然AtCPY-GFP被有效地运输到液泡中，AtCPY-GFP被保留在ER中，以蛋白酶体和Cdc 48依赖的方式降解。我们还发现AtCPY ERAD-GFP在酵母细胞中被ERAD降解，但其单个N-聚糖在酵母或植物细胞中不起降解信号的作用。因此，AtCPY β-GFP可以用作标记蛋白来分析植物细胞中的ERAD途径，可能是非糖基化底物。
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.