TY - JOUR
T1 - A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan
AU - Yamamoto, Masaya
AU - Kawanabe, Mitsuyoshi
AU - Hayashi, Yoko
AU - Endo, Toshiya
AU - Nishikawa, Shuh ichi
N1 - Funding Information:
We thank Y. Niwa, T. Ueda, A. Nakano, M.H. Sato and R. Tsien for materials, T. Tatsumi for assistance with plasmid constructions, and members of the Endo lab for discussion. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (Grant No. 16085202 ) from the MEXT Japan (No. 1685101 ) to S.N. and by Grants-in-Aid for Scientific Research from MEXT to T.E. M.Y. is a Research Fellow of JSPS.
PY - 2010/3/12
Y1 - 2010/3/12
N2 - 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.
AB - 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.
KW - Arabidopsis thaliana
KW - Endoplasmic reticulum (ER)
KW - ER-associated degradation (ERAD)
KW - Serine carboxypeptidase
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U2 - 10.1016/j.bbrc.2010.02.001
DO - 10.1016/j.bbrc.2010.02.001
M3 - Article
C2 - 20138839
AN - SCOPUS:77949272203
SN - 0006-291X
VL - 393
SP - 384
EP - 389
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -