Abstract
The quality control system of the endoplasmic reticulum (ER) ensures that only folded proteins are transported to the down stream organelles. Misfolded proteins accumulated in the ER are degraded by the ERAD (ER-associated degradation), where misfolded proteins are degraded by the ubiquitin-proteasome system in the cytosol. A mutant form of vacuolar carboxypeptidase Y (CPY*) has been used as a model ERAD substrate in yeast cells. Using an Arabidopsis ortholog of yeast CPY (AtCPY), we have constructed a model ERAD substrate in plant cells. When GFP-tagged AtCPY was expressed in Arabidopsis culture cells, it was transported to the vacuole. In contrast, AtCPY*-GFP containing a mutation orthologous to that of CPY* was retained in the ER and degraded in the proteasome-dependent manner. Degradation of AtCPY*-GFP was inhibited by co-expression of a dominant-negative mutant of CDC48 that is necessary for ERAD. We are also constructing a membrane bound ERAD substrate.
