Substrate Aggregation and Ubiquitination Dictate the Fate of Misfolded Proteins for Endoplasmic Reticulum Associated Degradation over Post Endoplasmic Reticulum Degradation
Protein folding is inherently dynamic and error prone. Even with an elaborate network of cellular factors, protein misfolding occurs frequently. To maintain protein homeostasis, eukaryotes have evolved a hierarchy of protein quality control checkpoints along the secretory pathway, including endoplasmic reticulum associated degradation (ERAD) and post-ER quality control (QC). Although most aberrant proteins are eliminated by ERAD, some misfolded proteins do exit the ER and are turned over by lysosomal proteases. To date, it remains elusive how misfolded membrane proteins are selected for different fates: ERAD versus post-ERQC. To address this question, a novel model substrate, SZ*, was designed and utilized in this study. SZ* is a single-pass membrane chimeric protein bearing cytosolic folding lesion. I first investigated its degradation fate in yeast and found that SZ* is eliminated by both the proteasome via the ERAD pathway and vacuolar proteases via the Golgi-QC pathway. The post-ER degradation of SZ* occurs after ER exit and requires the multivesicular body pathway. I then interrogated cells with different stress treatment to test how various conditions affect the fate of SZ*. My results showed that both heat-shock and substrate overexpression increase ERAD targeting, which both lead to substrate aggregation. Therefore, a misfolded membrane protein with a higher aggregation propensity is preferentially retained in the ER and targeted for ERAD. Next, I sequentially inhibited different steps in the ERAD pathway and tested SZ* ER export efficiency both in vivo and in vitro. I discovered that inhibiting steps required for ubiquitination, including substrate recognition and ubiquitination, facilitate ER exit of SZ* through coat protein complex (COP) II transport. These studies suggest that ERAD substrates can be rescued for ER export by eliminating ubiquitination. In line with this evidence, I fused SZ* to four tandem ubiquitin moieties that are not efficiently degraded by ERAD and found that this substrate could not exit the ER even when ERAD was inhibited. Together, these data provide evidence that substrate aggregation and ubiquitination can be sufficient for ER retention
