ERAD, which is essential for normal protein homeostasis, is orchestrated by the substrate receptors Hrd3 and Yos9 that target misfolded proteins for ubiquitylation by the E3 ligase Hrd1 on the cytoplasmic face of the ER membrane. Extraction of proteins from the ER lumen is thought to require a protein channel and several candidates have been put forward for this role, including the mammalian protein Derlin 1. Its homologue in budding yeast, Der1, is an integral membrane protein that oligomerizes within the Hrd complex and is required for the turnover specifically of soluble proteins.
Jarosch and colleagues set out to test how Der1 associates with the Hrd1 ligase complex. They first confirmed that Der1 oligomerizes and that its loss impaired the turnover of an established ER luminal ERAD substrate, CPY*. Using mutational analysis, they also showed that its predicted transmembrane domains are required for normal turnover of CPY* as well as CPY* ubiquitylation in the cytoplasm. In vivo photocrosslinking analysis determined that the ER-luminal domain of Der1 associates with the substrate receptor Hrd3, whereas the transmembrane domains are close to Hrd1, and that both of these regions can associate directly with ERAD substrates. If the transmembrane domain of Der1 was mutated at specific sites, it could still integrate into the ligase complex and associate with the CPY* substrate but no longer seemed to transfer substrates to the ligase, as indicated by the accumulation of these substrates at luminal regions of Der1.
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