UC San Diego

ER-associated degradation (ERAD) targets misfolded ER proteins for degradation. Retrotranslocation, a key feature of ERAD, entails removal of ubiquitinated substrates into the cytosol for proteasomal destruction. Recent work in S. Cerevisiae has revealed derlin rhomboid pseudoprotease Dfm1, related to the rhomboid superfamily, is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. Those studies also revealed a second, Hrd1-dependent pathway of ERAD-M retrotranslocation can arise in dfm1∆ null. In Chapter 1, we show the HRD complex undergoes remodeling to a form that mediates ERAD-M retrotranslocation in the dfm1∆ null. Specifically, we found neither Hrd1 autoubiquitination nor its cytosolic domain is required for suppressive ERAD-M retrotranslocation. Thus, the HRD complex displays remarkable functional flexibility in response to ER stress. In Chapter 2, we found that Dfm1’s conserved rhomboid residues are critical for membrane protein retrotranslocation. Specifically, we identified several retrotranslocation-deficient Dfm1 Loop1 mutants that display impaired binding to membrane substrates as well as TM2 mutants that retain lipid thinning functions of its rhomboid protease predecessors to facilitate in the removal of ER membrane substrates. This work reveals that derlin rhomboid pseudoproteases employ novel mechanisms of substrate engagement and lipid thinning for extracting multi-spanning membrane substrates. In Chapter 3, we sought to translate our studies of conserved rhomboid residues to mammalian rhomboid proteases by understanding preferential substrate specificity for rhomboid proteins RHBDL1 and RHBDL3. While the studies in this chapter were halted due to the pandemic, our studies provide valuable insight on the process of solubilizing and purifying these mammalian membrane proteins.