Hrd1 forms the retrotranslocation pore regulated by auto-ubiquitination and binding of misfolded proteins

Abstract

During endoplasmic-reticulum-associated protein degradation (ERAD), misfolded proteins are polyubiquitinated, extracted from the ER membrane and degraded by the proteasome 1-4. In a process called retrotranslocation, misfolded luminal proteins first need to traverse the ER membrane before ubiquitination can occur in the cytosol. It was suggested that the membrane-embedded ubiquitin ligase Hrd1 forms a retrotranslocation pore regulated by cycles of autoand deubiquitination 5-8. However, the mechanism by which auto-ubiquitination affects Hrd1 and allows polypeptides to cross the membrane and whether Hrd1 forms a membranespanning pore remained unknown. Here, using purified Hrd1 incorporated into different model membranes, we show that Hrd1 auto-ubiquitination leads to the opening of a pore. Substrate binding increases the pore size and its activity, whereas deubiquitination closes the pore and renders it unresponsive to substrate. We identify two binding sites for misfolded proteins in Hrd1, a low-affinity luminal site and a high-affinity cytoplasmic site formed following auto-ubiquitination of specific lysine residues in Hrd1's RING domain. We propose that the affinity difference between the luminal and cytoplasmic binding sites provides the initial driving force for substrate movement through Hrd1. In Saccharomyces cerevisiae, ERAD of luminal proteins (ERAD-L) requires the Hrd1 complex, which is composed of the ubiquitin ligase Hrd1, three other membrane proteins (Hrd3, Usa1 and Der1) and the lumen-soluble Yos9 (refs. 9-16). Ubiquitination depends on the ubiquitin-conjugating enzyme Ubc7 and its cofactor Cue1 (refs. 17-19) and recruits the Cdc48 complex, which is composed of the ATPase Cdc48 and its cofactors Npl4, Ufd1 and Ubx2. This complex catalyses the extraction of substrates from the ER membrane in an ATP-dependent manner 6,20-25. Overexpression of Hrd1 obviates the need for the other components of the Hrd1 complex 5,26. In further support of a crucial role of Hrd1 in retrotranslocation, reconstitution experiments with purified Hrd1 showed that a membrane-anchored version of a misfolded mutant of carboxypeptidase Y (CPY*) 27 is translocated across the membrane when specific lysine residues of Hrd1 are available for auto-ubiquitination 7. A cryo-electron microscopy structure of Hrd1 in complex with Hrd3 revealed a hydrophilic cavity on the cytoplasmic side of Hrd1 and it was speculated that it represents a closed state of the retrotranslocon 28. We hypothesized that retrotranslocation-competent Hrd1 forms a water-filled channel under appropriate conditions and is thus suitable for electrophysiological characterization.

HRD Complex Self-Remodeling Enables a Novel Route of Membrane Protein Retrotranslocation

“…Our findings reveal Hrd1-mediated translocation of membrane substrates is dispensable of its partner component, Hrd3, as well as its ubiquitination activity. Recent studies suggest Hrd1-mediated translocation of ERAD-L occurs through self-ubiquitination activity of Hrd1, which has also been shown to be regulated by Hrd3 ( Baldridge and Rapoport, 2016 ; Peterson et al., 2019 ; Vasic et al., 2020 ). This led us to posit whether HRD complex remodeling in the suppression pathway negatively affects ERAD-L. CHX-chase assay was used to monitor the degradation rate of ERAD-L substrates, CPY∗ and KHN, in dfm1Δ suppressed cells overexpressing SUS-GFP as well ( Figures S3 A and S3B).…”

“…As shown in our first studies of Dfm1, its dedicated route of ERAD-M even applies to Hrd1 itself: in conditions where Hrd1 self-ubiquitination leads to its very rapid degradation, Dfm1 is absolutely required for Hrd1 retrotranslocation, despite Hrd1's clearly demonstrated role as a ERAD-L retrotranslocon ( Neal et al., 2018 ). This strict boundary makes sense, considering that Hrd1 undergoes an elaborate cycle of self-ubiquitination and de-ubiquitination in the course of its dual roles as a ligase and a lumenal retrotranslocon ( Peterson et al., 2019 ; Vasic et al., 2020 ). If Hrd1 could mediate its own retrotranslocation in normal circumstances, that might lead to catastrophic loss of this key quality control factor in the course of its normal molecular duty cycle.…”

“…Because retrotranslocation of either lumenal (ERAD-L) or integral membrane (ERAD-M) substrates were expected to require a channel or other catalytic enhancement, much work has been focused on identifying candidate molecules involved ( Carvalho et al., 2010 ; Garza et al., 2009a ; Hampton and Sommer, 2012 ; Nakatsukasa et al., 2016 ; Plemper et al., 1997 ; Scott and Schekman, 2008 ; Wahlman et al., 2007 ). From in vitro , in vivo , and structural studies, it now appears that the multi-spanning E3 ligase Hrd1 serves as a channel for lumenal ERAD-L substrates ( Baldridge and Rapoport, 2016 ; Peterson et al., 2019 ; Schoebel et al., 2017 ; Vasic et al., 2020 ). Until recently, the identity of an analogous channel for ERAD-M substrates had remained unclear.…”

Inner-nuclear-membrane–associated degradation employs Dfm1-independent retrotranslocation and alleviates misfolded transmembrane-protein toxicity

“…More generally, this study and others suggest that a growing number of proteins possess the ability to retrotranslocate quality-control substrates out of or through lipid bilayers. These include, but as we show are not limited to, Hrd1, Dfm1, Doa10, and the Asi complex ( Baldridge and Rapoport, 2016 ; Schoebel et al ., 2017 ; Neal et al , 2018 , 2020; Natarajan et al , 2020 ; Schmidt et al , 2020 ; Vasic et al , 2020 ; Wu et al , 2020 ). While redundancy is a common feature of protein-quality-control pathways, it will be interesting to further dissect the biochemical and cell-biological nuances that necessitate these dedicated channels.…”

Derlin-3 Is Required for Changes in ERAD Complex Formation under ER Stress

“…HRD1, Derlin-1, and Sec61 have been considered potential components of the retrotranslocon [ 20 , 21 , 22 , 23 ]. Recent studies based on cryogenic electron microscopy [ 24 , 25 ] and electrophysiological analysis [ 26 ] revealed that yeast HRD1 forms a retrotranslocation pore. In mammals, the Derlin family consists of three members: Derlin-1, Derlin-2, and Derlin-3.…”