Endoplasmic reticulum (ER)-associated degradation (ERAD) removes misfolded proteins from the ER membrane and lumen by the ubiquitin-proteasome pathway. Retrotranslocation of ubiquitinated substrates to the cytosol is a universal feature of ERAD that requires the Cdc48 AAA-ATPase. Despite intense efforts, the mechanism of ER exit, particularly for integral membrane (ERAD-M) substrates, has remained unclear. Using a self-ubiquitinating substrate (SUS), which undergoes normal retrotranslocation independently of known ERAD factors, and the new SPOCK (single plate orf compendium kit) micro-library to query all yeast genes, we found the rhomboid derlin Dfm1 was required for retrotranslocation of both HRD and DOA ERAD pathway integral membrane substrates. Dfm1 recruited Cdc48 to the ER membrane with its unique SHP motifs, and it catalyzed substrate extraction through its conserved rhomboid motifs. Surprisingly, dfm1Δ can undergo rapid suppression, restoring wild-type ERAD-M. This unexpected suppression explained earlier studies ruling out Dfm1, and it revealed an ancillary ERAD-M retrotranslocation pathway requiring Hrd1.

Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of −310 mV via Mia40 with an intermediate midpoint potential of −290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of −150 mV. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX9C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.

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The HRD (HMG-CoA reductase degradation) pathway is a conserved route of endoplasmic reticulum-associated degradation (ERAD), by which misfolded ER proteins are ubiquitinated and degraded. ERAD substrates are ubiquitinated by the action of the Hrd1 RING-H2 E3 ligase. Hrd1 is always present in a stoichiometric complex with the ER membrane protein Hrd3, which is also required for HRD-dependent degradation. Despite its conserved presence, unequivocal study of Hrd3 function has been precluded by its central role in Hrd1 stability. Loss of Hrd3 causes unrestricted self-degradation of Hrd1, resulting in significant loss of the core ligase. Accordingly, the degree to which Hrd3 functions independently of Hrd1 stabilization has remained unresolved. By capitalizing on our studies of Usa1 in Hrd1 degradation, we have devised a new approach to evaluate Hrd3 functions in ERAD. We now show that Hrd3 has a direct and critical role in ERAD in addition to Hrd1 stabilization. This direct component of Hrd3 is phenotypically as important as Hrd1 in the native HRD complex. Hrd3 was required the E3 activity of Hrd1, rather than substrate or E2 recruitment to Hrd1. Although Hrd1 can function in some circumstances independent of Hrd3, these studies show an indispensable role for Hrd3 in living cells.highly conserved quality control pathway responsible for the degradation of both misfolded and normal proteins that limits cellular stress and cytotoxicity caused by an accumulation of misfolded ER proteins (1, 2). ERAD occurs through the ubiquitin proteasome pathway, wherein ER-localized ubiquitin ligases covalently modify substrates by attaching multiple copies of the protein ubiquitin, thus tagging them for degradation by cytosolic 26S proteasome (3-5).In Saccharomyces cerevisiae, the highly conserved HRD (HMGCoA † reductase degradation) pathway degrades a variety of ERAD substrates, including misfolded luminal proteins (ERAD-L substrates) such as CPY* and integral membrane proteins (ERAD-M substrates) such as Hmg2 (6, 7). Briefly, ERAD-L substrates are ubiquitinated by the HRD complex, which includes the ubiquitin RING-H2 ligase Hrd1, complex members Hrd3, Usa1, and Der1, and luminal factors implicated in substrate selection (3,(8)(9)(10)(11). Conversely, ERAD-M substrates require only Hrd1 and Hrd3 for in vivo degradation (12, 13). Although Hrd1 functions as the core catalytic subunit of the HRD E3 ligase, Hrd3's function has remained enigmatic. Both Hrd1 and Hrd3 are highly conserved, and Hrd1 is rate limiting for degradation of misfolded substrates (3,12,14,15). In yeast, the steady-state levels of Hrd1 strongly depend on Hrd3. At native levels of the HRD components, Hrd3 is required for Hrd1 stability: In a hrd3Δ, Hrd1 half-life drops to less than 15 min, resulting in very low levels (12). This concomitant loss of Hrd1 is due to rapid autodegradation, catalyzed by the RING domain of Hrd1 (16).Because of Hrd3's strong role in maintaining Hrd1 stability, it has remained unclear whether Hrd3 has any other independent ERAD functions. A ...

Highlights d Dfm1 selectively binds ERAD-targeted membrane substrates d Polyubiquitin chains bind directly to Cdc48 recruited by Dfm1 d Derlin lipid thinning facilitates removal of integral membrane substrates in the ER d Substrate engagement and lipid thinning are conserved derlin features