MHC class I molecules are preferentially ubiquitinated on endoplasmic reticulum luminal residues during HRD1 ubiquitin E3 ligase-mediated dislocation

Abstract

Misfolded MHC class I heavy chains (MHC I HCs) are targeted for endoplasmic reticulum (ER)-associated degradation (ERAD) by the ubiquitin E3 ligase HRD1, and E2 ubiquitin conjugating enzyme UBE2J1, and represent one of the few known endogenous ERAD substrates. The mechanism by which misfolded proteins are dislocated across the ER membrane into the cytosol is unclear. Here, we investigate the requirements for MHC I ubiquitination and degradation and show that endogenous misfolded MHC I HCs are recognized in the ER lumen by EDEM1 in a glycan-dependent manner and targeted to the core SEL1L/HRD1/UBE2J1 complex. A soluble MHC I HC lacking its transmembrane domain and cytosolic tail uses the same ERAD components and is degraded as efficiently as wildtype MHC I. Unexpectedly, HRD1-dependent polyubiquitination is preferentially targeted to the ER luminal domain of full-length MHC I HCs, despite the presence of an exposed cytosolic C-terminal tail. MHC I luminal domain ubiquitination occurs before p97 ATPase-mediated extraction from the ER membrane and can be targeted to nonlysine, as well as lysine, residues. A subset of integral membrane proteins, therefore, requires an early dislocation event to expose part of their luminal domain to the cytosol, before HRD1-mediated polyubiquitination and dislocation.T he assembly and regulated expression of plasma membrane and secreted proteins is fundamentally reliant on effective endoplasmic reticulum quality control (ERQC). Endoplasmic reticulum (ER)-associated degradation (ERAD) is central to ERQC, selectively disposing of misfolded or surplus proteins to maintain ER homeostasis. ERAD involves recognition, ubiquitination, and retrotranslocation of substrates from the ER to the cytosol for proteasome-mediated degradation (1). Polyubiquitination of the substrate is critical, both for facilitating ER membrane extraction by the p97 ATPase-ubiquitin fusion degradation 1 (Ufd1)-nuclear protein localization 4 (Npl4) complex (2-4) and for delivery to the proteasome. However, the precise role of ubiquitin in the actual dislocation event is unclear.The cellular ERAD machinery consists of multiprotein complexes, typically comprising a membrane-embedded ubiquitin E3 ligase, which engages substrates directly or via ER luminal adaptors (1). Different substrates degraded by the same E3 may use distinct ERAD cofactors that facilitate substrate delivery to the ligase and E2-conjugating enzyme for ubiquitination.Attempts to delineate distinct degradation pathways according to the site of the defect in a misfolded protein have been made in yeast. Proteins with cytosolic defects (ERAD-C) require Doa10p, whereas proteins with transmembrane (ERAD-M) or luminal (ERAD-L) lesions use Hrd1p (5, 6). The expanded repertoire of ERAD E3s in mammalian cells makes developing analogous rules more challenging. 3-hydroxy-3-methylglutaryl-CoA reductase degradation protein 1 (HRD1) and its homolog gp78/autocrine motility factor receptor (AMFR) are the best-characterized mammalian ERAD ligases. The association of HR...

Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase

“…Intriguingly, ubiquitylation of the non-secreted immunoglobulin light chain NS-1, a natural ERAD substrate of the Hrd1 ligase in mammals, occurs on lysine as well as serine and threonine residues, although the E2(s) involved in this process are not known (Shimizu et al, 2010). Moreover, degradation of MHC class I molecules via Hrd1 and UBE2J1, an additional mammalian Ubc6 homologue, and turnover of T-cell Antigen Receptor α-Chain involves the ubiquitylation of non-lysine residues within the substrate (Burr et al, 2013;Yu and Kopito, 1999). Finally, recent studies of the degradation of the yeast E3…”

Enterovirus 71 protease 2Apro and 3Cpro differentially inhibit the cellular endoplasmic reticulum-associated degradation (ERAD) pathway via distinct mechanisms, and enterovirus 71 hijacks ERAD component p97 to promote its replication

“…Previous studies reported that three E2 ubiquitin-conjugating enzymes function in mammalian ERAD: Ubc6e/UBE2J1, UBE2J2, and UBE2G2 [54]. Ubc6e forms an E2-E3 pair with Hrd1 and is considered the principal E2 in cellular ERAD [54][55][56]. To further clarify the precise mechanism by which Ubc6e is cleaved, we investigated whether EV71-encoded viral proteases 2A pro and 3C pro participate in this process.…”

The E3 Ubiquitin Ligase TMEM129 Is a Tri-Spanning Transmembrane Protein

“…Yeast Hrd1p, for example, contains six transmembrane domains [58]. Human HRD1 likely has an identical topology [59] and is involved in degradation of misfolded HLA class I molecules in the absence of US2 and US11 [60,61]. Experiments involving the in vitro reconstitution of retrotranslocation using proteoliposomes and purified Saccharomyces cerevisiae proteins suggest that Hrd1p forms a ubiquitin-gated protein-conducting channel [62,63].…”