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HRD1 and UBE2J1 Target Misfolded MHC Class I Heavy Chains for Endoplasmic Reticulum- Associated Degradation

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HRD1 and UBE2J1 Target Misfolded MHC Class I Heavy Chains for Endoplasmic Reticulum- Associated Degradation HRD1 and UBE2J1 target misfolded MHC class I heavy chains for endoplasmic reticulum- associated degradation Marian L. Burr, Florencia Cano, Stanislava Svobodova, Louise H. Boyle, Jessica M. Boname, and Paul J. Lehner1 Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom Edited* by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved December 21, 2010 (received for review October 29, 2010) The assembly of MHC class I molecules is governed by stringent orthologs of yeast Hrd1p, HRD1 (Synoviolin) and gp78 (AMFR), endoplasmic reticulum (ER) quality control mechanisms. MHC class I have distinct substrate specificities, exemplifying the increased heavy chains that fail to achieve their native conformation in complexity of mammalian ERAD (6, 7). HRD1 is implicated in the complex with β2-microglobulin (β2m) and peptide are targeted for pathogenesis of rheumatoid arthritis (10), and reported substrates ER-associated degradation. This requires ubiquitination of the MHC include α1-antitrypsin variants (11) and amyloid precursor protein class I heavy chain and its dislocation from the ER to the cytosol for (12). Three E2 ubiquitin-conjugating enzymes function in mam- proteasome-mediated degradation, although the cellular machin- malian ERAD: UBE2J1 and UBE2J2 (yeast Ubc6p orthologs) and ery involved in this process is unknown. Using an siRNA functional UBE2G2 (yeast Ubc7p ortholog) (7, 8, 13). screen in β2m-depleted cells, we identify an essential role for the E3 Many viruses down-regulate cell-surface MHC I to evade im- ligase HRD1 (Synoviolin) together with the E2 ubiquitin-conjugating mune detection (2). The human cytomegalovirus US2 and US11 gene products bind newly synthesized MHC I HCs and initiate enzyme UBE2J1 in the ubiquitination and dislocation of misfolded their rapid dislocation to the cytosol for proteasomal degradation MHC class I heavy chains. HRD1 is also required for the ubiquitina- and helped define mammalian ERAD (14, 15). US2-mediated tion and degradation of the naturally occurring hemochromatosis- β MHC I degradation requires signal peptide peptidase and the E3 associated HFE-C282Y mutant, which is unable to bind 2m. In the ligase TRC8 (9, 16), whereas US11 utilizes a distinct Derlin 1, absence of HRD1, misfolded HLA-B27 accumulated in cells with p97, and SEL1L-dependent pathway (17–19). HRD1 and gp78 a normal MHC class I assembly pathway, and HRD1 depletion pre- are found in complex with Derlin I, although no functional role vented the appearance of low levels of cytosolic unfolded MHC I for these ligases has been demonstrated in US11-mediated MHC heavy chains. HRD1 and UBE2J1 associate in a complex together I ERAD (6, 19). Despite the crucial role of US2 and US11 in with non-β2m bound MHC class I heavy chains, Derlin 1, and p97 identifying several ERAD components, whether MHC I degra- and discriminate misfolded MHC class I from conformational MHC I- dation normally uses the same cellular machinery is unknown. β2m-peptide heterotrimers. Together these data support a physio- We found no role for TRC8 in endogenous MHC I degradation logical role for HRD1 and UBE2J1 in the homeostatic regulation of (9), so we performed a targeted siRNA screen to identify the E3 MHC class I assembly and expression. ligase(s) responsible. In the absence of β2m, MHC I fails to traffic to the cell surface he MHC class I (MHC I) heterotrimer, consisting of heavy and is targeted to the cytosol for proteasomal degradation, where Tchain (HC), β2-microglobulin (β2m), and an 8- to 10-residue an intermediate form of soluble, deglycosylated MHC I HC can peptide ligand, plays a vital role in immune surveillance of in- be detected (3). Here we identify an absolute requirement for tracellular pathogens. Its assembly is governed by a stringent en- the E3 ligase HRD1 and E2-conjugating enzyme UBE2J1 in the doplasmic reticulum (ER) quality control process (1). The newly ubiquitination and degradation of misfolded MHC I, suggesting synthesized MHC I HC is cotranslationally inserted into the ER an important role for these proteins in MHC I regulation. and rapidly associates with BiP, calnexin, and ERp57 (2). Sub- Results sequent binding of the HC to β2m is an early regulatory step that assists HC folding and is essential for incorporation of MHC I into siRNA Screen Identifies HRD1 as the E3 Ligase Required for the the peptide-loading complex (PLC) (2), consisting of the trans- Dislocation of Free MHC I HC. To identify the cellular machinery porter associated with antigen processing (TAP), tapasin, calre- required for the ubiquitination of misfolded, β2m-deficient MHC ticulin, and ERp57. The PLC facilitates loading of high-affinity I HCs, we performed a flow cytometry-based siRNA optical peptides onto the HC–β2m heterodimer. This process is tightly screen. HeLa cells expressing a luminal GFP-tagged MHC I HC β regulated, and only correctly assembled MHC I complexes are (GFP-HLA-A2) (9) were stably depleted of 2m (HeLa GFP- β released from the PLC for export to the cell surface. MHC I HCs HLA-A2 sh 2m cells), resulting in low cell-surface MHC I (Fig. β failing to achieve their native conformation, for example in the S1). The rationale for the screen is that in the absence of 2m, the absence of β2m or peptide, are retained intracellularly and de- misfolded MHC I HC is dislocated from the ER to the cytosol and graded (3). degraded by the proteasome (3). Depletion of any essential Removal of misfolded proteins from the lumen of the ER is component of the MHC I ERAD pathway should rescue the essential for maintenance of cellular homeostasis. The accumu- GFP-HLA-A2 HC from proteasome-mediated degradation, with lation of misfolded proteins induces ER stress and activation of the a consequent gain in GFP signal. unfolded protein response (4). ER-associateddegradation (ERAD) involves protein substrate recognition, ubiquitination, and disloca- tion from the ER to the cytosol for proteasome-mediated degra- Author contributions: M.L.B., F.C., S.S., and P.J.L. designed research; M.L.B., F.C., S.S., and dation. The ubiquitination reaction requires activation of ubiquitin J.M.B. performed research; L.H.B. contributed new reagents/analytic tools; M.L.B., F.C., by an E1 enzyme, transfer to an E2 ubiquitin-conjugating enzyme, S.S., J.M.B., and P.J.L. analyzed data; and M.L.B. and P.J.L. wrote the paper. and targeting of ubiquitin to the substrate protein by the E3 ligase. The authors declare no conflict of interest. The E3 confers substrate specificity and is therefore the pivotal *This Direct Submission article had a prearranged editor. enzyme (5). In contrast to yeast, in which Hrd1p and Doa10p are the Freely available online through the PNAS open access option. only two known ERAD E3s, at least five mammalian ERAD E3 1To whom correspondence should be addressed. E-mail: [email protected]. – ligases are known (HRD1, gp78, RMA1, TEB4, and TRC8) (6 9) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and further ERAD E3s may still be identified. The two mammalian 1073/pnas.1016229108/-/DCSupplemental. 2034–2039 | PNAS | February 1, 2011 | vol. 108 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1016229108 Downloaded by guest on October 2, 2021 A targeted siRNA screen of 18 putative ER membrane E3 ligases HRD1 Associates with β2m-Deficient MHC I HCs. Because MHC I HCs (Table S1) was performed in the HeLa GFP-HLA-A2 shβ2m cells. are potential HRD1 substrates, do they interact? E3–substrate Depletion of a single ligase, HRD1, showed a significant gain in interactions are transient and often difficult to detect (5), so cells fluorescence (Z score 3.72) (Fig. 1 A and B), whereas depletion of were treated with IFN-γ and proteasome inhibitor to increase gp78, the other mammalian ortholog of yeast Hrd1p (7), had no endogenous MHC I expression and preserve any HRD1–MHC I effect on GFP-HLA-A2 levels (Fig. 1B). In HeLa GFP-HLA-A2 interaction. Endogenous HRD1 was readily visualized in associ- US11-expressing cells, depletion of HRD1 and gp78 had no effect ation with misfolded MHC I HCs from β2m-depleted but not wild- on GFP-HLA-A2 levels (Fig. S2). Deconvolution of the screening type HeLa cells (Fig. 2A). The increased levels of coprecipitated pool of four oligos (Fig. S3) showed effective HRD1 depletion with HRD1 seen after IFN-γ treatment likely reflect increased syn- oligo #1 (Fig. 1C), which was used in subsequent experiments. thesis of MHC I, although the low levels of HRD1 under these We validated our findings by showing that HRD1 regulates the conditions is as yet unexplained. degradation of endogenous misfolded MHC I. The absence of β2m is associated with a marked loss of MHC I HC, consistent Misfolded MHC I HCs Are Retained in the ER After HRD1 Depletion. with targeting for degradation (Fig. 1C) (3). Depletion of HRD1 After proteasome inhibition, dislocated MHC I HCs are detected effectively rescues MHC I from degradation (Fig. 1C), with little as non–membrane-bound, soluble, deglycosylated species (3, 15). effect on MHC I levels in wild-type cells where the majority of To determine whether the accumulation of soluble MHC I HCs is MHC I HCs are correctly folded (Fig. S4). HRD1 dependent, cells were fractionated into membrane and soluble (cytosolic) forms, detergent solubilized, and misfolded HRD1 RING Mutant Exerts a Dominant Negative Effect and Protects MHC I HCs immunoprecipitated with HC10 antibody. Dislocated β2m-Deficient MHC I from Degradation. HRD1 is a polytopic ER- MHC I HCs are detected as faster migrating (deglycosylated) resident E3 ligase with a C-terminal RING-H2 domain (6).
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