Defining Human ERAD Networks Through an Integrative Mapping Strategy

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Deﬁning human ERAD networks through an integrative mapping strategy

John C. Christianson1,2,5, James A. Olzmann1,5, Thomas A. Shaler3, Mathew E. Sowa4, Eric J. Bennett4,6, Caleb M. Richter1, Ryan E. Tyler1, Ethan J. Greenblatt1, J. Wade Harper4 and Ron R. Kopito1,7

Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identiﬁed, how these proteins are organized into a functional network that coordinates recognition, ubiquitylation and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organization of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics and the transcriptional response to ER stress. This analysis supports an adaptive organization for the mammalian ERAD machinery and reveals a number of metazoan-speciﬁc genes not previously linked to ERAD.

Approximately one-third of the eukaryotic proteome consists of conjugating enzymes and form functional complexes by scaffolding secreted and integral membrane proteins that are synthesized and shared ERAD-related factors12–16, seem to be sufficient to degrade all inserted into the ER, where they must correctly fold and assemble ERAD substrates16,17. ERAD substrates with luminal or membrane to reach functional maturity1. ER quality control refers to the folding lesions utilize Hrd1 (refs 16,18), whereas those with cytoplasmic processes simultaneously monitoring deployment of correctly folded lesions rely on Doa10 (refs 16,17,19). In contrast to yeast, at least ten proteins and assembled complexes to distal compartments, while different E3s have been implicated in mammalian ERAD (ref. 20), possi- diverting folding-incompetent, mutant or unassembled polypeptides bly reflecting an evolutionary adaptation to the broader substrate range for proteasomal degradation through the process of ERAD (reviewed in imposed by the more complex metazoan proteome. Three mammalian refs 2–4). An ever-growing list of sporadic and genetic human disorders E3s, gp78, Hrd1 and TEB4, share similar domain and topological have been associated with ER quality control, illustrating the pivotal organization, but scant sequence homology, with their yeast ortho- role these processes play in governance of protein trafficking5. logues Hrd1 (orthologue of gp78 and Hrd1) and Doa10 (orthologue of Many of the individual components thought to underlie ERAD have TEB4). Uncovering how the organization of E3-containing membrane been identified through genetic and biochemical analyses in Saccha- complexes allows them to access substrates in the ER lumen/membrane romyces cerevisiae and mammals4,6 and point towards a mechanism and recruit the cytoplasmic dislocation/extraction apparatus is crucial mediated by a network of topologically and compartmentally restricted, to establishing a comprehensive understanding of ERAD. partially redundant protein complexes2,4,7–9. ERAD is a vectorial process Here, we have employed a systematic, multilayered approach that whereby coordination of ERAD components across three subcellular integrates high-content proteomics, functional genomics and gene compartments (ER lumen, lipid bilayer and cytoplasm) must occur expression to elucidate the interconnectivity and organization of ERAD to effectively distinguish, target and deliver misfolded substrates for in mammals (Supplementary Fig. S1). These studies have allowed us degradation. Exclusion of the ubiquitin–proteasome system (UPS) to generate an integrated physical and functional map of the ERAD from the ER lumen necessitates that substrates traverse the ER system in the mammalian ER. membrane to be degraded, but the molecular identity and mechanism of the required dislocation apparatus remains controversial7,10,11. RESULTS Ubiquitin E3 ligases play central functional and organizational roles Mapping the mammalian ERAD interaction network in ERAD (ref. 9). In yeast, the E3s Hrd1 and Doa10, which contain We employed a high-content proteomics strategy to map the cytoplasmically oriented RING domains that recruit distinct ubiquitin- mammalian ERAD interaction network, starting with 15 S-tagged

1Department of Biology & Bio-X Program, Stanford University, Lorry Lokey Building, 337 Campus Drive, Stanford, California 94305, USA. 2Ludwig Institute for Cancer Research, University of Oxford, ORCRB, Headington, Oxford OX3 7DQ, UK. 3SRI International, Menlo Park, California 94025, USA. 4Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. 5These authors contributed equally to this work. 6Present address: Division of Biological Sciences, UC San Diego, La Jolla, California 92093, USA. 7Correspondence should be addressed to R.R.K. (e-mail: [email protected])

Received 18 April 2011; accepted 21 October 2011; published online 27 November 2011; DOI: 10.1038/ncb2383

RESOURCE baits consisting of proteins previously identified as ERAD pathway over-representation in folding, ubiquitin and catabolic processes components in biochemical studies or by orthology to components (Supplementary Fig. S3c,d). identified in yeast (Supplementary Table S1, Primary). After confirming ER localization in HeLa cells (Supplementary Fig. S2) and stable Overview of the mammalian ERAD interaction network expression of each full-length S-tagged bait in HEK293 cells (data Unbiased hierarchical clustering of all HCIPs identified for each not shown), protein complexes captured by S-protein affinity bait in both detergents was used to assemble interaction data into purification from detergent-solubilized lysates were analysed by a coherent network (Fig. 1). Four of the digitonin clusters define liquid chromatography–tandem mass spectrometry (LC–MS/MS). subnetworks organized around the E3s Hrd1 (clusters 1D and 6D) Interactions were initially assessed for all baits by independently and gp78 (clusters 3D and 8D), indicating a central role in organization analysing pulldowns from cells lysed in digitonin (Supplementary of the mammalian ERAD system. Both Hrd1 and gp78 clustered Table S2) or the more stringent detergent Triton X-100 (Supplementary with established integral membrane, luminal and cytoplasmic ERAD Table S3). Total spectral counts for each captured protein were components (clusters 1D and 8D) as well as, separately, with most subsequently evaluated with the Comparative Proteomics Analysis 26S proteasome subunits (clusters 3D and 6D). Cluster 2D defines a Software Suite21,22 (CompPASS; Supplementary Methods). CompPASS macromolecular complex of previously uncharacterized proteins that employs a database of interacting proteins (including data from we have designated the mammalian ER membrane complex (mEMC, baits in this study and 102 unrelated proteins described previously21) discussed below) to reflect its orthology to a complex associated with the and comparative metrics to determine the likelihood of validity unfolded protein response (UPR) in yeast25. Cluster 4D confirms the of interactors. The CompPASS parameter WDN-score22, which previously reported interactions of the AAA+ ATPase VCP (also known integrates the abundance, uniqueness and reproducibility of an as p97) with an integral membrane binding partner VIMP (refs 26,27) interacting protein, was used to identify high-confidence candidate and cytosolic NGly1 (ref. 28), while revealing interactions of VIMP and interacting proteins (HCIPs) for the ERAD network. Previous studies UBXD2 with the VCP accessory protein UBE4A, a Ufd2 orthologue demonstrated that >68% of identified HCIPs were validated in implicated in ubiquitin chain extension29. In addition to confirming subsequent biochemical analyses21, a rate of validation that is well the ERFAD–SEL1L interaction30, cluster 5D validated the recently above other high-throughput approaches to study protein–protein reported interaction of ERFAD with TXD16 (ref. 24), reinforcing the interactions23. In our study, interacting proteins surpassing a connection between ERAD and oxidative protein folding/unfolding. stringent threshold score of WDN > 1.0 were designated as HCIPs Cluster 7D contains several HCIPs in complex with Derlin-1 and + (Supplementary Tables S2a and S3a). Interacting proteins scoring Derlin-2, including the Ca2 -sensing protein extended-synaptotagmin below this cutoff may still represent bona fide interactions (full list 1 (E-Syt1; ref. 31), the Ras superfamily member ARL6IP and YIF1B, in Supplementary Tables S2b and S3b). both implicated in protein trafficking32,33. In addition to revealing interconnections among primary baits, Of the eight prominent clusters identified in digitonin-solubilized this analysis uncovered 10 HCIPs that had no previous relationship cells, only cluster 2 remained intact with Triton X-100 lysis. Four with ERAD. Seven (FAM8A1, UBAC2, KIAA0090, TTC35, C15orf24, clusters (1, 3, 5 and 8) were fragmented into discrete subclusters, TMEM111 and COX4NB) are functionally uncharacterized open and three (4, 6 and 7) were fully disrupted. On the basis of these reading frames and two (E-Syt1 and MMGT1, also known as TMEM32) clusters (Fig. 1), we merged individual interactomes (Supplementary are implicated in cellular processes unrelated to quality control. The Fig. S4) to construct a topologically rendered, detailed interaction HCIP TXD16 (also known as ERp90) was recently suggested to map of the mammalian ERAD network in digitonin and Triton X-100 be involved in ERAD (ref. 24). On the basis of their identification (Fig. 2). The interaction network for ERAD (INfERAD) was arranged as HCIPs with multiple ERAD components in both digitonin and around clusters identified for Hrd1–SEL1L, gp78 and the mEMC Triton X-100, high spectral counts and predicted ER localization subnetworks, with those components located centrally reflecting shared (criteria described in Methods), these 10 HCIPs were introduced interactions between clusters. into the proteomic workflow to iteratively expand and validate the As with any systems-based analysis of interaction networks, our network (Supplementary Fig. S1, Secondary). Three proteins previously analysis was not exhaustive, and therefore we sought to integrate implicated in mammalian ERAD (TEB4, RNF5 and HERP) could data from public protein–protein interaction resources (STRING). not be sufficiently expressed and were omitted. Ultimately, our However, as ERAD components are poorly represented in this database ERAD network analysis included 25 baits, of which 9 seem to be (Supplementary Table S5) and other online resources (BIOGRID and unique to metazoans. No correlation was observed between bait MINT), interactions identified in this study were instead mapped abundance and the total number of interactions and HCIPs identified together with pairwise interactions reported previously (Supplementary (Supplementary Fig. S3a). From 3,325 individual proteins identified by Table S5 and Fig. S5). These combined data sets contain over MASCOT in digitonin and 2,971 in Triton X-100 (Supplementary 250 interactions, reflecting the organizational complexity of the Tables S2b and S3b), CompPASS identified 320 and 202 HCIPs, mammalian ERAD system. respectively, for the 25 baits (Supplementary Tables S2a and S3a). These HCIPs correspond to 143 and 97 non-redundant proteins The Hrd1–SEL1L subnetwork with 71 HCIPs of interest, previously uncharacterized for a role in Our proteomic analysis confirmed the E3 Hrd1 and its established ERAD (see Methods for selection criteria, Supplementary Table S4 cofactor SEL1L as a prominent nexus for ERAD. Nearly all previously and Fig. S3b). Over 50% of HCIPs are ER/membrane localized, and reported interactions of the Hrd1–SEL1L complex (Supplementary gene ontology analysis indicates diverse functionality with significant Table S5) were validated by our data set, which also uncovered

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1 10+ Triton X-100 solubilization Digitonin solubilization WDN -score

AUP1 AUP1 OS-9 1T 1T OS-9 SEL1L b c SEL1L 6D XTP3-B XTP3-B 1D FAM8A1 5T 1T FAM8A1 5D Hrd1 a Hrd1 EDEM1 EDEM1 ERFAD ERFAD TXD16 TXD16 Derlin-2 Derlin-2 7D

Derlin-1 Derlin-1 Baits* FAM62A Baits FAM62A C15orf24 2T C15orf24 2D KIAA0090 KIAA0090 COX4NB COX4NB TMEM111 TMEM111 4D MMGT1 MMGT1 TTC35 TTC35 NGly1 3T NGly1 3D VIMP a 3T VIMP UBXD2 b UBXD2 8D gp78 8T gp78 ERLIN2 ERLIN2 UBAC2 UBAC2 UBXD8 UBXD8 HCIPs HCIPs *Baits are arranged in the same hierarchical C15orf24 1T 1T cluster as for digitonin treatment 1D AUP1 2D a c OS-9 KIAA0090 SEL1L COX4NB FAM8A1 AUP1 XTP3-B TMEM111 Hrd1 UBE2G2SURF4PRKDCCKAP5AUP1 FAM8A1 MMGT1 Hrd1FAM8A1 Hrd1 TTC35 2T UBE2J1XTP3-BSEL1LSLC27A3CPVLHrd1FAM8A1PSMD7PSMD1HMGCRFDFT1OS-9MYH9LONP2GRP94UBE2G2DIP2BAUP1CAPRIN2 TTC35TMEM111TMEM93C19orf63MMGT1C15orf24COX4NBKIAA0090 C15orf24 1T KIAA0090 b OS-9 COX4NB SEL1L TMEM111 gp78 MMGT1 3D XTP3-B NGly1 SERPINH1GRP94RPN1LONP2GGHOS-9SEL1LCPVLXTP3-B TTC35 ERLIN2 4D TTC35MMGT1C15orf24TMEM85TMEM111KIAA0090COX4NBTMEM93C19orf63C14orf122 UBAC2 VIMP UBXD8 UBXD2 VCP VIMPDerlin-2SELKUBXD6KLHDC2UBXD2UBE4A TM PSMPSMPSMPSMPSMPSMPSMOSBPL8MYH10BRI3BPgp78PSMB7PSMA1PSMA7PSMA2PSMB6PSMB1PSMB4PSMB2PSMA6PSMB3PSMA5PSMA3ERLIN1ERLIN2 UB1 D4 D6 D3 D14C2 B5 A4 3T 3T gp78 a gp78 VCPPSME4PSMB6PSMB4PSMB3PSMB2PSMB1PSMA7PSMA6PSMA5PSMPSMA3PSMA2PSMA1gp78UBE2G2 b ERLIN2 6D A4 UBAC2 ERFAD UBXD8 5D Hrd1 ERLIN2ERLIN1 TXD16 PSMA1PSMB7PSMA7PSMA2PSMB6PSMB1PSMB4PSMB2PSMA6PSMB3PSMA5PSMA3ERLIN1ERLIN2 MCM2ENO1TXD16TAGLN2RPL35PPIBMARCKSHMRMPUHMRMPA2B1FLNAATP5F1USH2ANIPSNAP1LIN7CGANABGALK1DPY30CASKACTG2ERFADCANX

EDEM1 UBAC2 5T 8T 8D ERFAD UBXD8 gp78 TXD16 Derlin-2UBXD8 RAB9AKRT18EMDACTG2TXD16MCM2FLNASLC25A10NIPSNAP1LIN7CCASKERFADACTA1MTCH1EDEM1CANXE-Syt1 7D Derlin-2 ERLIN2 Derlin-1 UBAC2 E-Syt1 UBXD8 YIF1BARL6IP5SYNJ2BPE-Syt2E-Syt1RTN4PGRMC2EPHX1HMOX1Derlin-1POR UBAC2TUBA1ATMEM43TMED9RAB7APEX19GCN1L1HNRNPMSEC22BVCPVIMPDerlin-2

Figure 1 Hierarchical cluster analysis of CompPASS-identified Triton X-100 (right). Prominent HCIP clusters identified in digitonin high-confidence candidate interaction proteins (HCIPs). Hierarchical (1–8D) and Triton X-100 (1–3, 5 and 8T) were manually selected and clustering of HCIPs for interactions present in digitonin (left) and are highlighted below. The colour of the square indicates the WDN-score. several unreported interactors including FAM8A1; LONP2, a putative The gp78 subnetwork Lon-protease (with OS-9); CPVL, a putative carboxypeptidase (with Cluster analysis exposed a reciprocally co-precipitating complex XTP3-B); the stress-inducible haem oxygenase HMOX1 (also known consisting of the E3 gp78, an uncharacterized UBA domain-containing as HO1); and two components of the sterol biosynthetic pathway, polytopic protein UBAC2, the membrane-embedded, VCP-binding HMG-CoA reductase (HMGCR) and squalene synthetase (FDFT1). protein UBXD8, and Derlin-1 and Derlin-2. The high degree of The rate-limiting enzyme in cholesterol synthesis, HMGCR, is subject interconnectivity indicates that gp78 and its cognate E2 (UBE2G2) to strict feedback regulation whereby sterol end products induce its and UBAC2 comprise a transmembrane pathway for ERAD that shares degradation by ERAD (refs 12,34,35). Although evidence supports a essential cytoplasmic (for example, VCP and 26S proteasomes) and role for gp78 in the degradation of HMGCR in mammalian cells36, integral membrane components (UBXD8 and Derlin-2) with the the Hrd1 pathway degrades HMGCR in both yeast (Hmg2; ref. 12) Hrd1–SEL1L cluster. The recently described protein TMUB1 (ref. 40) and Drosophila37. Identification of HMGCR as a Hrd1 HCIP lends was found in the gp78 cluster, as was BRI3BP, hitherto unlinked to ER. strength to the possibility that Hrd1 also plays a role in HMGCR Signal peptide peptidase (SPP, also known as HM13) and a number degradation in mammals38. of poorly characterized integral membrane proteins (TMEM201, The integrity of the Hrd1–SEL1L subnetwork was strongly influenced TMEM43, LRRC59 and CLPTM1) were also linked through UBAC2. by solubilization conditions. All Hrd1 HCIPs except FAM8A1 (Fig. 1a, In contrast to the Hrd1–SEL1L cluster, most HCIPs associated with cluster 1Ta) were lost in Triton X-100, whereas the complexes the gp78 subnetwork were stable in both detergents (Supplementary containing SEL1L, OS-9 and other luminal components were Table S2, S3 and S6). Disruption of the Hrd1–SEL1L cluster in Triton preserved (Fig. 1a, cluster 1Tb), consistent with all upstream (luminal) X-100 caused the shared cytoplasmic interactors VCP and UBE2G2 interactions being mediated through SEL1L, which is linked to Hrd1 to cluster with gp78, probably reflecting their direct binding to the by a Triton X-100-labile association39. carboxy-terminal cytoplasmic domain of gp78 (refs 41,42). These data

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Protein Length Locale SC SGD ID name (aa) GRP94 KIAA0090 993 IMP Emc1 YCL045C CPVL LONP2 MMGT1 131 IMP Emc5 YIL027C TXD16 Unidirectional (only in digitonin) Reciprocal TMEM85 183 IMP Emc4 YGL231C In digitonin and Triton X-100 OS-9 TMEM111 261 IMP Emc3 YKL207W Only in Triton X-100 ERFAD TTC35 297 Cyto. Emc2 YJR088C Bait Interactor XTP3-B TMEM93 110 IMP Emc6 YLL014W EDEM1 CANX C15orf24 242 IMP - - SEL1L C14orf122 208 Cyto. - - Lumen COX4NB 210 Cyto. - - FAM8A1 C19orf63 262 - Hrd1 IMP -

Derlin-2 Derlin-1 UBAC2 KIAA0090 C15orf24

TMEM111 MMGT1 UBE2J1 VIMP ERLIN2

UBXD2 UBXD8 C19orf63 TMEM85 AUP1 ERLIN1 E-Syt1 E-Syt2 SPP TMEM93 UBE2G2 Cytoplasm PSMA 1–7 TTC35 PSMB 1–7 C14orf122 hHR23B PSMC 2 COX4NB NGly1 UBE4A PSMD 1–3,6,7,14 PSME4 VCP ER localized Other

YIF1Bc19orf6 CLPTM1 TMUB1 TMEM201 SURF4* NIPSNAP1*DPY30 TBC1D15* UBXD6 CAPRIN2 BRI3BP FDFT1 TMED9 ARL6IP5 TMEM43RTN4* CASK* SYNJ2BP SELK DIP2B TBC1D17* HMOX HMGCRSERPINH1* RPN1* LRRC59 GGH*LIN7*DUSP3 KLHDC2 OSBPL8 CIB2*

Figure 2 The INfERAD. Interaction network for ERAD isolated in digitonin lists the determined constituents of the mammalian ER membrane and Triton X-100 represented by baits (squares) and their HCIPs (circles). complex (mEMC), their size (in amino acids, aa), cellular localization Unidirectional (dashed, single arrow) and reciprocal (solid black, double (IMP, integral membrane protein; Cyto., cytosolic), and corresponding arrows) interactions are shown. Each bait protein is rendered in a unique yeast orthologues (SC) and ID in the Saccharomyces Genome Database colour and line colour reﬂects the bait protein used to identify the (SGD). For clarity, a selection of additional digitonin HCIPs not included interaction with the HCIP. Dotted lines marked with a circle indicate in the map is shown on the bottom, with a circle’s colour corresponding interactions detected in both digitonin and Triton X-100, and long to the bait for which the HCIP was observed and asterisks denoting an dashed lines represent those found only in Triton X-100. The inset table HCIP also detected in Triton X-100. establish gp78 as the core of a detergent-stable E3 subnetwork that The gp78 HCIP PSME4 (also known as PA200) was identified in a shares several components with the Hrd1 complex, and identify UBAC2 screen for 26S proteasome activators and originally reported as a nu- as a central element in the gp78 complex. clear protein with a possible role in DNA repair45. PA200 can assemble with 20S and 19S subunits to form hybrid 26S proteasomes46 and a ERAD E3s are associated with the 26S proteasome crystal structure of the apparent yeast orthologue Blm10 indicates that Strikingly, nearly all subunits of the 26S proteasome were captured it interacts directly with proteasome α-subunits47. The functional signif- with Hrd1 and gp78 in digitonin lysates. Although not all of these icance of the PA200 interaction is unclear, but its presence in gp78 (but interactions reached our stringent criteria to qualify as HCIPs (19/32 not Hrd1) complexes indicates that there may be heterogeneous 26S for gp78; 15/31 for Hrd1; Supplementary Table S6), the fact that proteasome populations associated with the ER membrane and ERAD. gp78 captured all subunits of the 20S core particle and most subunits of the 19S regulatory particle indicates a significant connection The mEMC subnetwork between the ERAD E3s and 26S proteasomes (Supplementary The detergent-stable mEMC (Fig. 1, cluster 2) was initially identified Table S6 and Fig. S6). Persistence of these interactions in Triton through KIAA0090, an uncharacterized, putative type I integral X-100, together with the observation that proteasome subunits membrane glycoprotein detected as a Derlin-1/2 HCIP (Fig. 1 and were not identified as HCIPs of other ERAD interaction network Supplementary Table S2). With KIAA0090 as bait, we identified components, indicates an intimate, perhaps direct interaction of five additional HCIPs (TTC35, MMGT1, TMEM85, C15orf24 and gp78 (and possibly Hrd1) with the 26S proteasome. Proteasome COX4NB), which reciprocally co-precipitated each other, and four stability requires ATP and without it, dissociation into 20S core additional proteins (TMEM111, C19orf63, C14orf122 and TMEM93; and 19S regulatory particles can occur43,44. The excess of 20S Supplementary Fig. S4). The mEMC comprises ten unique subunits, core particle subunits over 19S regulatory subunits captured with whereas its yeast counterpart seems to contain six (Fig. 2). Although gp78 and Hrd1 (Supplementary Fig. S6) raises the possibility that the function of the mEMC is unknown, three subunits (KIAA0090, E3–proteasome connections may be linked independently of the 19S TMEM111 and TTC35) were identified as HCIPs of UBAC2 and Derlin- regulatory particle, perhaps through direct interactions with 20S or 2, indicating a close link between this complex and ERAD components through alternative adaptors. implicated in ubiquitin recognition and protein dislocation.

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a Input AP: S-prot beInput AP: S-prot OS-9 OS-9 XTP3-B–S ++++ XTP3-B–S ++++ FF Luc shRNA + ––+ myc–UBE2J1 ++++ XTP3-B d XTP3-B SEL1L shRNA ––+ + FF Luc shRNA + ––+ a ––+ + Mr (K) SEL1L shRNA c SEL1L SEL1L 100 SEL1L Mr (K) 75 Hrd1 50 myc–UBE2J1 FAM8A1 FAM8A1 75 XTP3-B–S Hrd1 Hrd1 50 100 SEL1L 75 b 75 XTP3-B–S c Input AP: S-prot 50 UBE2J1 UBE2J1 UBE2G2 XTP3-B–S ++++ d AUP1 AUP1 UBE2G2 FF Luc shRNA + – + – Input AP: S-prot ––+ + SEL1L shRNA S–OS9.2 ++++ Mr (K) FF Luc shRNA + ––+ 50 FAM8A1 SEL1L shRNA ––+ + Mr (K) 100 SEL1L 100 SEL1L 75 75 Hrd1 75 XTP3-B–S 100 S–OS9.2 50 75 f Input AP: S-prot g Input AP: S-prot h Input AP: S-prot S–FAM8A1 ++++++ Hrd1–S + +++ S–SEL1L ++++ FF Luc shRNA + ––– + – myc–UBE2J1 + +++ myc–UBE2J1 ++++ SEL1L shRNA ––+ – + – FF Luc shRNA + – + – FF Luc shRNA + ––+ Hrd1 shRNA –––– ++ SEL1L shRNA – + – + Hrd1 shRNA ––+ + Mr (K) Mr (K) Mr (K) SEL1L 75 75 SEL1L 75 Hrd1 50 50 75 Hrd1 myc–UBE2J1 myc–UBE2J1

S–FAM8A1 75 Hrd1–S S–SEL1L 35 75 i j k OS-9 OS-9 Input AP: S-prot Input AP: S-prot AUP1–S ++++ AUP1–S ++++ XTP3-B XTP3-B FF Luc shRNA + ––+ myc–UBE2G2 ++++ Hrd1 shRNA ––+ + FF Luc shRNA + --+ SEL1L SEL1L Hrd1 shRNA --+ + Mr (K) f g 75 Mr (K) Hrd1 FAM8A1 FAM8A1 75 Hrd1 Hrd1 Hrd1 SEL1L 20 75 myc–UBE2G2 h i AUP1-S UBE2J1 UBE2J1 AUP1–S 35 35 j AUP1 UBE2G2 AUP1 UBE2G2 l Input AP: S-prot m Input AP: S-prot n Input AP: S-prot UBAC2–S ++ ++ UBAC2–S ++++ FF Luc shRNA + ––+ UBXD8–S ++++ FF Luc shRNA + ––+ + – + UBXD8 shRNA ––+ + gp78 shRNA – FF Luc shRNA + ––+ gp78 shRNA ––+ + M (K) M (K) r r M (K) 75 gp78 50 UBXD8 r 35 UBAC2 50 UBXD8 75 gp78 gp78 35 UBAC2–S 35 UBAC2–S 75 UBAC2 UBAC2 50 UBXD8–S o Input AP: S-prot p UBXD8–S ++++ Input AP: S-prot FF Luc shRNA + ––+ q ––+ + UBXD8–S ++++ gp78 gp78 UBAC2 shRNA m M (K) FF Luc shRNA + --+ Derlin-2 Derlin-2 r Derlin-2 shRNA --+ + UBAC2 UBAC2 75 gp78 l Mr (K) l 35 UBAC2 75 gp78 p n o 25 25 Derlin-2 Derlin-2 UBE2G2 UBXD8 UBE2G2 UBXD8 50 UBXD8–S 50 UBXD8–S VCP VCP

Figure 3 shRNA-mediated refinement of ERAD E3 ligase subnetworks. SEL1L. (h) Co-expression of myc–UBE2J1 and S–SEL1L, probe for (a–q) S-tagged ERAD baits were transiently co-expressed with the myc and Hrd1. (i) AUP1–S expression, probe for Hrd1 and SEL1L. indicated shRNAs in HEK293 cells. All complexes were affinity (j) Co-expression of myc–UBE2G2 and AUP1–S, probe for myc and purified (AP) in 1% digitonin and analysed by immunoblotting. Hrd1. (k) Refined interaction map for Hrd1 complex. (l) UBAC2–S S-prot, S-protein; FF, firefly. (a) XTP3-B–S expression, probe for Hrd1 expression with UBXD8 knockdown, probe for gp78. (m) UBAC2–S and SEL1L simultaneously. (b) Co-expression of myc–UBE2J1 and expression with gp78 knockdown, probe for UBXD8. (n) UBXD8–S XTP3-B–S, probe for myc and SEL1L; (c) XTP3-B–S expression, probe expression with gp78 knockdown, probe for UBAC2. (o) UBXD8–S for FAM8A1 and SEL1L. (d) S–OS-9 expression, probe for Hrd1 and expression with UBAC2 knockdown, probe for Derlin-2, UBAC2 and SEL1L simultaneously. (e) Incorporation of refinements (a–d) to the gp78. (p) UBXD8–S expression with Derlin-2 knockdown, probe for Hrd1 complex. (f) S–FAM8A1 expression, probe for Hrd1 and SEL1L. gp78. (q) Refined interaction map for the gp78 complex. Uncropped (g) Co-expression of myc–UBE2J1 and Hrd1–S, probe for myc and images of blots are shown in Supplementary Fig. S12.

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Deconvolving the ERAD interaction network with RNA effects that might alter UPS function, reporter gene expression or GFP interference fluorescence intensity. To begin to decipher the organization within the mammalian ERAD All substrate reporter lines responded to proteasome inhibition interaction network, we systematically analysed the Hrd1–SEL1L and with time-dependent increases in mean GFP fluorescence (Fig. 4b). gp78 subnetworks. Co-expression of S-tagged proteins with short Expression of a dominant-negative VCP mutant (H317A; ref. 52) hairpin RNAs (shRNAs) targeting central subnetwork nodes was severely impaired degradation of only the ERAD substrates, but not used to ascertain the requirement of each component to maintain GFPu (Fig. 4c), in agreement with the strong dependence of ERAD individual interactions (Fig. 3). Following SEL1L knockdown, XTP3-B pathways on VCP and 26S proteasomes. The mannosidase inhibitor interactions with Hrd1 (Fig. 3a), UBE2J1 (Fig. 3b) and FAM8A1 kifunensine selectively inhibited the degradation of A1ATNHK (Fig. 4c), (Fig. 3c) were abolished, and OS-9 lost its connection to Hrd1 (Fig. 3d). consistent with an established requirement for mannose trimming of LC–MS/MS analyses confirmed that XTP3-B and OS-9 affinity-purified this glycoprotein for ERAD (refs 55,56). GluR1 and CFTR1F508 are complexes from cells lacking SEL1L lost their interactions with all also glycoproteins (Supplementary Fig. S7c), but were unaffected by downstream membrane and cytosolic components (for example Hrd1, kifunensine (Fig. 4c), indicating that mannose trimming is unlikely data not shown). These data verify the essential role that SEL1L to be the dominant signal committing them to degradation, or that plays in scaffolding luminal, substrate-recognition elements to the there is redundant, glycan-independent targeting for these polytopic Hrd1 transmembrane complex30,39,48,49, and reveal the independent proteins. These data reflect an implicit requirement for multiple, interactions of XTP3-B and OS-9 with the Hrd1–SEL1L node (Fig. 3e). substrate-specific recognition elements within the ERAD interaction Hrd1–SEL1L subnetwork connections to integral membrane and network to deliver substrates to shared degradation machinery. cytosolic ERAD components differed in that SEL1L knockdown did To identify the individual factors required for substrate degradation, not affect the Hrd1–FAM8A1 (Fig. 3f) or Hrd1–UBE2J1 interactions we generated an shRNA library targeting genes implicated in ERAD (Fig. 3g). Similarly, loss of Hrd1 failed to sever the connections between (Fig. 4d and Supplementary Table S5) and monitored their impact on SEL1L–UBE2J1 (Fig. 3h), SEL1L–AUP1 (Fig. 3i) or AUP1–UBE2G2 the mean GFP fluorescence of reporter cell lines (Fig. 4e and Supple- (Fig. 3j). Thus, both Hrd1 and SEL1L bind to UBE2J1, either mentary Table S7). Any shRNA that significantly stabilized an ERAD directly or through a factor not identified in our proteomic analysis. reporter was selected for further validation by re-screening through Hrd1 knockdown abolished the SEL1L–FAM8A1 interaction (Fig. 3f), all other reporter lines and confirmation of knockdown (see Methods indicating that SEL1L and FAM8A1 independently bind to Hrd1. This and Supplementary Fig. S8). Each substrate seemed to rely on a unique conclusion is reinforced by the maintenance of the Hrd1–FAM8A1 set of individual ERAD components for degradation (Supplementary interaction in Triton X-100 where SEL1L is lost (Fig. 1). These data Table S7), which is illustrated as a hierarchically clustered heat map for refine the molecular topology of the Hrd1–SEL1L complex, and identify comparison (Fig. 4f). Of the 59 components our library targeted, only FAM8A1 as an obligate, SEL1L-independent partner of Hrd1 (Fig. 3k). the non-ATPase subunit of the 19S regulatory particle PSMD2 and VCP A second prominent, highly interconnected subnetwork is composed were essential for all ERAD substrates (Fig. 4f). GFPu was stabilized by of gp78, Derlin-2, UBAC2 and UBXD8 (Fig. 2). Knockdown of knockdown of PSMD2, but not VCP, mimicking the effects of MG132 UBXD8 did not disrupt the gp78–UBAC2 interaction (Fig. 3l), nor and VCPH317A (Fig. 4b,c) and validating the strategy of using shRNA- did knockdown of gp78 affect UBXD8–UBAC2 (Fig. 3m,n). Although mediated gene silencing with ERAD reporters to interrogate the contri- gp78 binding was lost, maintenance of the UBXD8–UBAC2 interaction bution of individual components to the overall degradation process. Hi- in Triton X-100 indicates that their organization occurs independently erarchical cluster analysis demonstrated that substrates were segregated of gp78 (Fig. 1). However, the UBXD8–gp78 interaction was abrogated by topology (luminal versus integral membrane), but not by glycosy- by knockdown of UBAC2 (Fig. 3o) but not Derlin-2 (Fig. 3p). These lation (Fig. 4f). Moreover, a surprising degree of heterogeneity within data allow refinement of the gp78 subnetwork topology (Fig. 3q) each substrate’s requirement profile was observed, especially for sub- and identify a role for UBAC2 in the recruitment of UBXD8 to the strates utilizing the same central ERAD components (for example Hrd1, gp78 complex. discussed below). These characteristic patterns indicate that the ERAD system operates largely as an adaptive network, in which unique combi- Functional genomic analysis of ERAD components nations of common components process individual substrates. Such an To assess their functional roles in substrate degradation, we monitored adaptive mechanism could be explained by the formation of substrate- the effect of RNA interference (RNAi)-mediated knockdown of specific subcomplexes or by a multisubunit complex that utilizes individual ERAD components on steady-state fluorescence levels of discrete sets of components to achieve substrate-specific degradation. fluorescent ERAD substrate reporters21,50–53. Cell lines stably expressing GFP fusions representing three major topological classes of ERAD An adaptive mechanism for Hrd1-dependent degradation substrates: luminal-glycosylated (null Hong Kong variant of α1-anti- We merged the heat map of shRNA-mediated impairment for each trypsin (A1ATNHK)), luminal-non-glycosylated (A1ATNHK-QQQ and substrate (Fig. 4f) with the comprehensive ERAD interaction network mutant transthyretin TTRD18G) and integral membrane-glycosylated (Supplementary Fig. S5) to generate integrated substrate-specific (CFTR1F508; Fig. 4a) substrates were employed. We also included the snapshots of the physical and functional networks responsible for degra- AMPA-type glutamate receptor subunit GluR1, as it is retained in dation (Supplementary Figs S9–S11). Loss of either E3 in the network the ER and degraded in a UPS-dependent manner (Supplementary impacted degradation in a substrate-specific manner. Hrd1 knockdown Fig. S7). Cell lines expressing the cytosolic proteasome substrate stabilized topologically disparate substrates including A1ATNHK, GFPu (ref. 54) and GFP served as controls for ERAD-independent A1ATNHK-QQQ, TTRD18G and GluR1, but had little effect on GFPu or

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a bc GFP Degradation score 4.0 MG132 –3.0 +3.0 0 h Enhanced Impaired GFP GFP 1 h NHK 2 h Integral D18G A1AT –GFP 3.0 TTR –GFP 3 h Cyto. membrane Luminal NHK-QQQ GFP A1AT –GFP 2.0

ER lumen F508 Δ NHK-QQQ NHK u 1.0 D18G CFTR GFP GluR1 TTR A1AT A1AT Fold change in mean

GFP fluorescence intensity Wild-type VCP Cytosol 0 u VCPH317A GFP NHK F508

GFP–GluR1 D18G Δ GFP ΔF508 GluR1 GFP– CFTR NHK-QQQ 0 TTR

GFP GFP A1AT

u CFTR 2

GFP GFP (h) 4 A1AT 6 d Cyto. Integral Luminal Kifunensine Total number of shRNA constructs 309 membrane Total number of target genes 59 Reported ERAD components 45 g XTP3-B XTP3-B Potential ERAD components 14 OS-9 OS-9 f Average number of shRNAs per target ~5 SEL1L SEL1L

FAM8A1 Hrd1 FAM8A1 Hrd1 F508 NHK-QQQ Δ

e NHK u Transfect shRNA D18G

TTR A1AT A1AT GFP GluR1 AUP1 AUP1 library into ERAD CFTR UBE2J1 UBE2J1 59 targets reporter lines 309 shRNA Derlin-2 UBE2G2 UBE2G2 NGly1 UBE2G2 GFPu A1ATNHK ERFAD Incubate cells OS-9 for 72 h Empty Derlin-3 XTP3-B OS-9 XTP3-B OS-9 JAMP UBE4B Aha1 SEL1L SEL1L Control FF Luc ERAD Measure GFP impairment UBE4A FAM8A1 Hrd1 FAM8A1 Hrd1 fluorescence intensity ERDJ5 Cells by flow cytometry XTP3-B UBXD2 GFP fluorescence intensity AUP1 UBE2J1 AUP1 UBE2J1 AUP1 TTC35 gp78 UBE2G2 UBE2G2 VIMP TXD16 TTRD18G A1ATNHK-QQQ Select positive E-Syt1

score EDEM1

z hits KIAA0090 XTP3-B C15orf24 OS-9 XTP3-B OS-9 shRNA PSMD2 VCP Hrd1 Luc SEL1L SEL1L UBAC2 Control FF shRNA 1 shRNA 2 shRNA FAM8A1 Mr (K) FAM8A1 FAM8A1 ERAD HERP Hrd1 Hrd1

Cells 100 Target impairment VCIP135 Lysate SVIP LONP2 UBE2J1 AUP1 UBE2J1 AUP1 GFP fluorescence intensity 50 Tubulin UBXD8 COX4NB UBE2G2 UBE2G2 Re-screen Confirm knockdown UBE2J1 SEL1L positive hits of target ΔF508 Derlin-1 GluR1 CFTR

Figure 4 Functional genomic screen to identify essential substrate- treatment, and thus a degradation score of 3 is equivalent to the impairment specific ERAD components. (a) Localization and topology of GFP induced by 3 h MG132 treatment. (d) Target composition of the shRNA reporters (TTRD18G–GFP, A1ATNHK–GFP, A1ATNHK-QQQ–GFP, GFP–GluR1, library. (e) Overview of the functional genomic screen. (f) Hierarchically GFP–CFTR1F508 and GFPu) and GFP. (b) Time course of relative mean GFP clustered heat map of the normalized fold change in mean GFP fluorescence fluorescence intensity levels for each ERAD reporter cell line treated with intensity of ERAD reporter lines in response to shRNA-mediated knockdown MG132 (10 µM). Cyto., cytosolic. (c) Heat maps reflecting the normalized of ERAD components. The normalization and colour scale are the same as in fold change in mean GFP fluorescence intensity of ERAD reporter lines c. FF, firefly. (g) Functional data from the heat map shown in f were mapped transfected with wild-type or dominant-negative VCP (wild-type or H317A, onto the refined Hrd1 physical interaction network (Fig. 3k) to provide top panel) and time course of treatment with kifunensine (30 µM, bottom an integrated snapshot of substrate-specific functional requirements for panel). Fold change in mean GFP fluorescence intensity was normalized Hrd1 network components. Uncropped images of blots are shown in to the levels measured for each reporter at the 3 h time point of MG132 Supplementary Fig. S12.

CFTR1F508 (Fig. 4f,g). Instead, CFTR1F508 was stabilized following gp78 share a common dependence on Hrd1–SEL1L subnetwork components. knockdown, as previously reported57. Substrates utilizing Hrd1 did not Whereas FAM8A1 and SEL1L were essential for degradation of

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a 6

HERP 5 BiP )

2 3 Derlin-3 GRP94 Hrd1 COX4NB OS-9 TTC35 FAM8A1 2 CANX VIMP KIAA0090 Derlin-1 XTP3-B MMGT1 Derlin-2 CNX SPP ORP150 EDEM2 UBE2J1 C15orf24 ERLIN2 EDEM3 TMEM85 E-Syt2 SIL1 UBAC2 C14orf122 CPVL Parkin Fold induction (log UBE4B Aha1 1 EDEM1 hHR23B Fbx6 TMEM111 NGly1 UFD1 AUP1 UBE2G2 USP13 ccdc47 JAMP p47 SVIP CHIP VCIP135 PSMD2 VCP UBXD8 RNF5 TMEM93 E-Syt1 ERFAD UGT2 UBXD2 hHR23A gp78 ATX3 BAP31 TXD16 ERMan1 YOD1 0 TMUB1 TRC8 ERLIN1 UGT1 Npl4 C19orf63 LONP2 GLT25D1 RFP2 UBE4A TEB4

–1 EMC Redox Other Ubiquitin binding E2 enzymes E3 enzymes factors VCP cofactorsVCP recruiting ER chaperonesGlycan binding Deubiquitylatingenzymes and processing

b Unidirectional GRP94 Membrane Cytoplasm Lumen CPVL LONP2 Reciprocal TXD16 Derlin-3 Fbx6 UGT1

OS-9 Bait Interactor TRC8 CHIP UGT2 ERdj5 TMUB1 AHA1 ERMan1 ERFAD XTP3-B Parkin EDEM2

EDEM1 CANX UBE4B EDEM3

SEL1L RFP2 ORP150 Lumen HACE1 SIL1 FAM8A1 gp78 Hrd1 RNF5 BiP BAP31 JAMP

HERP Derlin-2 Derlin-1 UBAC2 KIAA0090 C15orf24 TEB4 TMEM111 MMGT1 UBE2J1 VIMP ERLIN2 C19orf63 TMEM85 UBXD2 UBXD8 AUP1 ERLIN1 E-Syt1 E-Syt2 SPP SVIP TMEM93 Cytoplasm UBE2G2 TTC35 PSMD2 C14orf122 COX4NB hHR23B UBE4A VCIP135 NGly1 VCP Ufd1 –3 3 ATX3 Npl4 Fold induction (log ) YOD1 2

Figure 5 Coordinated ER stress response of ERAD genes. qRT–PCR results changes in ERAD genes plotted as groups according to: (a) fold induction of for validated and suspected ERAD components following treatment of gene expression represented by functional category, and (b) fold induction HEK293 cells with tunicamycin (10 µg ml−1, 6 h). Data are presented as of gene expression from a mapped onto the ERAD interactome from Fig. 2. fold induction (log2) normalized to β-actin. Tunicamycin-induced expression Additional genes of interest are presented alongside the induction map.

A1ATNHK and TTRD18G and dispensable for GluR1, the converse was Coordinate regulation of ERAD genes by the unfolded true for AUP1 and UBE2G2 (Fig. 4f,g). Furthermore, despite sharing protein response a common requirement for VCP, ERAD substrates were differentially Expression of more than half of the ERAD genes in our network, dependent on VCP-interacting proteins such as SVIP, UBE4A and including the Hrd1–SEL1L subnetwork (Fig. 5) and other known VCIP135 (Fig. 4f), perhaps indicating additional heterogeneity among UPR targets (for example, BiP and HERP), was induced by the VCP-containing complexes employed for dislocation. tunicamycin (Fig. 5). In contrast, gp78 and other ER-resident

RESOURCE abcCyto. Lumen TMD d IP NaClNa2CO3 SDS FAM8A1 Mr (K) SPSPSP RDD 1.0 50 413 aa FAM8A1 CONSENSUS

M (K) 10% input Control FAM8A1 TM1: 255–275 OS-9 r 0.5 XTP3-B 100 SEL1L TM2: 306–326 100 VCP 100 Reliability TM3: 371–391 Hrd1 0 100 0 100 200 300 400 SEL1L SEL1L 50 FAM8A1 FAM8A1 Amino-acid position 50 Tubulin Hrd1 ef g Cytosolic and 40% Cytosolic epitopes luminal epitopes 10% GFPu GluR1 TTR D18G S–FAM8A1 Hrd1–S S–FAM8A1 Hrd1–S Fractions1 2 3 4 5 6 7 8 9 10 11 12 10% AP Wild-type Hrd1 Mr (K) Hrd1C294A FAM8A1 Wild-type Hrd1 + SEL1L KDEL KDEL KDEL KDEL 35 100 SEL1L Wild-type FAM8A1 DFP 75 Hrd1–S DFP–FAM8A1Δ1–229 Overlay Overlay Overlay Overlay FAM8A1Δ230–413 Degradation score -3.0 +3.0 Enhanced Impaired

Figure 6 Characterization of the Hrd1-binding partner FAM8A1. antibody access to cytosolic epitopes or cytosolic and luminal epitopes, (a) Domain structure and interaction network of FAM8A1. aa, amino respectively, immunostained and analysed by fluorescence microscopy. acids. (b) Immunoprecipitation (IP) with anti-FAM8A1 from HEK293 Scale bar, 10 µm. (f) Hrd1–S-expressing HEK293 cell lysates separated digitonin-soluble lysates was analysed by immunoblotting with the on a continuous 10–40% sucrose gradient. S-tagged Hrd1 protein indicated antibodies. (c) Consensus TOPCONS prediction of FAM8A1 complexes were affinity purified from each 1 ml fraction (fractions 1–12) membrane orientation (http://topcons.cbr.su.se). The reliability index or from 150 mg whole-cell lysate (10% AP), and analysed by western indicates the likelihood for consensus prediction at each position using blotting for Hrd1 (S-tag), SEL1L and FAM8A1. (g) Heat map representing a sliding 21 amino-acid window. Cyto., cytosolic; TMD, transmembrane the normalized change in mean GFP fluorescence intensity (20,000 cells, domain. (d) HEK293 membrane fractions incubated with 1 M NaCl, n =3) of the indicated ERAD reporter cell lines following transfection with

0.1 M Na2CO3 at pH 12 or 1% SDS. Following 100,000g centrifugation, the indicated Hrd1, SEL1L and FAM8A1 plasmids. DFP indicates dead equal volumes of soluble (S) and pellet (P) fractions were analysed by ﬂuorescent protein, a non-ﬂuorescent GFP variant. Data are represented western blotting with anti-FAM8A1. (e) HeLa cells expressing S–FAM8A1 as a normalized heat map as in Fig. 4c. Uncropped images of blots are or Hrd1–S were permeabilized with digitonin or Triton X-100 to allow shown in Supplementary Fig. S12.

E3s responded only weakly (Fig. 5). All but one of the mEMC impaired degradation of TTRD18G while enhancing that of GluR1. components were transcriptionally upregulated by tunicamycin TTRD18G degradation was restored or enhanced when Hrd1 was (Fig. 5); in yeast, only EMC3 is upregulated by the UPR (refs 25,58). co-expressed with SEL1L (Fig. 6g). Similarly, FAM8A1 overexpression The selective response to ER stress indicates a previously unrecognized, (or its RDD domain, amino acids 230–413) impaired TTRD18G coordinate transcriptional regulation of this physically and functionally but not GluR1 degradation, whereas a cytoplasmic N-terminal integrated network. The contributions of the Hrd1–SEL1L and mEMC fragment (FAM8A11230-413) affected neither (Fig. 6g). The dominant- subnetworks to the cellular response to ER stress underscore the negative effect of FAM8A111–229) on TTRD18G stability implies that important role ERAD plays in this process. Hrd1 interacts with FAM8A1 through its RDD domain and that its cytoplasmic N-terminal region is required for Hrd1-mediated ERAD components identiﬁed within the Hrd1–SEL1L and degradation of luminal substrates. Collectively, our results establish gp78 subnetworks FAM8A1 as a binding partner and potential regulator of Hrd1- FAM8A1 was identified as a previously uncharacterized component dependent ERAD. of the Hrd1–SEL1L subnetwork (Fig. 6a). Immunoprecipitation of UBAC2, identified as a UBXD8 interaction partner (Fig. 7a), is endogenous FAM8A1 captured Hrd1 and SEL1L (Fig. 6b), confirming predicted to be a rhomboid family pseudoprotease similar to the that FAM8A1 is a bona fide interactor of both components. Derlin proteins59 that also contains a putative C-terminal UBA domain. Resistance to extraction from purified microsomes by high salt A native interaction between the two was validated by endogenous concentration or pH conditions support predictions for FAM8A1 as an co-precipitation (Fig. 7b). Functionally, UBAC2 knockdown stabilized integral membrane protein with three membrane-spanning domains the Hrd1 substrate TTRD18G–GFP (Fig. 7c), indicating potential (TOPCONS, Fig. 6c,d), and limited proteolysis of FAM8A1-containing coordination between the two ubiquitin ligase complexes. Both the microsomes (data not shown) and immunodetection of an amino- UBAC2 C terminus (amino acids 304–344) and the N terminus of terminal epitope tag in semipermeabilized cells (Fig. 6e) established UBXD8 (2–52) show a high degree of conservation with residues the cytoplasmic localization of the N terminus. A complex isolated essential for ubiquitin binding in UBA domains (Fig. 7d), and with S-tagged Hrd1 contained both FAM8A1 and SEL1L, confirming their predicted cytosolic localization positions them appropriately FAM8A1 as a component of this E3 ligase complex (Fig. 6f). for ubiquitin binding (Fig. 7e). Whereas a recombinant UBAC2 Disrupting the stoichiometry of the Hrd1 E3 complex by FAM8A1 C-terminal fragment (amino acids 293–344) was sufficient to capture knockdown (Fig. 3f) or wild-type Hrd1 overexpression (Fig. 6g) polyubiquitin chains from HEK293 cell lysates at a level comparable

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a UBAC2 b IP c UBA 344 aa 2.0

KIAA0090 10% input Control UBXD8 1.5 C15orf24 Mr (K) TMEM111 50 UBXD8 gp78 MMGT1 1.0 UBAC2 C19orf63 25 UBAC2 TMEM85 0.5 50 Tubulin

TMEM93 Fold change in mean 0

TTC35 GFP fluorescence intensity

C14orf122 Luc COX4NB shRNA: Hrd1 FF UBAC2-1 UBAC2-2

ERLIN1 ERLIN2 UBXD8 E-Syt1 SPP

d α1 α2 α3

UBAC2_HUMAN/304–344 ...EVSEEQVARLMEM.GFSR.GDALEALRASNNDLNVATNFLLQH UBAC2 DSK2_YEAST/327–371 PPEERYEHQLRQLNDM.GFFDFDRNVAALRRSGGSVQGALDSLLNG Dsk2 UBQL2_HUMAN/581–621 ....RFQQQLEQLNAM.GFLNREANLQALIATGGDINAAIERLLGS hPlic-2 RD23A_HUMAN/161–201 ...SEYETMLTEIMSM.GYER.ERVVAALRASYNNPHRAVEYLLTG hHR23A-1 RD23A_HUMAN/318–358 ...PQEKEAIERLKAL.GFPE.SLVIQAYFACEKNENLAANFLLSQ hHR23A-2 RD23B_HUMAN/364–404 ...PQEKEAIERLKAL.GFPE.GLVIQAYFACEKNENLAANFLLQQ hHR23B-2 RD23B_HUMAN/190–228 ...QSYENMVTEIMSM.GYER.EQVIAALRASFNNPDRAVEYLLMG hHR23B-1 UBXD8_HUMAN/11–53 ...QEQTEKLLQFQDLTGIESMDQCRHTLEQHNWNIEAAVQDRLNE UBXD8 CONSENSUS

e Cytosolic epitopes Cytosolic and luminal epitopes f Input Pulldown UBAC2–S gp78–S UBAC2–S gp78–S Bead UBAC2 UBXD8 hPlic2 Bead UBAC2 UBXD8 hPlic2 alone 293–344 1–77 575–624 alone 293–344 1–77 575–624 MG132 –+–+–+–+ –+–+–+–+ Mr (K) KDEL KDEL KDEL KDEL 250 150 Anti-ubiquitin 100 75 Overlay Overlay Overlay Overlay 50

50 Anti-tubulin

Figure 7 Characterization of UBAC2, a ubiquitin-binding ERAD UBA domains. (e) HeLa cells expressing C-terminally S-tagged UBAC2 or component. (a) Predicted domain structure and interaction network of gp78 were permeabilized, immunostained and analysed by fluorescence UBAC2. aa, amino acids. (b) Immunoprecipitation (IP) with anti-UBXD8 microscopy as in Fig. 6e. Scale bar, 10 µm. (f) Recombinantly expressed from HEK293 digitonin-soluble lysates was analysed by western blotting UBA domains of hPlic2, UBXD8 and UBAC2 were coupled to Affi-Gel with the indicated antibodies. (c) Analysis of multiple UBAC2-targeting and incubated with HEK293 cell lysates (±10 µM MG132, 6 h). Samples shRNAs on the Hrd1 substrate TTRD18G–GFP by flow cytometry. FF, firefly. were separated by SDS–PAGE, and ubiquitin binding was determined by (d) Sequence alignment of the predicted UBA domains from UBAC2 immunoblotting with anti-ubiquitin. Uncropped images of blots are shown (304–344) and UBXD8 (8–53) with characterized human and yeast in Supplementary Fig. S12. to the well-characterized hPlic2 UBA (ref. 60), under these conditions combinatorial interactions of the two central E3s, Hrd1 and gp78, with the UBXD8 UBA domain was not (Fig. 7f). Thus, it is UBAC2 rather a palette of accessory factors (Fig. 8). than UBXD8 that adds polyubiquitin-binding capabilities to the Although many individual components of the mammalian ERAD gp78 subnetwork. system have been previously identified, so far there has been no systematic effort to place them into an integrated interaction landscape. DISCUSSION Our study confirmed many of the interactions previously reported in The application of high-content proteomics to identify interconnec- mammals62 and those inferred from yeast16,63 (shown in Fig. 8; black tivity within defined functional networks has been used with success lines), validating our approach and allowing us to arrange components to map high-resolution interaction landscapes for several complex into a topologically and functionally coherent model (Fig. 8). This mammalian protein networks21,22,61. In this study, we have integrated analysis also identified 71 HCIPs that are either uncharacterized or the mammalian ERAD interaction landscape with gene expression have not previously been linked to ERAD (Fig. 8, only selected nodes data and substrate-specific functional ‘fingerprints’ of the mapped and interactions shown), illustrating the ability of focused proteomic components to generate a multidimensional view of this dynamic and strategies to uncover new components. Our analysis integrates complex network. Our data indicate that the mammalian ERAD system interaction and functional data from the present study into a framework may accommodate the diverse array of potential substrates by using consisting of six functional modules that execute the principal ERAD

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Legend Gene induction Substrate recognition and processing Functional requirement UPR induced GFPu A1ATNHK NHK-QQQ ERFAD complex XTP3-B complex OS-9 complex GluR1 A1AT Interactions D18G ΔF508 ∗ ∗ TTR CFTR Intra-module TXD16 ERdj5 BiP CPVL BiP GRP94 Thioredoxin Reductase Chaperone Protease Chaperone Chaperone Impact scale Inter-module 1.1–1.5× >1.5× ERFAD Reductase XTP3-B LectinOS-9 Lectin

Lumen EDEM1 complex

CANX EDEM1 Lectin Lectin Membrane Ubiquitin ligase Ubiquitin ligase SEL1L Adaptor HRD1 complex gp78 complex Dislocation Trafficking? Derlins ERLIN2 SPFH HERP UBE2J1 Derlin-3 ESYT1 C2 Processing UBL E2 enzyme ERLIN1 SPFH protease SPP RING ESYT2 C2 Hrd1 ERLIN2 mEMC complex Derlin-1 Derlin-2 E3 ligase UBAC2 SPFH UBA C15orf24 KIAA0090 WD40 ERLIN1 repeat gp78 SPFH TMEM93 TMEM111 Rab5IP CUE / RING Recruitment factors C19orf63 TMEM85 E3 ligase AUP1 FAM8A1 MMgT CUE Hrd1 regulator? MMGT1 UBXD8 VIMP UBXD UBA/UBX UBX UBE2G2 UBE2G2 E2 enzyme E2 enzyme TTC35 C14orf122 MPN Substrate extraction + VCP TPR AAA repeat ATPase COX4NB MPN Ubiquitin Proteasome extension Proteasome 19S regulatory particle Deubiquitylating 19S regulatory particle Cytosol U-box UBE4A E4enzyme PSMD1*,2*,3,4*,6*,7*, OTU/UBXL VCIP135 PSMD1,2*,3*,4*,6*,7, 8*,11*,12*,13*,14 8*,11*,12*,13*,14* PSMC1*,2,3*,4*,5*,6* PSMC1*,2*,3*,4*,5*,6* UBP/UBA Usp13 Deglycosylating Glycanase / 20S core particle Josephine / NGly1 20S core particle ATX3 PUB PSMA1,2,3,4,5,6,7 UIM PSMA1,2,3,4*,5,6,7* hHR23B Shuttling factor? PSMB1,2,3,4,5,6,7,8 OTU/UBXL YOD1 UBA/UBL PSMB1,2,3,4,5*,6,7,8

Activator PSME4

Figure 8 Functional integration of mammalian ERAD networks. The interactions, UPR induction and functional requirements are indicated schematic model of the ERAD protein interaction network is topologically in the legend. Inter-module interactions represented terminate either organized with respect to the ER membrane and arranged as an at the specific node within a module that establishes the link with the array of six colour-coded functional modules. Individual components module periphery or at the module itself (where there are interactions from this study (baits or HCIPs) are indicated as nodes with reported with multiple components and that module is a single complex; for components (black) and previously unknown components (red). Similarly, example, the mEMC or proteasome). Asterisks indicate components that reported interactions confirmed in this study (black lines) and previously were identified by proteomics, but exhibited a subthreshold CompPASS unknown interactions (red lines) are shown. Symbols for protein–protein score (WDN-score < 1.0). activities: substrate recognition, dislocation, extraction, ubiquitylation through SEL1L, which connects Hrd1 to the upstream luminal substrate and degradation (proteasome), as well as the EMC whose function is recognition machinery, as well as to the downstream dislocation unknown at present. Submodules are grouped together on the basis module through Derlin-2 and the substrate extraction module through of predicted structural and topological features, and on an unbiased UBXD8. Direct interactions between the last two proteins and VCP analysis of network interconnectivity of the proteins represented in provide an extended pathway from the luminal substrate-binding each group. The ERAD system can thus be viewed as a distributed lectins OS-9 and XTP3-B to cytoplasmic VCP. The third connection is network, organized around central ubiquitin ligase modules for Hrd1 a direct link between this E3 and the 26S proteasome. and gp78 that cooperate with components of the membrane-embedded The gp78 submodule seems to connect to the ERAD network through dislocation and the cytoplasmically-oriented substrate extraction UBAC2. This protein interacts with UBXD8 and Derlin-1/2, and has modules. These interconnections are likely to ensure secure coupling a functional polyubiquitin-binding domain, indicating that it may between substrate dislocation/extraction and ubiquitin conjugation. function as a membrane nexus integrating ubiquitin conjugation, The Hrd1 and gp78 complexes contain submodule-specific factors and dislocation and extraction. A VCP-binding site within the cytoplasmic share interactions with ERLIN1/2 and UBE2G2. The Hrd1 submodule domain of gp78 (ref. 42) means that this complex can associate with has four main connections to other modules. Three are mediated VCP in at least two ways. VCP interacts directly with multiple compo-

RESOURCE nents of the ERAD machinery including Derlin-1 and Derlin-2 (refs 27, We thank the members of the Kopito laboratory for helpful discussion, and M. 59,64), VIMP (refs 26,27), gp78 (ref. 42), UBXD2 (ref. 65) and UBXD8 Pearce, J. Hwang and C. Beveridge for critical reading of the manuscript. (refs 66,67). With at least six different recruitment sites for VCP within AUTHOR CONTRIBUTIONS the ERAD network, it is not surprising that disruption or silencing of The manuscript was written collectively by J.C.C., J.A.O. and R.R.K. Experiments this cytoplasmic AAA+ ATPase has a more universal effect on the degra- and data analysis were carried out by J.A.O. and J.C.C. with assistance from C.M.R. R.E.T. and E.J.G. LC–MS/MS analysis was carried out by T.A.S. CompPASS analysis dation of diverse ERAD substrates when compared with the loss of an was carried out by M.E.S. and E.J.B with support from J.W.H. individual factor (Fig. 4f). Multiple recruitment avenues at the ER mem- COMPETING FINANCIAL INTERESTS brane may reflect an acquired adaptability of VCP to accommodate and The authors declare no competing financial interests. engage the diverse substrates it encounters. Moreover, VCP accessory Published online at http://www.nature.com/naturecellbiology factors (for example, UBE4A, VCIP135 and SVIP), functionally essen- Reprints and permissions information is available online at http://www.nature.com/ tial for specific substrates (Fig. 4f), could confer an added level of speci- reprints ficity or may reflect a requirement for different VCP configurations at 1. Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, different steps of the dislocation and membrane extraction processes. 737–741 (2003). 2. Hebert, D. N., Bernasconi, R. & Molinari, M. ERAD substrates: which way out? ‘Input’ of luminal substrates into the Hrd1 submodule occurs Semin. Cell Dev. 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Nature 443, 827–831 (2006). recognition, recruitment, dislocation, extraction, ubiquitylation and 20. Mehnert, M., Sommer, T. & Jarosch, E. ERAD ubiquitin ligases: multifunctional tools for protein quality control and waste disposal in the endoplasmic reticulum. degradation of the diverse classes of secretory pathway proteins. This Bioessays 32, 905–913 (2010). work should provide a resource for future analysis of this cellular 21. Sowa, M. E., Bennett, E. J., Gygi, S. P. & Harper, J. W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009). quality-control system. 22. Behrends, C., Sowa, M. E., Gygi, S. P. & Harper, J. W. Network organization of the human autophagy system. Nature 466, 68–76 (2010). METHODS 23. Braun, P. et al. An experimentally derived confidence score for binary protein–protein interactions. Nat. Methods 6, 91–97 (2009). Methods and any associated references are available in the online 24. Riemer, J., Hansen, H. G., Appenzeller-Herzog, C., Johansson, L. & Ellgaard, L. Identification of the PDI-family member ERp90 as an interaction partner of ERFAD. version of the paper at http://www.nature.com/naturecellbiology PLoS One 6, e17037 (2011). 25. Jonikas, M. C. et al. Comprehensive characterization of genes required for protein Note: Supplementary Information is available on the Nature Cell Biology website folding in the endoplasmic reticulum. Science 323, 1693–1697 (2009). 26. Ye, Y., Shibata, Y., Yun, C., Ron, D. & Rapoport, T. A. A membrane protein complex ACKNOWLEDGEMENTS mediates retro-translocation from the ER lumen into the cytosol. Nature 429, This work was supported by grants from the NIH to R.R.K. and J.W.H. J.C.C. was 841–847 (2004). supported by funding from the Ludwig Institute for Cancer Research. J.A.O. and 27. Lilley, B. N. & Ploegh, H. L. Multiprotein complexes that link dislocation, R.E.T were supported by NRSA fellowships from NIH. 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METHODS DOI: 10.1038/ncb2383

METHODS Cell culture, transfection and stable cell lines. HEK293 and HeLa cells were Plasmids and constructs. ERAD component complementary DNAs (Supple- maintained in DMEM (Mediatech) +10% animal serum complex (Gemini Bio- ◦ mentary Table S10) were cloned into the pcDNA3.1 vector in frame with an Products) at 37 C and 5% CO2. Cells were transfected by the calcium-phosphate S-tag (KETAAAKFERQHMDS) either at the N or C terminus. For some, the co-precipitation technique or with FuGENE6 (Roche). Stable HEK293 clones/pools endogenous signal sequence was replaced by bovine preprolactin followed by the expressing S-tagged ERAD components were selected by G418 resistance and S-peptide. ERAD reporters were constructed by fusing eGFP in frame with the limiting dilution. Clonal HEK293 cell lines expressing GFP-fusion ERAD reporters C termini of transthyretin (TTRD18G), an α1-anti-trypsin NHK variant (A1ATNHK) are described below. and an NHK variant lacking consensus glycosylation sites (A1ATNHK-QQQ), gift from N. Hosokawa, Kyoto University, Japan). An N-terminal eGFP fusion of rat Functional genomic analysis of the ERAD network. Clonal HEK293 cell GluR1 (GFP–GluR1–flop) was subcloned into pcDNA3.1(-) (gift from R. Malinow, lines expressing GFP-tagged substrates (GFPu, GFP–GluR1, TTRD18G–GFP, University of California, San Diego, USA). GFP–CFTR1F508; ref. 71) and GFPu A1ATNHK–GFP, A1ATNHK-QQQ–GFP and GFP–CFTR1F508) were obtained by G418 (ref. 54) have been described previously. HA–GluR1 (rat) was a gift from W. Green, selection followed by limiting dilution and/or sorting by FACS as described University of Chicago, USA. previously52. Cell line selection was based on multiple criteria including: expression of full-length protein; a single GFP peak as measured by flow cytometry; and Primary baits were selected on the basis of previous reports or Bait selection. accumulation with MG132 (10 µM for 12 h). For primary screening, reporter on orthology to known yeast ERAD components. Secondary baits were selected cells were seeded in 96-well plates (15,000 cells per well) and reverse-transfected using multiple criteria, including: identification as an HCIP with several primary using FuGENE6 with individually arrayed pSUPERSTAR-shRNA plasmids baits; identification as an HCIP in both digitonin and Triton X-100; presence in (175 ng) and pcDNA3–mCherry (ChFP) (25 ng). After 72 h, cells were analysed LC–MS/MS analyses with high total spectral counts; a predicted ER localization; and by high-throughput flow cytometry (LSR-II, Becton Dickinson). Mean GFP a domain structure or previous reports suggesting a potential function of relevance fluorescence intensity was determined for ∼2,000 ChFP-positive cells and to ER quality control. normalized to the mean GFP fluorescence intensity of pSUPERSTAR empty Mass spectrometry. Cells were collected in PBS and solubilized in lysis buffer vector (n = 3). Potential positive hits (Z scores > 1.5) were re-screened (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA) containing Complete through all reporters, and the mean GFP fluorescence intensity for 20,000 protease inhibitor cocktail (Roche), and either 1% digitonin or 1% Triton X-100. cells was measured. shRNAs scoring positive in three independent experiments Lysates were spun twice, first at 1,000g and the supernatant was respun at 20,000g.A and also depleted for the target transcript were considered bona fide hits. quantity of 1–1.5 mg of total protein from cleared lysates was affinity purified with S- The heat map (Fig. 4f) used mean GFP fluorescence values for each shRNA protein agarose (Novagen). Bead-bound complexes were washed three times in lysis that were normalized to values resulting from a 3 h MG132 treatment, so buffer containing 0.1% digitonin or Triton X-100 then twice in 50 mM ammonium as to facilitate comparison of each shRNAs effect between reporter cell lines. bicarbonate, at pH 8. Bound proteins were eluted by overnight treatment with Rapigest (Waters) and subjected to trypsin digestion (Promega) before injection into the mass spectrometer. Samples were analysed in duplicate/triplicate on a system Immunopurification, affinity purification and immunoblotting. Cells were consisting of a CTC-PAL autosampler (Leap Technologies), a capillary gradient collected and affinity purified (S-protein) as described above. For immunoprecipi- HPLC pump (Agilent Model 1100) and a linear-ion-trap mass spectrometer (Model tations, anti-FAM8A1 or anti-UBXD8 antibodies were conjugated to beads using the LTQ, ThermoFisher Scientific). The acquired MS/MS spectra were searched using Pierce direct immunoprecipitation kit (Thermo Scientific) and incubated with cell the MASCOT protein database search program (Matrix Science) against a full lysates. All samples were washed three times in lysis buffer, resuspended in Laemmli database of human protein sequences to which the set of S-tagged protein sequences buffer +10 mM dithiothreitol, separated by SDS–PAGE and transferred to PVDF and S-protein sequence were added. membrane for western blotting.

Data deposition. The spectral files reported in this article have been Antibodies. Experiments used the following antibodies: anti-S-tag (1:5,000), deposited in the Proteome Commons Tranche repository (http://tranche. anti-myc (9E10; 1:2,000), anti-Hrd1 (1:50; gift from R. Wojcikiewicz, SUNY proteomecommons.org) and can be accessed using the following hash: Upstate Medical University, USA), anti-SEL1L, anti-UBXD8, anti-AUP1 (gifts from ZPwKzx2i + VY1M2U0GB450u9kH7MFcrfOjXFam9/xha4QUN/6O5 + KLj/ H. Ploegh, Whitehead Institute, USA), anti-VCP (1:1,000; Novus), anti-HA (12CA5; NnuEYMBTJPSwId/rJFvjsYbPbQrg8mDys7F8AAAAAAABMXA ==. 1:1,000), anti-KDEL (1:500; Stressgen), anti-calnexin (1:500; Assay Designs), anti- GFP (1:1,000; Roche), anti-tubulin (gift from T. Stearns, Stanford University, USA) CompPASS analysis. A MASCOT-generated list of all interacting proteins and and anti-ubiquitin (FK2; 1:2,000; BioMol). The polyclonal rabbit anti-FAM8A1 their corresponding total spectral counts from duplicate MS analyses for each bait (1:20,000) antibody was generated against a synthetic human FAM8A1 peptide affinity complex was merged with a database containing 102 unique baits analysed (residues 65–78, CDKLEPPRELRKRGE) by conjugation to KLH and immunization previously21. CompPASS output metrics were then adjusted using both a weighting of two rabbits (Proteintech Group). factor and normalization such that a WDN score greater than 1 signifies a HCIP. Briefly, the CompPASS-calculated WD score assesses interacting protein abundance (peptide number), uniqueness (number of baits that interact with the protein) and Measurement of shRNA efficacy by qRT–PCR and western blotting. For replication (number of experiments the interaction is observed in) to determine target gene messenger RNA depletion, cells seeded in 12-well plates were transfected HCIPs (ref. 21). To analyse data sets containing high numbers of shared interactors with the pSUPERSTAR-shRNA construct. After 24 h, cells were divided between (for example, autophagy network and ERAD network), a normalized WD score two plates. Following an additional 48 h of growth, total RNA from plate one (WDN) was developed and has been described in extensive detail previously22. This was isolated using RNeasy columns (Qiagen). HEK293 cells from the duplicate score includes a normalization factor based on the standard deviation of the scan plate were collected, stained with anti-CD4-allophycocyanin and analysed using a number for the interactor across the bait proteins, which was found to correlate with FACSCalibur (Becton Dickenson) to determine transfection efficiency. Transcript bona fide interacting proteins22. All MASCOT and CompPASS data can be accessed levels were subsequently measured by quantitative real-time PCR with reverse through INfERAD, an interactive web-based portal at http://falcon.hms.harvard. transcription (qRT–PCR) with an IQ5 Real Time PCR Detection System (Bio-Rad), edu/ipmsmsdbs/comppass.html. The hierarchically clustered heat map representing using the iScript One-Step RT-PCR kit with SYBR green (Bio-Rad), in triplicate HCIP data was generated using MultiExperimental Viewer v4.7. It should be noted reactions according to the manufacturer’s instructions. Primer sequences are listed that the high WDN score for the bait protein could be due to self-identification in Supplementary Table S9. or a self-interaction (Fig. 1 and Supplementary Tables S2 and S3) and these two For analysis of shRNA-mediated effects on target protein levels, HEK293 cells possibilities could not be distinguished in the present study. HCIP clusters were were transfected with the expression plasmid encoding S-tagged target protein along selected manually by encompassing the largest number of HCIPs proximal to each with the corresponding control or shRNA-targeting plasmid at a ratio of 1:3. Levels bait with a minimum of two HCIPs. of the S-tagged target protein were subsequently analysed by immunoblotting of either SDS-solubilized cell lysates or of S-protein agarose affinity-purified proteins. ERAD shRNA library. Target gene selection was based on a reported/suspected role in ERAD or on identification as an HCIP in proteomic analyses. shRNA target Measurement of UPR induction of the ERAD network. HEK293 cells were sequences were selected from the literature, the RNAi codex online repository, or incubated in the absence or presence of 10 µg ml−1 tunicamycin and total RNA generated using siRNA selection programs as indicated (Supplementary Table S7). was isolated using RNeasy columns (Qiagen). Transcript levels were analysed by shRNAs were cloned into the pSUPERSTAR expression vector52. The library qRT–PCR with an IQ5 Real Time PCR Detection System (Bio-Rad) using the iScript contains 309 shRNA constructs, including 3 negative control constructs, 222 One-Step RT-PCR kit with SYBR green (Bio-Rad), in triplicate reactions, according constructs targeting 45 reported ERAD components, and 87 constructs targeting 14 to the manufacturer’s instructions. Individual reaction efficiencies were determined potential ERAD components. Each gene is targeted by an average of ∼5 shRNAs. using the LinRegPCR software and fold change in gene expression was calculated

DOI: 10.1038/ncb2383 METHODS using the Pfaffl method with β-actin as the reference gene. Primer sequences are down non-specifically by S-protein agarose from untransfected HEK293 cells (bead listed in Supplementary Table S9. control). The result yielded 267 (digitonin) and 153 (Triton X-100) interactions (cellular HCIPs/ERAD interactions) with 143 (digitonin) and 97 (Triton X-100) Immunofluorescence microscopic analysis of protein localization and corresponding to unique HCIPs. Proteins whose gene ontology assignments were topology. HeLa cells grown on poly-l-lysine-coated glass coverslips were fixed with cytoskeletal, mitochondrial, peroxisomal or nuclear (for example, MYH9, KRT8, 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked by 1% PEX19, TOMM20 and MCM2) were deemed unlikely to be ERAD-related and bovine serum albumin. Primary antibody incubation (2 h at room temperature) was removed from subsequent analysis (likely false positives) and indicated as such followed by incubation with AlexaFluor secondary antibodies (Invitrogen, 1 h at in individual interactomes (Supplementary Fig. S4). To determine the number room temperature). Nuclei were stained with 10 µg ml−1 bisbenzamide for 10 min of new HCIPs identified, all primary and secondary baits were subtracted (other before mounting. For analysis of protein topology, HeLa cells grown and fixed as primary/secondary baits as prey), as well as proteins implicated in ERAD (for above were permeabilized with either 20 µM digitonin for 1.5 min or 0.1% Triton example, VCP, GRP94 and proteasome subunits) but not used as bait (reported X-100 for 3 min at room temperature to allow permeabilization of the plasma ERAD factors). Ultimately, we identified 59 (digitonin) and 30 (Triton X-100) membrane or permeabilization of both the plasma and ER membranes, respectively. uncharacterized proteins of interest with a wide range of topologies and functional Cells were washed three times with PBS and immunostained as described above. domains, and a potential role within the ERAD network (Supplementary Fig. S3d, Stained cells were visualized on a Zeiss Axiovert 200M (×40 air objective) and the not including the 10 secondary baits). resulting images were acquired digitally (Roper Scientific). Comparison and integration of proteomic data set with reported Sucrose gradient fractionation. HEK293 cells were lysed as described above. A interactions. A list of reported pairwise interactions for ERAD components from quantity of 1.5 mg of total protein from cleared lysates was adjusted to 5% sucrose published data was manually curated and used to generate a more comprehensive and loaded onto 10–40% continuous sucrose gradients (containing 0.1% digitonin) picture of the mammalian ERAD interaction network (Supplementary Table S5). prepared using a Gradient Master (BioComp). Samples were centrifuged in an SW41 This table includes: an assigned ‘interaction number’; bait and prey defining rotor at 39,000 r.p.m. for 14.25 h at 4 ◦C. Fractions of 1 ml in volume were collected, the interaction and whether our study used the bait (red); the method(s) of sucrose concentrations were adjusted to approximately 20% with lysis buffer, and identification (for example, affinity capture-MS, two-hybrid, and so on) and the S-tagged proteins were affinity purified with S-protein agarose. relevant reference(s) for the reported interaction; whether the reciprocal interaction was shown and its corresponding ‘interaction number’; detection of the reported Metabolic labelling and pulse-chase assay. Pulse-chase assays of GFP–GluR1 interaction in our digitonin or Triton X-100 interaction networks; and the presence reporter cells were carried out as described previously39. of this interaction in the STRING online protein interaction database. STRING lists only 33/131 reported interactions (∼25%), supporting the use of a manually Proteomic interaction data set analysis. LC–MS/MS analysis of replicate affinity- curated list rather an online resource. Of the 81 reported interactions using the purified protein complexes from 25 bait proteins identified a total of 10,481 proteins 25 primary/secondary baits, 31 were confirmed by our proteomic analysis with an in digitonin and 8,262 in Triton X-100. Approximately two-thirds were redundant additional 3 being identified in a reciprocal interaction not previously reported. entries, with the number of proteins having unique GenInfo Identifier numbers Seven more reported interactions were observed but were subthreshold (WDN < 1). reduced to 3,325 (digitonin, 31.7%) and 2,917 (Triton X-100, 35.9%) for the We identified 21 reciprocal and 36 unidirectional interactions between baits and entire analysis. A total of 320 (digitonin) and 202 (Triton X-100) entries were ERAD components, and interactions with 71 uncharacterized proteins of interest designated as HCIPs by CompPASS (WDN-score > 1) and included redundant that scored above the threshold for HCIP classification (Supplementary Table S5 entries representing shared interactors (Supplementary Tables S2 and S3). A and Fig. S3d). Combining reported pairwise interactions (Supplementary Table breakdown of digitonin and Triton X-100 HCIPs is presented in Supplementary S5) with INfERAD (Fig. 2) and the RNAi-mediated ‘epistasis-like’ experiments Table S4, including specific analyses for primary and secondary baits. The total (Fig. 3) yielded an up-to-date, comprehensive view of the ERAD interaction network number of CompPASS-identified HCIPs represents all bait-interacting proteins. (Supplementary Fig. S5). Bait proteins often had WDN-score > 10, but as we could not distinguish bait from an endogenous, homo-oligomerized counterpart, HCIPs representing their identical bait proteins were not considered (bait self-identification), neither were 71. Ward, C. L., Omura, S. & Kopito, R. R. Degradation of CFTR by the ubiquitin- trypsin (introduced during the experimental method (trypsin)) and proteins pulled proteasome pathway. Cell 83, 121–127 (1995).

Kopito Supplemental Figure S1

SUPPLEMENTARY INFORMATION

DOI: 10.1038/ncb2383

PROTEOMICS FUNCTIONAL GENE GENOMICS EXPRESSION Validated ERAD component

1° baits (15) RNAi ER Stress 2° baits (10) S-tagging and 59 targets Tunicamycin affinity capture 309 shRNA

Stable S expression in Functional assay with HEK293 cells Quantitative RT-PCR S-tag ERAD reporters shotgun LC-MS/MS

GFP fluorescence Cycle duplicate or triplicate runs PSMD2 VCP UBE4A ERDJ5 XTP3-B UBXD2 AUP1 Derlin2 NGLY1 UBE2G2 ERFAD OS9 Empty Derlin3 JAMP UBE4A AHA1 Luc. F. GP78 VIMP TXD16 ESYT1 HRD1 UBAC2 FAM8A1 HERP VCPIP1 SVIP LON2P UBXD8 COX4NB UBE2J1 SEL1L TTC35 TMEM33 Derlin1 Identification of EDEM1 KIAA0090 c15orf24 Interactors

CompPASS Biochemical Functional Coordinate gene analysis interactions interactions induction

Novel component identification

ERAD functional networks

Figure S1 Strategy to elucidate functional complexes of the mammalian ERAD pathway. Flow chart illustrating the strategy used to identify functional ERAD complexes by integrating high-content proteomics, functional genomics and gene expression data sets (see Methods).

SUPPLEMENTARY INFORMATION Kopito Supplemental Figure S2

A Hrd1-S KDEL Overlay Derlin2-S KDEL Overlay

gp78-S KDEL Overlay S-SEL1L KDEL Overlay

UBXD8-S KDEL Overlay NGly1-S KDEL Overlay

UBXD2-S KDEL Overlay VIMP-S KDEL Overlay

EDEM1-S KDEL Overlay ERLIN2-S KDEL Overlay

XTP3-B-S KDEL Overlay S-ERFAD KDEL Overlay

S-OS9.2 KDEL Overlay AUP1-S KDEL Overlay

Derlin1-S KDEL Overlay

B UBAC2-S KDEL Overlay TMEM111-S KDEL Overlay

S-FAM8A1 KDEL Overlay S-TTC35 KDEL Overlay

TXD16-S KDEL Overlay C15orf24-S KDEL Overlay

S-E-Syt1 KDEL Overlay COX4NB-S KDEL Overlay

KIAA0090-S KDEL Overlay

Figure S2 Subcellular localisation of S-tagged ERAD components. HeLa antibodies, with nuclei identified by bisbenzamide (blue). (a) Primary baits cells, transiently expressing individual S-tagged ERAD components, were employed for initial proteomic analyses. (b) Secondary baits identified as immunostained simultaneously with anti-S-tag (green) and anti-KDEL (red) HCIPs from initial proteomic screening (MMGT1-S not shown)

SUPPLEMENTARYKopito Supplemental INFORMATION Figure S3

A Digitonin TritonX-100 800 40 600 30

700 35 Total Interactors Total Interactors 25 500 HCIPs 600 HCIPs 30 400 20 HCIPs 500 25 HCIPs

400 20 300 15

300 15 200 10 Total Interactors Total Total Interactors Total 200 10 100 5 100 5

0 0 0 0

gp78 OS-9 VIMPHrd1 gp78 OS-9 VIMPHrd1 AUP1 Erlin2 NGly1 AUP1 Erlin2E-Syt1 NGly1 SEL1L TTC35TXD16 UBXD2UBXD8 E-Syt1AM8A1ERFAD SEL1L TTC35TXD16UBAC2UBXD2UBXD8 XTP3-B Derlin-1Derlin-2EDEM1 ERFAD UBAC2 c15orf24XTP3-B Derlin-1Derlin-2EDEM1 F c15orf24 FAM8A1 COX4NB TMEM111TMEM32 COX4NB KIAA0090 TMEM111TMEM32 bead CTRL KIAA0090 bead CTRL

Uncharacterized HCIPs of interest identified B ARF5 DIP2B HMGCR RAB7A TMED9 by CompPASS including the 10 HCIPs ARL6IP1 DPY30 HMOX1 RPN2 TMEM201 that became secondary bait (grey) ARL6IP5 DUSP3 KLHDC2 SEC22B TMEM43 BRI3BP EPHX1 KIAA0746 SELK TMUB1 18 C19orf6 FAM62B LRRC59 SLC27A3 UBXD6 41 12 CAPRIN2 FDFT1 MARCK5 STT3A USH2A (28) CLPTM1 GALK1 OSBPL8 SYNJ2BP VAPA CLPTM1L GANAB PGRMC2 TAGLN2 VAPB YIF1B ARF3 Digitonin FAM8A1 COX4NB DNAJA1 UBAC2 TTC35 FAM186B FAM62A C15orf24 GGH TXN1DC16 TMEM111 C !"#$%&'()*)#+,#-*.*/+%*%#0123)# !"#"$#%&%' HSP90AB2P (&#)*+*%&%' KIAA0090 MMGT1 (&#%,+$-*./' RAB9A /!$!(#0&/' C14orf122 NIPSNAP1 RPN1 1*2*0#"3*+$' 1&4*5*+.!.#+' C19orf63 NPEEPS SERPINH1 678'1!3!)*' 678'9*"0&/!.#+' CAND1 PFKL SLC25A10 :#01&+)' 3*$!(#0&/' CASK PFKP SURF4 3&$#%&%' CPVL RTN4 TBC1D17 ;<=>9' "-#%"-#5,0!.#+' EMD SAE1 TMEM85 "5#$*#0,%&%' 978'?5#/*%%&+)' ENO1 TBC1D15 %&)+!0'$5!+%1@/.#+' $5!+%/5&".#+' LIN7C TMEM93 TX-100 $5!+%0!.#+' @(&A@&.+' LONP2 UBQLN2 2*%&/0*'$5!+%"#5$' Both

'&" <-/-+*1-1" D '%" B;-+*1HI'!!" !"#$%&'()*)#+,#-.*/+%01233#4567)# !"#"$#%&%' '$" (&#)*+*%&%' (&#%,+$-*./' '#" /!$!(#0&/' 1*2*0#"3*+$' '!" 1&4*5*+.!.#+' 678'1!3!)*' &" 678'9*"0&/!.#+' :#01&+)' %" 3*$!(#0&/' 3&$#%&%' ;<=>9' $" "-#%"-#5,0!.#+' "5#$*#0,%&%' #" 978'"5#/*%%&+)' %&)+!0'$5!+%1?/.#+' Fold change in representation !" $5!+%/5&".#+' $5!+%0!.#+' ?(&@?&.+' 2*%&/0*'$5!+%"#5$' @*67-1/" 9-+*,-,"ABCD?" ()*)+*,-," 5(+(.*6-5" F.-GF-41" .-*/010,-," 90+(.*6-5" );*+0*62,-," +;(1,6(4*1" .-*,21+3045" <=>"7(9(/0" +;(1,5;-)4*1" 70806*)901+"7-:0;014(4*1" <=>"?0)6-5(4*1" )3*,)3*;26(4*1"?=>"E;*50,,-1/" 80,-560"+;(1,)*;+" ,-/1(6"+;(1,7F54*1"

Figure S3 Analysis of HCIP abundance and gene ontology (GO). (a) The the 10 HCIPs boxed in grey selected and designated as secondary baits. (c) total number of interactors (red) and identified HCIPs (blue) are plotted Individual pie graphs reflecting the relative percentage of HCIPs belonging for individual baits solubilised in DIG (top) or TX-100 (bottom). (b) Venn to each designated GO annotation in DIG (top) or TX-100 (bottom). (d) diagram showing the uncharacterised HCIPs obtained in DIG (red), TX-100 Enrichment of HCIPs in GO defined categories. Determined as the ratio of (blue), or in both (purple). 28 HCIPs were identified in both detergents, with the representative percentage of HCIPs vs. entire proteome.

SUPPLEMENTARY INFORMATION

Kopito Supplemental Figure S4

PSMB5 PSMD14 PSMD6 PSME4 PSMC2 PSMD3 gp78 PSMA4 CAPRIN2 E-Syt1 STT3A PSMA1 Derlin-1 Derlin-2 ERLIN1 UBE2G2 E-Syt2 UBXD2 gp78 PSMA2 EDEM1 MYH10 AUP1 DIP2B HMOX1 TTC35 PSMA3 MYH9 SEL1L EPHX1 VIMP PSMA5 TMUB1 SURF4 POR POR PSMA6 MTCH1 UBE2G2 PRKDC YIF1B Derlin-1 PSMA7 E-Syt1 ERLIN2 CKAP5 RTN4 ARL6IP5 PSMB1 CANX OSBPL8 SEC22B VCP PSMB2 UBAC2 ARL6IP5 SEL1L NGly1 PSMB3 UBXD8 TMEM111 PSMB4 VCP BRI3BP PGRMC2 UBXD8 VCP PSMB6 E-Syt1 PSMB7 KIAA0090 UBXD8 RTN4 RPN2 PSMD1 PSMD7 OS-9 hHR23B ERLIN2 FAM8A1 E-Syt1 UBXD2 VCP

GRP94 SYNJ2BP UBE4A HNRNPM MYH9 Hrd1 E-Syt2 C19orf6 SEL1L OS-9 ARL6IP5 DUSP3 LONP2 CAPRIN2 YIF1B ERLIN1 SERPINH1 SEL1L HMGCR GGH XTP3-B FAM8A1 RPN1 SEL1L3 XTP3-B PSMA1 ERLIN1 TXD16 Hrd1 PSMA2 VDAC1 SEL1L SEL1L PSMA3 ERFAD VIMP MYH9 DPY30 PSMA5 EMD FDFT1 SELK PSMA6 UBE2J1 GALK1 OS-9 FLNA KLHDC2 PSMA7 ERLIN2 NIPSNAP1 UBE2J1 C2orf30 UBXD6 PSMB1 OS-9 CASK XTP3-B PSMB2 VDAC2 Derlin-1 POR RPL10 PSMB3 KRT18 Derlin-2 AUP1 ACTG2 PSMB4 PRKDC VCP HMOX1 CANX UBXD8 PSMB5 RAB9A PSMD7 LIN7C UBAC2 UBE4A PSMB6 PSMD1 PSMB7 ACTA1 SEL1L SURF4 USH2A GANAB TOMM40 RAB35 VAPA SEC22B HSP90AB2P TBC1D17 RPN2 CAND1 LRRC59 ACTA1 XTP3-B KIAA0090 TUBA8 UBXD8 TUBA4Q TMEM201 KRT18 CAND1 COX2 COX4I1 SLC27A3 TXD16 CLPTM1 VDAC1 SERPINH1 ERLIN1 TAGLN2 Hrd1 PEX11B E-Syt1 TMEM43 HNRNPM SEL1L TMEM43 FLNA OS-9 CSE1L UBXD8 TBC1D15 UBAC2 Derlin-2 VCP RPL35 ANK2 ENO1 PEX19 UBE2G2 TUBA1A PPIB EMD SEL1L IPO9 TMEM111 GCN1L1 CPVL TOMM40 ENO1 ERLIN1 RAB7A FAM8A1 ATP5B HNRNPU GCN1L1 RPL10 VDAC3 HNRNPA2B1 TUBA1A HM13 TUBA1B1 OAT VDAC2 ATP5F1 TMED9 TTC35 NPEPPS ABCD3 MCM2 ERFAD ERLIN2 MLL2 gp78 MARCKS PFKP ARF5 Derlin-2 ACTG2 C14orf122 C14orf122 ERLIN2 EMD TMEM85 C15orf24 COX4NB OS-9 ARL6IP1 KRT6A TMEM85 Derlin-1 RAB7A TTC35 KIAA FAM186B TTC35 PFKL TOMM22 TMEM111 COX4 0090 LARS VAPB TTC35 TMEM111 NB KRT8 TOMM20 C19orf63 ACTG1 TMEM111 C15orf24 RAB9A VIMP CLPTM1L ARF3 TMED9 C19orf63 ENO1 UQCRC2 SAE1 MCM2 TMEM93 C15orf24 COX4NB TMEM32 TMEM93 TMEM32 KIAA0090 UBQLN2 TMEM32 VDAC2 HCIPs of interest TMEM85 KIAA0090 CANX UBQLN2 ERAD or ER related (RED) Non-ER/Suspected Contaminants C15orf24 C14orf122 C14orf122 cytoplasmic/nuclear (GREY) MMGT1 TTC35 TMEM COX4NB C15orf24 TMEM111 111 TTC35 TTC35 COX4NB peroxisomal (GREEN) C19orf63 C15orf24 TMEM111 mitochondrial (BLUE) C14orf122 C19orf63 C19orf63 TMEM93 TMEM85 TMEM85 DIG and TX-100 COX4NB TMEM32 TMEM93 KIAA0090 DIG only KIAA0090 TMEM32 UBQLN2 UBQLN2 KIAA0090 TX-100 only CIB2

Figure S4 Interaction maps for individual ERAD baits. Interactomes for each Fig. 2. HCIPs of interest are depicted as dark brown circles with either red or individual S-tagged ERAD component are shown, representing HCIPs detected black text, depending on their predicted localisation. HCIPs with alternative in DIG only (dashed-green line), TX-100 only (dashed-purple line), and both GO localisation (i.e. non-ER) and suspected non-specific contaminants are DIG and TX-100 (solid-black line). ER-localised / ERAD-related HCIPs are shown as: cytoplasmic/nuclear (grey circles, grey text), peroxisomal (beige indicated with red text and where appropriate, by their assigned colour from circles, green text) and mitochondrial (light brown circles, light blue text).

SUPPLEMENTARY INFORMATION

Kopito Supplemental Figure S5

GRP94 CPVL LONP2 unidirectional (dashed) MS analysis recipricol (solid) TXD16 Reported Both BiP OS-9 ERdj5 BAIT INTERACTOR REPORTED INTERACTOR ERFAD XTP3-B All Interactions-Pruned CANX EDEM1

SEL1L Derlin-3 JAMP BAP31 LUMEN

FAM8A1 gp78 Hrd1 RNF5

Herp Derlin-1 UBAC2 Insig1 Derlin-2 KIAA 0090 c15orf24

TMEM VIMP MMGT1 Ube2j1 ERLIN1 ERLIN2 111 c19orf63 UbxD2 UbxD8 TMEM AUP1 E-Syt1 E-Syt2 SPP 85

Ube2g2 TMEM PLAP TTC35 93 Cul2 COX4 c14orf122 Parkin hHR23B VCIP135 PSMA1-7 NGly1 VCP PSMB 1-7 NB PSMC 2 UBE4A PSMD 1-3,6,7,14 PSME4 CYTOPLASM UBQLN1 Npl4 Ufd1 hHR23A RFP2 SVIP Usp13 ATX3 YOD1

Figure S5 Integration of previously reported interactions with the determined the appropriate refinements from Fig. 2. Shown are reported interactions ERAD network. The previously reported interactions (Table S5) for ERAD that were identified in our study (green), interactions only identified in our baits and HCIPs were mapped onto the ERAD interactome from Fig. 1b with study (red), and additional reported interactions (blue).

SUPPLEMENTARY INFORMATION

Kopito Supplemental Figure S6

a gp78 proteasome interactions (DIG) b gp78 (DIG) 0.18 0.12 0.16 0.10 0.14 0.12 0.08 0.10 0.06 0.08 0.06 0.04 0.04 Average spectral Average abundance factor 0.02 0.02

Spectral abundance factor 0 0 20S 19S PSMA6 PSMA2 PSMA4 PSMA7 PSMA5 PSMA1 PSMA3 PSMB6 PSMB7 PSMB3 PSMB2 PSMB5 PSMB1 PSMB4 PSME4 PSMD6 PSMD7 PSMC2 PSMC1 PSMC4 PSMC6 PSMC3 PSMC5 PSMD2 PSMD4 PSMD8 PSMD1 PSMD3 PSMD11 PSMD13 PSMD14 PSMD12

20S Core subunits Act. 19S cap subunits

c gp78 proteasome interactions (TX) d gp78 (TX) 0.16 0.10 0.14 0.09 0.08 0.12 0.07 0.10 0.06 0.08 0.05 0.06 0.04 0.03 0.04 Average spectral Average

abundance factor 0.02 0.02 0.01

Spectral abundance factor 0 0 20S 19S PSMA6 PSMA2 PSMA4 PSMA7 PSMA5 PSMA1 PSMA3 PSMB6 PSMB7 PSMB3 PSMB2 PSMB5 PSMB1 PSMB4 PSME4 PSMD2 PSMD4 PSMD8 PSMD1 PSMD3 PSMD6 PSMD7 PSMC2 PSMC1 PSMC4 PSMC6 PSMC3 PSMC5 PSMD11 PSMD14 PSMD12 PSMD13

20S Core subunits Act. 19S cap subunits

e Hrd1 proteasome interactions (DIG) f Hrd1 (DIG) 0.10 0.10 0.09 0.09 0.08 0.08 0.07 0.07 0.06 0.06 0.05 0.05 0.04 0.04 0.03 0.03 Average spectral Average 0.02 abundance factor 0.02 0.01 0.01

20S Core subunits Act. 19S cap subunits

g Hrd1 proteasome interactions (TX) h Hrd1 (TX) 0.06 0.05 0.03 0.04 0.02 0.03 0.02 0.01 Average spectral Average 0.01 abundance factor

20S Core subunits Act. 19S cap subunits

Figure S6 Analysis of gp78 and Hrd1-associated proteasome average spectral abundance factor for 20S core subunits (PSMA and compositions. (a, c, e, g) The calculated spectral abundance factor PSMB subunits) and 19S core subunits (PSMC and PSMD subunits) co- (Supplementary Table S6) for proteasome subunits co-precipitated precipitated with gp78 or Hrd1 in the indicated detergent is plotted with with gp78 or Hrd1 in the indicated detergent is plotted. (b, d, f, h) The the standard error.

SUPPLEMENTARYKopito Supplemental INFORMATION Figure S7

A B GFP GFP-GluR1 ER lumen

cytosol GluR1 calnexin overlay

C EndoH - + - + E MG132 - + MG132 - - + + IP: α-HA (Ub)n IB: α-HA IB: α-Ub

D CTRL MG132 hrs. 0 2 4 6 6 GFP-GluR1 IB: α-GFP IP: anti-GFP

F 150 4.0 MG132 3.5 120 MG132 CTRL 3.0

90 2.5 2.0

counts 60 1.5 CTRL 1.0 30 MG132 Fold change in mean F mean in change Fold 0.5 emetine +emetine 0 0.0 100 101 102 103 104 0 2 4 6 8 10 12 GFP fluorescence post-drug addition (hrs.)

Figure S7 Unassembled GluR1 is a bona fide ERAD substrate. (a) calculated to be ~2.5 hrs. and its degradation is mitigated by MG132. Illustration of GFP-GluR1 fusion protein topology. (b) Colocalisation (e) GluR1 is ubiquitinated upon addition of MG132. GFP-GluR1 of GFP-GluR1 (green) expressed in HEK293 cells with the ER marker was immunoprecipitated with anti-GFP antibody, and Western blots calnexin (red). Nuclei are stained with DAPI (blue). (c) HA-GluR1 were probed with anti-Ub (top) and anti-GFP antibodies (bottom). (f) migration in the absence and presence of Endo H and MG132 (10 Fluorescence histograms of the GFP-GluR1 stable cell line untreated mM). EndoH sensitivity indicates that GluR1 is retained in the ER in (CTRL) and treated with MG132 (6 hrs, 10 mM) as measured by flow its core glycosylated form and proteasome inhibition does not promote cytometry (left). Time course of GFP-GluR1 degradation after treatment its maturation. (d) GFP-GluR1 pulse-chase assay. A t1/2 for GluR1 was with MG132 (10 mM) and/or emetine (10 mM) over 12 hrs (right).

Kopito Supplemental Figure S8 SUPPLEMENTARY INFORMATION

UBXD8-S myc-UBE2J1 A B Hrd1-S C

HEK Empty 48 51 226 229 230 231 232 233 shRNA HEK Empty 170 171 173 shRNA

HEK Empty 31 32 65 66 85 86 95 shRNA UBXD8-S myc-UBE2J1 Hrd1-S (51kDa) (39kDa) (75kDa) tubulin tubulin tubulin (lysate) D E F G H F.F. Luc. F.F. 64 132 F.F. Luc. F.F. 36 shRNA shRNA F.F. Luc. F.F. 38 102 shRNA Luc. F.F. 72 73 shRNA F.F. Luc. F.F. 24 shRNA S-VIMP S-UBE2G2 mNGly1 S-SEL1L EDEM1-S (22kDa) (20kDa) (73kDa) (100kDa) (73kDa) tubulin tubulin tubulin tubulin tubulin

I J K L M

F.F. Luc. F.F. 37 shRNA F.F. Luc. F.F. 319 320 F.F. Luc. F.F. 159 234 shRNA Luc. F.F. 223 224 shRNA shRNA F.F. Luc. F.F. 142 143 shRNA Ubn-gp78-S C15orf24-S S-E-Syt1 UBAC2 UBXD2-S (>250kDa) (23kDa) (120kDa) (35kDa) (71kDa) gp78-S tubulin tubulin (73kDa) tubulin UBXD8-S (51kDa) tubulin N O P Q R F.F. Luc. F.F. 196 F.F. Luc. F.F. 313 316 F.F. Luc. F.F. 217 shRNA Luc. F.F. 161 shRNA shRNA Luc. F.F. 178 179 180 shRNA shRNA

FAM8A1 ERFAD-S S-TTC35 AUP1-S TXD16-S (50kDa) (90kDa) (35kDa) (45kDa) (92kDa) Hrd1-S tubulin tubulin tubulin tubulin

S T U V F.F. Luc. F.F. 148

shRNA HEK Empty 74 83 F.F. Luc. F.F. 327 329 shRNA shRNA

F.F. Luc. F.F. 5 67 88 shRNA Derlin2-S COX4NB-S S-JAMP S-OS9.2 (23kDa) (25kDa) (27kDa) (~90kDa) tubulin tubulin tubulin tubulin

W 100

25 Normalized expression level

0 11 53 37 65 94 25 26 70 42 63 80 81 38 311 196 109 123 125 200 341 353 148 348 127 136 166 188 102 307 309 310 312 131 132 143 TMEM111 Derlin-1 Derlin-2 VCP Hrd1 gp78 SVIP Aha1 VIMP ERdj5 HERP SEL1L LONP2 UBXD2 PSMD2 Derlin-3 XTP3-B FAM8A1 KIAA0090

Figure S8 Validation of shRNA knockdown by Western blotting and qRT- antibodies and anti-tubulin antibodies as a loading control. (w) To determine PCR. To determine the efficacy of shRNA constructs targeting the each the efficacy of shRNA constructs at depleting the target transcript, a control gene, plasmids encoding the corresponding S-tagged or myc-tagged target shRNA plasmid targeting firefly luciferase or shRNA plasmids targeting protein were transiently co-expressed in HEK293 cells with a control shRNA potential ERAD components (indicated by the library reference number; construct or the targeting shRNA construct (indicated by the library reference Table S7a) were transiently expressed in HEK293 cells. Transcript levels for number; Table S7a). SDS-solubilised cellular lysates (a-c) or S-protein the indicated components were determined by qRT-PCR, normalised to cells agarose affinity-purified proteins from TX-100 lysates (d-v) were separated by transfected with the control plasmid targeting firefly luciferase and to the SDS-PAGE and analysed by Western blotting with anti-S-peptide or anti-myc percentage of transfection, and presented as the normalised expression level.

SUPPLEMENTARYKopito Supplemental INFORMATION Figure S9

A TTR(D18G) Integrated degradation map

GRP94 CPVL LON2P TXD16 unidirectional (dashed) MS analysis recipricol (solid) Reported BiP OS-9 Both ERdj5 ERFAD BAIT INTERACTOR no effect XTP3-B

CANX not determined EDEM1 SEL1L Derlin-3 JAMP BAP31

FAM8A1 Hrd1 gp78 RNF5

HERP UBAC2 Insig1 Derlin-2 Derlin-1 KIAA 0090 C15orf24

TMEM MMGT1 VIMP UBE2J1 ERLIN1 ERLIN2 111 C19orf63 UBXD2 UBXD8 TMEM AUP1 E-Syt1 E-Syt2 SPP 85

TMEM UBE2G2 PLAP TTC35 93 Cul2 C14orf122 PARK2 COX4 hHR23B VCIP135 PSMA1-7 NB NGly1 VCP PSMB 1-7 PSMC 2 UBE4A PSMD 1-3,6,7,14 PSME4 UBQLN1 Npl4 Ufd1 hHR23A RFP2 SVIP USP13 ATX3 YOD1

B GluR1 Integrated degradation map

GRP94 CPVL LON2P TXD16 unidirectional (dashed) MS analysis recipricol (solid) Reported BiP OS-9 ERdj5 Both ERFAD BAIT INTERACTOR no effect XTP3-B

CANX not determined EDEM1 SEL1L Derlin-3 JAMP BAP31

FAM8A1 Hrd1 gp78 RNF5

HERP UBAC2 Insig1 Derlin-2 Derlin-1 KIAA 0090 C15orf24

TMEM MMGT1 VIMP UBE2J1 ERLIN1 ERLIN2 111 C19orf63 UBXD2 UBXD8 TMEM AUP1 E-Syt1 E-Syt2 SPP 85

TMEM UBE2G2 PLAP TTC35 93 Cul2 C14orf122 PARK2 COX4 hHR23B VCIP135 PSMA1-7 NB NGly1 VCP PSMB 1-7 PSMC 2 UBE4A PSMD 1-3,6,7,14 PSME4 UBQLN1 Npl4 Ufd1 hHR23A RFP2 SVIP USP13 ATX3 YOD1

Figure S9 Integrated substrate-specific degradation snapshots. The comprehensive ERAD interactome (Fig. S5) to construct an integrated normalised fold change in mean GFP fluorescence of ERAD reporter snapshot of substrate-specific functional ERAD networks for two lines in response to shRNA-mediated knockdown of ERAD components example substrates, TTR(D18G)-GFP (panel (a)) and GFP-GluR1 (panel represented as a heat map (Fig. 3f) was directly mapped onto the (b)).

SUPPLEMENTARY INFORMATION Kopito Supplemental Figure S10

A GFPu Integrated degradation map

GRP94 CPVL LON2P TXD16 unidirectional (dashed) MS analysis recipricol (solid) Reported BiP OS-9 Both ERdj5 ERFAD BAIT INTERACTOR no effect XTP3-B

CANX not determined EDEM1 SEL1L Derlin-3 JAMP BAP31

FAM8A1 Hrd1 gp78 RNF5

HERP UBAC2 Insig1 Derlin-2 Derlin-1 KIAA 0090 C15orf24

TMEM MMGT1 VIMP UBE2J1 ERLIN1 ERLIN2 111 C19orf63 UBXD2 UBXD8 TMEM AUP1 E-Syt1 E-Syt2 SPP 85

TMEM UBE2G2 PLAP TTC35 93 Cul2 C14orf122 PARK2 COX4 hHR23B VCIP135 PSMA1-7 NB NGly1 VCP PSMB 1-7 PSMC 2 UBE4A PSMD 1-3,6,7,14 PSME4 UBQLN1 Npl4 Ufd1 hHR23A RFP2 SVIP USP13 ATX3 YOD1

B CFTR(∆F508) Integrated degradation map

GRP94 CPVL LON2P TXD16 unidirectional (dashed) MS analysis recipricol (solid) Reported BiP OS-9 ERdj5 Both ERFAD BAIT INTERACTOR no effect XTP3-B

CANX not determined EDEM1 SEL1L Derlin-3 JAMP BAP31

FAM8A1 Hrd1 gp78 RNF5

HERP UBAC2 Insig1 Derlin-2 Derlin-1 KIAA 0090 C15orf24

TMEM MMGT1 VIMP UBE2J1 ERLIN1 ERLIN2 111 C19orf63 UBXD2 UBXD8 TMEM AUP1 E-Syt1 E-Syt2 SPP 85

TMEM UBE2G2 PLAP TTC35 93 Cul2 C14orf122 PARK2 COX4 hHR23B VCIP135 PSMA1-7 NB NGly1 VCP PSMB 1-7 PSMC 2 UBE4A PSMD 1-3,6,7,14 PSME4 UBQLN1 Npl4 Ufd1 hHR23A RFP2 SVIP USP13 ATX3 YOD1

Figure S10 Integrated substrate-specific degradation snapshots for GFPu and CFTR(∆F508). Integrated snapshots of substrate-specific functional ERAD networks as described in Fig. S9 are shown for GFPu (a) and CFTR(∆F508) (b).

SUPPLEMENTARYKopito Supplemental INFORMATION Figure S11

A A1AT(NHK) Integrated degradation map

GRP94 CPVL LON2P TXD16 unidirectional (dashed) MS analysis recipricol (solid) Reported BiP OS-9 Both ERdj5 ERFAD BAIT INTERACTOR no effect XTP3-B

CANX not determined EDEM1 SEL1L Derlin-3 JAMP BAP31

FAM8A1 Hrd1 gp78 RNF5

HERP UBAC2 Insig1 Derlin-2 Derlin-1 KIAA 0090 C15orf24

TMEM MMGT1 VIMP UBE2J1 ERLIN1 ERLIN2 111 C19orf63 UBXD2 UBXD8 TMEM AUP1 E-Syt1 E-Syt2 SPP 85

TMEM UBE2G2 PLAP TTC35 93 Cul2 C14orf122 PARK2 COX4 hHR23B VCIP135 PSMA1-7 NB NGly1 VCP PSMB 1-7 PSMC 2 UBE4A PSMD 1-3,6,7,14 PSME4 UBQLN1 Npl4 Ufd1 hHR23A RFP2 SVIP USP13 ATX3 YOD1

B A1AT(NHK-QQQ) Integrated degradation map

GRP94 CPVL LON2P TXD16 unidirectional (dashed) MS analysis recipricol (solid) Reported BiP OS-9 ERdj5 Both ERFAD BAIT INTERACTOR no effect XTP3-B

CANX not determined EDEM1 SEL1L Derlin-3 JAMP BAP31

FAM8A1 Hrd1 gp78 RNF5

HERP UBAC2 Insig1 Derlin-2 Derlin-1 KIAA 0090 C15orf24

TMEM MMGT1 VIMP UBE2J1 ERLIN1 ERLIN2 111 C19orf63 UBXD2 UBXD8 TMEM AUP1 E-Syt1 E-Syt2 SPP 85

TMEM UBE2G2 PLAP TTC35 93 Cul2 C14orf122 PARK2 COX4 hHR23B VCIP135 PSMA1-7 NB NGly1 VCP PSMB 1-7 PSMC 2 UBE4A PSMD 1-3,6,7,14 PSME4 UBQLN1 Npl4 Ufd1 hHR23A RFP2 SVIP USP13 ATX3 YOD1

Figure S11 Integrated substrate-specific degradation snapshots for A1AT(NHK) and A1AT(NHK-QQQ). Integrated snapshots of substrate-specific functional ERAD networks as described in Fig. S9 are shown for A1AT(NHK) (a) and A1AT(NHK) (b).

SUPPLEMENTARY INFORMATION Kopito Supplemental Figure S12

Figure 3A Figure 3B Figure 3C Figure 3D

250 250 150 150 250 250 100 100 150 150 75 75 100 100 75 75 50 50 50 50 37 37 37 37 25 25 IB: SEL1L and Hrd1 25 20

250 20 20 150 100 IB: myc IB: SEL1L and S-peptide IB: SEL1L and Hrd1 75

50 250 250 150 150 250 37 150 100 100 100 75 75 75 IB: SEL1L 25 50 50 20 37 75 37 50 25 20 37 25

25 20 IB: FAM8A1 IB: S-peptide 20

IB: S-peptide

Figure 3F Figure 3H Figure 3I Figure 3G 250 250 250 150 250 150 150 100 150 100 100 75 100 75 75 75 50 50 50 50 37 37 37 37 25 25 25 25 20 20 IB: SEL1L 20 IB: Hrd1 IB: S-peptide IB: SEL1L 250 250 150 150 250 250 100 100 150 150 75 75 100 100 75 75 50 50 50 37 50 37 37 37

25 25 25

25 20 IB: S-peptide 20 IB: S-peptide IB: Hrd1 250 IB: S-peptide 150 100 250 250 250 75 150 150 150 100 100 100 50 75 75 75

37 50 50 50

37 37 37 25

25 25 25 IB: Hrd1 20 20 20 IB: myc IB: myc IB: SEL1L

Figure S12 Full scans of immunoblots.

SUPPLEMENTARYKopito Supplemental Figure INFORMATION S12 (cont.)

Figure 3J Figure 3L Figure 3M Figure 3N 250 150 250 250 100 150 250 150 100 150 100 100 75 75 75 75 50 50 50 50 37 37 37 37 25

25 20 25 25 20 20 IB: UBAC2 IB: S-peptide IB: UBAC2 IB: UBAC2 250 250 150 150 250 250 100 150 100 150 75 100 100 75 75 75 50 50 37 50 50 37 37 37 25

20 25 25 25 20 20 IB: S-peptide IB: gp78 and UBXD8 IB: gp78 IB: myc 250 250 150 150 250 100 150 100 75 100 75 50 75 50 37 50 37 25 37 25 20

25 IB: gp78 20 IB: UBXD8 IB: Hrd1

Figure 3O Figure 3O Figure 3P Figure 3P

250 250 250 250 150 150 150 150 100 100 100 100 75 75 75 75 50 50 50 37 50 37 37

25 37 20 25 25

20 20 25 IB: S-peptide 20 IB: gp78 IB: Derlin-2 IB: UBAC2 250 150 250 100 150 250 100 75 150 75 100 50 50 75 37 37 50 25 37 25 20

20 IB: S-peptide 25 IB: Derlin-2 20 IB: gp78

Figure S12 Full scans of immunoblots continued

SUPPLEMENTARY INFORMATION Kopito Supplemental Figure S12 (cont.) Kopito Supplemental Figure S12 (cont.)

Figure 6B Figure 6B Figure 6F Figure 7B 250 250 150 150 250 250 150 100 100 150 75 75 100 100 75 75 50 Figure S8C Figure S8D Figure S8E Figure S8F Figure S8G 50 250 250 250 50 50 37 37 150 150 150 250 100 100 250 150 IB: Hrd1 75 100 37 25 75 75 150 100 25 100 75 250 20 50 75 50 20 150 50 IB: UBXD8 37 37 50 100 25 50 IB: FAM8A1 75 250 IB: S-peptide 150 25 37 100 37 37 50 250 250 75 25 150 150 20 100 37 100 20 25 50 25 75 75 15 IB: S-peptide 25 25 37 250 IB: S-peptide 50 50 150 IB: S-peptide IB: S-peptide 20 IB: myc 100 37 25 75 250 IB: Tubulin 37 20 250 150 50 100 25 250 150 IB: UBAC2 150 250 75 150 100 20 25 100 37 250 75 100 75 75 50 IB: SEL1L IB: FAM8A1 150 100 50 50 50 25 75 37 250 37 Figure 6D Figure 6D 150 37 100 50 37 IB: tubulin 250 250 75 25 25 150 150 37 25 100 100 20 50 25 IB: tubulin 25 20 75 75 20 15 37 IB: tubulin IB: Tubulin IB: tubulin 50 50 IB: tubulin

25 IB: p97/VCP and FAM8A1 IB: SEL1L IB: SEL1L Figure S8H Figure S8I Figure S8J Figure S8K Figure S8L 250 250 150 150 75 250 100 100 250 150 Figure S7F Figure S8A Figure S8B 100 75 75 50 150 100 75 250 250 75 150 150 50 50 250 100 100 37 75 75 50 50 150 37 37 37 37 100 50 50 25 37 25 75 25 25 37 25

25 IB: S-peptide IB: S-peptide 50 25 IB: S-peptide 20 IB: S-peptide IB: S-peptide 250 37 20 250 150 IB: S-peptide 150 100 250 250 100 75 150 150 IB: ubiquitin IB: S-peptide 75 250 100 250 100 150 75 150 50 100 75 75 250 100 50 250 150 75 37 50 37 50 50 150 100 37 75 50 37 100 37 25 25 37 50 75 25 25 37 25 IB: tubulin IB: tubulin 25 IB: tubulin 50 20 25 IB: tubulin IB: tubulin 37 20 IB: tubulin IB: tubulin

IB: ubiquitin

Figure S12 Full scans of immunoblots continued

Kopito SupplementalSUPPLEMENTARY Figure S12 (cont.)INFORMATION

Figure S8C Figure S8D Figure S8E Figure S8F Figure S8G 250 250 250 150 150 150 250 100 100 250 150 75 100 75 75 150 100 100 75 50 75 50 50 37 37 50 50 25 37 37 37 25 20 20 25 25 15 IB: S-peptide 25 250 IB: S-peptide 150 IB: S-peptide IB: S-peptide IB: myc 100 75 250 250 150 50 100 250 150 150 250 75 150 100 100 37 75 100 75 75 50 50 50 25 50 37 37 37 37 IB: tubulin 25 25 25 20 20 25 IB: tubulin 15 IB: tubulin IB: tubulin IB: tubulin

Figure S8H Figure S8I Figure S8J Figure S8K Figure S8L 250 250 150 150 75 250 100 100 250 150 100 75 75 50 150 100 75 75 50 50 37 50 50 37 37 37 37 25 25 25 25 25

IB: S-peptide IB: S-peptide IB: S-peptide IB: S-peptide IB: S-peptide 250 250 150 150 100 250 250 100 75 150 150 75 250 100 100 150 75 50 75 100 50 75 37 50 37 50 50 37 37 25 25 37 25 25 IB: tubulin IB: tubulin 25 IB: tubulin

IB: tubulin IB: tubulin

Figure S12 Full scans of immunoblots continued

Kopito Supplemental Figure S12 (cont.)

SUPPLEMENTARY INFORMATION

Figure S8M Figure S8N Figure S8O Figure S8P Figure S8Q 250 250 100 250 150 150 150 100 100 100 75 75 75 150 75 100

50 50 50 50 75 37 37 37 37 50 25 25 37 25 20 20 25

IB: S-peptide 25 IB: FAM8A1 IB: S-peptide IB: S-peptide IB: S-peptide

250 250 250 150 150 250 150 150 100 150 100 100 100 75 100 75 75 75 75 50 37 50 50 50 50 37 37 25 37 37 25 20 25 25 25 IB: tubulin IB: S-peptide IB: tubulin IB: tubulin IB: tubulin

Figure S8R Figure S8S Figure S8T Figure S8U Figure S8V 250 250 250 250 250 150 150 150 150 100 100 150 100 100 100 75 75 75 75 75 50 50 50 50 50 37 37 37 37 37 25 25 25 25 20 20 IB: S-peptide 25 IB: S-peptide IB: S-peptide IB: S-peptide 250 IB: S-peptide 150 100 250 250 250 150 150 75 250 100 100 150 150 75 100 100 75 50 75 75 50 50 37 50 37 37 50 25 37 37 25 IB: tubulin 25 20 25 20 25 IB: tubulin IB: tubulin IB: tubulin IB: tubulin

Figure S12 Full scans of immunoblots continued

SUPPLEMENTARY INFORMATION

Supplementary Tables

Table S1 Listing of primary and secondary bait proteins. Primary (top) and secondary (bottom) S-tagged proteins employed as baits in the proteomic analyses are shown with relevant information including alternative names, known functions, protein domains/repeats, cellular localisation (ER-L: ER luminal, ER-M: ER integral membrane, C: cytoplasmic), and identified yeast orthologs (Standard and Systematic name) are also shown.

Table S2 CompPASS-identified HCIPs and complete MASCOT list of interactors in DIG. (a) Listing of proteins interacting in DIG that displayed a WDN-score above the threshold score of 1 and were therefore designated as HCIPs. Also shown are additional statistical measures of interactor confidence, including Z-score, WS-score, and ZD-score. Self-identification of the bait protein identified is shown in red. (b) Listing of all MASCOT-identified protein interactors and their respective number of peptide scans found in affinity purifications of bait proteins from DIG-solubilised cell lysates.

Table S3 CompPASS-identified HCIPs and complete MASCOT list of interactors in TX-100. (a) Listing of proteins interacting in TX-100 that displayed a WDN-score above the threshold score of 1 and were therefore designated as HCIPs. Also shown are additional statistical measures of interactor confidence, including Z-score, WS-score, and ZD-score. The bait protein identified in each purification is shown in red. (b) Listing of all MASCOT-identified protein interactors and their respective number of peptide scans found in affinity purifications of bait proteins from TX-100-solubilised cell lysates.

Table S4 Analysis of proteomic interaction data. (a) Analysis and breakdown of proteomic data obtained in DIG. The total number of HCIPs identified by CompPASS is shown as well as the number of scans contributed by Bait self-identification, Trypsin, and Bead controls that were removed to yield the total number of HCIPs/ERAD interactions. The total number HCIPs is further broken down into likely false positives, primary / secondary baits obtained as prey, reported ERAD factors, and uncharacterised proteins of interest. The number of HCIPs is also shown separately for primary and secondary baits, as well as the overlap between these two data sets. (b) Same as in panel (a), but for data obtained in TX-100.

Table S5 Previously reported ERAD component interactions. Listing of reported pairwise interactions. Each interaction between a bait and prey was assigned an ‘interaction number’. The method by which the interaction was identified and the relevant reference (blue text) are indicated. Proteins used as baits in this study are shown (red text), and gray shading indicates proteins that were not employed as baits or identified as HCIPs in this study. The identification of the reported interaction in our DIG or TX-100 analyses or in the STRING database is indicated as an ‘O’ (identified) or as an ‘X’ (not identified). A comparative analysis of the reported interactions literature and those identified in our study is shown in the inset table.

Table S6 E3-proteasome subunit interactions. Listing of all MASCOT-identified proteasome subunits, their number of peptide scans, and their spectral abundance factor (SAF) from affinity purifications of the E3s Hrd1 and gp78 in DIG or TX-100 as indicated. Proteasome subunits determined by CompPASS to be HCIPs (WDN-score > 1) are highlighted in blue.

Table S7 Raw substrate fluorescence data from the primary ERAD reporter screen. (a). Complete listing of all shRNA target sequences, library reference numbers, and their effects on the fluorescence levels of the ERAD reporter cell lines tested in the initial round of screening, including GFPu, GFP-GluR1, TTR(D18G)-GFP, A1AT(NHK)-GFP, and GFP-CFTR(∆F508). (b) shRNA constructs employed in the second round of screening in the following cell lines: GFPu, GFP-GluR1, TTR(D18G)-GFP, A1AT(NHK)-GFP, A1AT(NHK-QQQ)-GFP, GFP-CFTR(∆F508), and GFP cell lines. These data are represented as a heat map in Fig. 4f.

Table S8 ERAD component data and links. Listing of ERAD components and identified HCIPs of interest along with relevant information, including gene and protein names, known function, localisation, UNIPROT ID, and known yeast orthologs.

Table S9 qRT-PCR primers. Listing of qRT-PCR primer sequences used to assess the affect of UPR induction on transcript levels in Fig. 5 and to assess shRNA knockdown of target transcript in Supplementary Fig. S8.

Table S10 ERAD cDNA sources. Listing of cDNA sources for expression of the bait proteins employed in this study.