IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ stein-2014-5 Key steps in ERAD of luminal ER proteins reconstituted with purified components. in vitro In vitro pull down pull down weight by staining molecular weight estimation by staining weight by staining Stein A., Ruggiano A., Carvalho P., Rapoport T.A. alexander_stein@hms.harvard.edu, tom_rapoport@hms.harvard.edu Cell (0092-8674) 2014 Only protein-protein interactions imex curation stein-2014-2 Key steps in ERAD of luminal ER proteins reconstituted with purified components. in vitro In vitro pull down pull down tag fluorescence tag visualisation by fluorescence tag fluorescence Stein A., Ruggiano A., Carvalho P., Rapoport T.A. alexander_stein@hms.harvard.edu, tom_rapoport@hms.harvard.edu Cell (0092-8674) 2014 Only protein-protein interactions imex curation stein-2014-3 Key steps in ERAD of luminal ER proteins reconstituted with purified components. in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column weight by comassie molecular weight estimation by coomasie staining weight by comassie Stein A., Ruggiano A., Carvalho P., Rapoport T.A. alexander_stein@hms.harvard.edu, tom_rapoport@hms.harvard.edu Cell (0092-8674) 2014 Only protein-protein interactions imex curation stein-2014-4 Key steps in ERAD of luminal ER proteins reconstituted with purified components. in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Stein A., Ruggiano A., Carvalho P., Rapoport T.A. alexander_stein@hms.harvard.edu, tom_rapoport@hms.harvard.edu Cell (0092-8674) 2014 Only protein-protein interactions imex curation stein-2014-1 Key steps in ERAD of luminal ER proteins reconstituted with purified components. yeast Saccharomyces cerevisiae Baker's yeast molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Stein A., Ruggiano A., Carvalho P., Rapoport T.A. alexander_stein@hms.harvard.edu, tom_rapoport@hms.harvard.edu Cell (0092-8674) 2014 Only protein-protein interactions imex curation stein-2014-6 Key steps in ERAD of luminal ER proteins reconstituted with purified components. in vitro In vitro deubiquitinase assay deubiquitinase assay deubiquitinase assay tag fluorescence tag visualisation by fluorescence tag fluorescence Stein A., Ruggiano A., Carvalho P., Rapoport T.A. Hrd1 was ubiquitinated by fluorescent-labelled ubiquitin. Deubiquitiation was monitored by fluorescent scanning. alexander_stein@hms.harvard.edu, tom_rapoport@hms.harvard.edu Cell (0092-8674) 2014 Only protein-protein interactions imex curation hrd1_yeast ERAD-associated E3 ubiquitin-protein ligase HRD1 HMG-CoA reductase degradation protein 1 HRD1 DER3 RING-type E3 ubiquitin transferase HRD1 YOL013C protein protein yeast Saccharomyces cerevisiae Baker's yeast MVPENRRKQLAIFVVVTYLLTFYCVYSATKTSVSFLQVTLKLNEGFNLMVLSIFILLNSTLLWQLLTKLLFGELRLIEHEHIFERLPFTIINTLFMSSLFHERYFFTVAFFGLLLLYLKVFHWILKDRLEALLQSINDSTTMKTLIFSRFSFNLVLLAVVDYQIITRCISSIYTNQKSDIESTSLYLIQVMEFTMLLIDLLNLFLQTCLNFWEFYRSQQSLSNENNHIVHGDPTDENTVESDQSQPVLNDDDDDDDDDRQFTGLEGKFMYEKAIDVFTRFLKTALHLSMLIPFRMPMMLLKDVVWDILALYQSGTSLWKIWRNNKQLDDTLVTVTVEQLQNSANDDNICIICMDELIHSPNQQTWKNKNKKPKRLPCGHILHLSCLKNWMERSQTCPICRLPVFDEKGNVVQTTFTSNSDITTQTTVTDSTGIATDQQGFANEVDLLPTRTTSPDIRIVPTQNIDTLAMRTRSTSTPSPTWYTFPLHKTGDNSVGSSRSAYEFLITNSDEKENGIPVKLTIENHEVNSLHGDGGEQIAKKIVIPDKFIQHI cdc48_yeast Cell division control protein 48 CDC48 Cell division cycle protein 48 Transitional endoplasmic reticulum ATPase homolog YDL126C protein protein yeast Saccharomyces cerevisiae Baker's yeast MGEEHKPLLDASGVDPREEDKTATAILRRKKKDNMLLVDDAINDDNSVIAINSNTMDKLELFRGDTVLVKGKKRKDTVLIVLIDDELEDGACRINRVVRNNLRIRLGDLVTIHPCPDIKYATRISVLPIADTIEGITGNLFDVFLKPYFVEAYRPVRKGDHFVVRGGMRQVEFKVVDVEPEEYAVVAQDTIIHWEGEPINREDEENNMNEVGYDDIGGCRKQMAQIREMVELPLRHPQLFKAIGIKPPRGVLMYGPPGTGKTLMARAVANETGAFFFLINGPEVMSKMAGESESNLRKAFEEAEKNAPAIIFIDEIDSIAPKRDKTNGEVERRVVSQLLTLMDGMKARSNVVVIAATNRPNSIDPALRRFGRFDREVDIGIPDATGRLEVLRIHTKNMKLADDVDLEALAAETHGYVGADIASLCSEAAMQQIREKMDLIDLDEDEIDAEVLDSLGVTMDNFRFALGNSNPSALRETVVESVNVTWDDVGGLDEIKEELKETVEYPVLHPDQYTKFGLSPSKGVLFYGPPGTGKTLLAKAVATEVSANFISVKGPELLSMWYGESESNIRDIFDKARAAAPTVVFLDELDSIAKARGGSLGDAGGASDRVVNQLLTEMDGMNAKKNVFVIGATNRPDQIDPAILRPGRLDQLIYVPLPDENARLSILNAQLRKTPLEPGLELTAIAKATQGFSGADLLYIVQRAAKYAIKDSIEAHRQHEAEKEVKVEGEDVEMTDEGAKAEQEPEVDPVPYITKEHFAEAMKTAKRSVSDAELRRYEAYSQQMKASRGQFSNFNFNDAPLGTTATDNANSNNSAPSGAGAAFGSNAEEDDDLYS 02ADDB9A227614D8 npl4_yeast Nuclear protein localization protein 4 NPL4 YBR170C YBR1231 HRD4 HMG-CoA reductase degradation protein 4 protein protein yeast Saccharomyces cerevisiae Baker's yeast MLIRFRSKNGTHRVSCQENDLFGTVIEKLVGNLDPNADVDTFTVCEKPGQGIHAVSELADRTVMDLGLKHGDMLILNYSDKPANEKDGVNVEIGSVGIDSKGIRQHRYGPLRIKELAVDEELEKEDGLIPRQKSKLCKHGDRGMCEYCSPLPPWDKEYHEKNKIKHISFHSYLKKLNENANKKENGSSYISPLSEPDFRINKRCHNGHEPWPRGICSKCQPSAITLQQQEFRMVDHVEFQKSEIINEFIQAWRYTGMQRFGYMYGSYSKYDNTPLGIKAVVEAIYEPPQHDEQDGLTMDVEQVKNEMLQIDRQAQEMGLSRIGLIFTDLSDAGAGDGSVFCKRHKDSFFLSSLEVIMAARHQTRHPNVSKYSEQGFFSSKFVTCVISGNLEGEIDISSYQVSTEAEALVTADMISGSTFPSMAYINDTTDERYVPEIFYMKSNEYGITVKENAKPAFPVDYLLVTLTHGFPNTDTETNSKFVSSTGFPWSNRQAMGQSQDYQELKKYLFNVASSGDFNLLHEKISNFHLLLYINSLQILSPDEWKLLIESAVKNEWEESLLKLVSSAGWQTLVMILQESG 6EF35D21338EE07E ufd1_yeast Ubiquitin fusion degradation protein 1 UFD1 PIP3 Polymerase-interacting protein 3 YGR048W protein protein yeast Saccharomyces cerevisiae Baker's yeast MFSGFSSFGGGNGFVNMPQTFEEFFRCYPIAMMNDRIRKDDANFGGKIFLPPSALSKLSMLNIRYPMLFKLTANETGRVTHGGVLEFIAEEGRVYLPQWMMETLGIQPGSLLQISSTDVPLGQFVKLEPQSVDFLDISDPKAVLENVLRNFSTLTVDDVIEISYNGKTFKIKILEVKPESSSKSICVIETDLVTDFAPPVGYVEPDYKALKAQQDKEKKNSFGKGQVLDPSVLGQGSMSTRIDYAGIANSSRNKLSKFVGQGQNISGKAPKAEPKQDIKDMKITFDGEPAKLDLPEGQLFFGFPMVLPKEDEESAAGSKSSEQNFQGQGISLRKSNKRKTKSDHDSSKSKAPKSPEVIEID 198E0CD48B863B98 ubx2_yeast UBX domain-containing protein 2 SEL1 YM9571.05 YML013W UBX2 Secretion lowering protein 1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MPVVNHEDSEFHLSHTEEDKLNEFQVITNFPPEDLPDVVRLLRNHGWQLEPALSRYFDGEWKGEPDQMGEPTQTSTPMAETLVPPALGPRPLLFTASLPVVRPLPANFRNDFRTIGLNGRSNTVWSMFESFSYDGNPFLFILLLIPRIINRLSATIFTFFCTLLSLHSISGGGNSGKPKISKVPKAPTRETHIPLAEILGDTKDKDAFCELKSFKPDISFNEALRIAKEEFKFMLLILVGDTYDTDTDTVDVNSKLLLEKILLNKKTLQYLRKIDNDLIIYLKCVHELEPWLVARQLGVRNTPEIFLIANVANKASHSETLPSQRLSILGKLKVNSLNRFLQSLTNVVEKYTPELVVNKTEMHELRMSREIKKLQEDAYKKSLEMDRIKAIEKEKSLKHAQDLKLNSTARQLKWLKACIDEIQPFETTGKQATLQFRTSSGKRFVKKFPSMTTLYQIYQSIGCHIYLAVYSSDPAEWSNALQDKIRQLSADDDMLCFKEGQLETATATTIEELGHIINNELTSFDLERGKLEFDFELVSPFPKYTVHPNEHMSVDQVPQLWPNGSLLVEALDEEDEEDEENEEQ DA4DDD3D624C17B8 cbpy_yeast Carboxypeptidase Y PRC1 YMR297W Carboxypeptidase YSCY protein protein yeast Saccharomyces cerevisiae Baker's yeast MKAFTSLLCGLGLSTTLAKAISLQRPLGLDKDVLLQAAEKFGLDLDLDHLLKELDSNVLDAWAQIEHLYPNQVMSLETSTKPKFPEAIKTKKDWDFVVKNDAIENYQLRVNKIKDPKILGIDPNVTQYTGYLDVEDEDKHFFFWTFESRNDPAKDPVILWLNGGPGCSSLTGLFFELGPSSIGPDLKPIGNPYSWNSNATVIFLDQPVNVGFSYSGSSGVSNTVAAGKDVYNFLELFFDQFPEYVNKGQDFHIAGESYAGHYIPVFASEILSHKDRNFNLTSVLIGNGLTDPLTQYNYYEPMACGEGGEPSVLPSEECSAMEDSLERCLGLIESCYDSQSVWSCVPATIYCNNAQLAPYQRTGRNVYDIRKDCEGGNLCYPTLQDIDDYLNQDYVKEAVGAEVDHYESCNFDINRNFLFAGDWMKPYHTAVTDLLNQDLPILVYAGDKDFICNWLGNKAWTDVLPWKYDEEFASQKVRNWTASITDEVAGEVKSYKHFTYLRVFNGGHMVPFDVPENALSMVNEWIHGGFSL 7227F3489CBDD952 otu1_yeast Ubiquitin thioesterase OTU1 OTU1 OTU domain-containing protein 1 YFL044C protein protein yeast Saccharomyces cerevisiae Baker's yeast MKLKVTGAGINQVVTLKQDATLNDLIEHINVDVKTMRFGYPPQRINLQGEDASLGQTQLDELGINSGEKITIESSDSNESFSLPPPQPKPKRVLKSTEMSIGGSGENVLSVHPVLDDNSCLFHAIAYGIFKQDSVRDLREMVSKEVLNNPVKFNDAILDKPNKDYAQWILKMESWGGAIEIGIISDALAVAIYVVDIDAVKIEKFNEDKFDNYILILFNGIHYDSLTMNEFKTVFNKNQPESDDVLTAALQLASNLKQTGYSFNTHKAQIKCNTCQMTFVGEREVARHAESTGHVDFGQNR 32ABA3CFEA4BB5EB hrd1-cdc48-7 1763678 n/a 1763684 unspecified role unspecified role bait bait purified purified region (required) ubiquitinylated lysine ubiquitinylated lysine undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763685 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig. 3 Only low levels of Cdc48 bound to Hrd1 in the absence of Ufd1/Npl4. Ubiquitination of Hrd1 was also required. High salt disrupted binding. hrd1-cdc48-6 1763678 n/a 1763686 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1763685 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1763687 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1763684 unspecified role unspecified role bait bait purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region (required) ubiquitinylated lysine ubiquitinylated lysine undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false yeast Saccharomyces cerevisiae Baker's yeast association association false false false Fig. 3 Ubiquitination of Hrd1 was also required. High salt disrupted binding. hrd1-cdc48-8 1763678 n/a 1763686 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1763684 unspecified role unspecified role bait bait purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region (required) ubiquitinylated lysine ubiquitinylated lysine undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763687 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) association association false false false Fig. 3 Ubiquitination of Hrd1 was also required. High salt disrupted binding. hrd1-ubx2 1763678 n/a 1763684 unspecified role unspecified role bait bait purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region (required) ubiquitinylated lysine ubiquitinylated lysine undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763688 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig. 3 Only low levels of Cdc48 bound to Hrd1 in the absence of Ufd1/Npl4. Ubiquitination of Hrd1 was also required. High salt disrupted binding. hrd1-cdc48-9 1763678 n/a 1763684 unspecified role unspecified role bait bait purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region (required) ubiquitinylated lysine ubiquitinylated lysine undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763686 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1763687 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1763685 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1763688 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) association association false false false Fig. 3 Ubiquitination of Hrd1 was also required. High salt disrupted binding. hrd1-prc1-6 1763678 n/a 1763689 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal fluorescent dye fluorescent dye label fluorescent dye c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763684 unspecified role unspecified role bait bait purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast direct interaction direct interaction false false false Fig. 4. Supp Fig. 4 Non-ubiquitinated Prc1 was released from Hrd1 by Cdc48 activity. No release was seen for substrate bound to non-ubiquitinated Hrd1. The release was significantly reduced with two Lys-to-Arg mutations in different regions but was stimulated by a third. The addition of Cdc48, Ufd1/Npl4, and ATP led to the dissociation of approx. 80% of labeled poly-ubiquitinated Hrd1. Significantly less Hrd1 was released when ATP was depleted or in the absence of Ufd/Npl4. These results indicate that the Cdc48 ATPase causes the dissociation of poly-ubiquitinated Hrd1 oligomers. hrd1-prc1-7 1763679 n/a 1763689 unspecified role unspecified role prey prey purified purified P00729:p.Gly255Arg mutation mutation certain certain sequence position certain certain certain sequence position certain false c-terminal fluorescent dye fluorescent dye label fluorescent dye c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763684 unspecified role unspecified role bait bait purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast direct interaction direct interaction false false false 1763679 Fig. 1A, 1B Wild-type Prc1 does not bind to Hrd1, only the mis-folded mutant. hrd1-prc1-9 1763679 n/a 1763684 unspecified role unspecified role bait bait purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763689 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal fluorescent dye fluorescent dye label fluorescent dye c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast direct interaction direct interaction false false false 1763679 Fig. 1C, 1D hrd1-prc1-8 1763679 n/a 1763684 unspecified role unspecified role bait bait purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763689 unspecified role unspecified role prey prey c-terminal fluorescent dye fluorescent dye label fluorescent dye c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false dhfr tag fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false yeast Saccharomyces cerevisiae Baker's yeast direct interaction direct interaction false false false Fig. 1E hrd1-prc1-10 1763679 n/a 1763684 unspecified role unspecified role bait bait purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763689 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c-terminal fluorescent dye fluorescent dye label fluorescent dye c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast direct interaction direct interaction false false false Fig. 1E Wild-type Prc1 does not bind to Hrd1 hrd1-prc1-11 1763679 n/a 1763689 unspecified role unspecified role prey prey c-terminal fluorescent dye fluorescent dye label fluorescent dye c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763684 unspecified role unspecified role bait bait purified purified c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast 1763762 1763759 direct interaction direct interaction false false false Fig. 1F cdc48-npl4-1 1763680 n/a 1763686 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1763687 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1763685 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 6.0 physical association physical association false false false Supp. Fig. 3B cdc48-npl4-1 1763680 n/a 1763685 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 6.0 n/a 1763688 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Supp. Fig. 3B cdc48-npl4-1 1763680 n/a 1763687 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1763686 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1763688 unspecified role unspecified role neutral component neutral component ubx region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) physical association physical association false false false Supp. Fig. 3B cdc48-npl4-1 1763680 n/a 1763688 unspecified role unspecified role neutral component neutral component ubx region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1763687 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1763685 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 6.0 n/a 1763686 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 physical association physical association false false false Supp. Fig. 3B cdc48-1 1763680 n/a 1763685 unspecified role unspecified role neutral component neutral component Stoichiometry: 6.0 direct interaction direct interaction false false false Supp. Fig. 3B cdc48-npl4-1 1763681 n/a 1763685 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 6.0 n/a 1763686 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1763687 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 physical association physical association false false false Supp. Fig. 3A cdc48-1 1763681 n/a 1763685 unspecified role unspecified role neutral component neutral component Stoichiometry: 6.0 direct interaction direct interaction false false false Supp. Fig. 3A cdc48-npl4-1 1763681 n/a 1763688 unspecified role unspecified role neutral component neutral component ubx region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1763685 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 6.0 n/a 1763687 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1763686 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 physical association physical association false false false Supp. Fig. 3A cdc48-npl4-1 1763681 n/a 1763685 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 6.0 n/a 1763688 unspecified role unspecified role neutral component neutral component ubx region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Supp. Fig. 3A cdc48-npl4-1 1763681 n/a 1763688 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1763687 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1763686 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 physical association physical association false false false Supp. Fig. 3A hrd1-hrd1 1763682 n/a 1763684 unspecified role unspecified role neutral component neutral component purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n/a 1763684 unspecified role unspecified role neutral component neutral component purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false direct interaction direct interaction false false false Fig. 1G, Supp. Fig. 1D hrd1-prc1-5 1763682 n/a 1763684 unspecified role unspecified role neutral component neutral component purified purified c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast n/a 1763689 unspecified role unspecified role neutral component neutral component P00729:p.Gly255Arg mutation mutation certain certain sequence position certain certain certain sequence position certain false dhfr tag fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false c-terminal fluorescent dye fluorescent dye label fluorescent dye c-term range C-terminal range c-term range c-term range C-terminal range c-term range false yeast Saccharomyces cerevisiae Baker's yeast direct interaction direct interaction false false false Fig. 1G otu1-prc1 1763683 n/a 1763690 enzyme enzyme unspecified role unspecified role ecoli Escherichia coli (strain K12) n/a 1763689 enzyme target enzyme target substrate substrate unspecified role unspecified role purified purified c-terminal fluorescent dye fluorescent dye label fluorescent dye c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region ubiquitinylated lysine ubiquitinylated lysine undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P00729:p.Gly255Arg mutation mutation certain certain sequence position certain certain certain sequence position certain false yeast Saccharomyces cerevisiae Baker's yeast deubiquitination deubiquitination reaction deubiquitination false false false Fig. 6C Otu1 deubiquitinated modified fluorescently labeled Hrd1 or Prc1 efficiently only when Cdc48 and Ufd1/Npl4 were present. hrd1-otu1-1 1763683 n/a 1763690 enzyme enzyme unspecified role unspecified role P43558:p.Cys120Ser mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1763684 enzyme target enzyme target substrate substrate unspecified role unspecified role purified purified region fluorescent dye fluorescent dye label fluorescent dye undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region (required) ubiquitinylated lysine ubiquitinylated lysine undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false c-terminal sbp sbp c-term range C-terminal range c-term range c-term range C-terminal range c-term range false Q08109:p.Asp255Arg mutation mutation certain certain sequence position certain certain certain sequence position certain false yeast Saccharomyces cerevisiae Baker's yeast deubiquitination deubiquitination reaction deubiquitination false false false Fig. 6, Supp. Fig. 6 Otu1 deubiquitinated modified fluorescently labeled Hrd1 efficiently only when Cdc48 and Ufd1/Npl4 were present. Much less deubiquitination was observed with Otu1 and Cdc48 alone.