FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2000555535> ?p ?o ?g. }

• W2000555535 endingPage "17487" @default.
• W2000555535 startingPage "17475" @default.
• W2000555535 abstract "During endoplasmic reticulum (ER)-associated degradation (ERAD), a relatively small number of ubiquitin ligases (E3) must be capable of ubiquitinating an assortment of substrates diverse in both structure and location (ER lumen, membrane, and/or cytosol). Therefore, mechanisms that operate independently of primary sequence determinants must exist to ensure specificity during this process. Here we provide direct evidence for adapter-mediated substrate recruitment for a virus-encoded ERAD E3 ligase, mK3. Members of an ER membrane protein complex that normally functions during major histocompatibility complex class I biogenesis in the immune system are required for mK3 substrate selection. We demonstrate that heterologous substrates could be ubiquitinated by mK3 if they were recruited by these ER accessory molecules to the proper position relative to the ligase domain of mK3. This mechanism of substrate recruitment by adapter proteins may explain the ability of some E3 ligases, including cellular ERAD E3 ligases, to specifically target the ubiquitination of multiple substrates that are unrelated in sequence. During endoplasmic reticulum (ER)-associated degradation (ERAD), a relatively small number of ubiquitin ligases (E3) must be capable of ubiquitinating an assortment of substrates diverse in both structure and location (ER lumen, membrane, and/or cytosol). Therefore, mechanisms that operate independently of primary sequence determinants must exist to ensure specificity during this process. Here we provide direct evidence for adapter-mediated substrate recruitment for a virus-encoded ERAD E3 ligase, mK3. Members of an ER membrane protein complex that normally functions during major histocompatibility complex class I biogenesis in the immune system are required for mK3 substrate selection. We demonstrate that heterologous substrates could be ubiquitinated by mK3 if they were recruited by these ER accessory molecules to the proper position relative to the ligase domain of mK3. This mechanism of substrate recruitment by adapter proteins may explain the ability of some E3 ligases, including cellular ERAD E3 ligases, to specifically target the ubiquitination of multiple substrates that are unrelated in sequence. Ubiquitin-regulated pathways intersect with virtually all aspects of cell biology. This is certainly true of protein quality control pathways, including those that operate to degrade proteins from the ER 2The abbreviations used are: ERendoplasmic reticulumERADER-associated degradationMHCmajor histocompatibility complexHCMHC class I heavy chainRINGreally interesting new geneMARCHmembrane-associated RING-CHTAPtransporter associated with antigen processingTMtransmembrane domainE3ubiquitin ligasemAbmonoclonal antibodyPBSphosphate-buffered salineβ2mβ2-microglobulinhhuman. 2The abbreviations used are: ERendoplasmic reticulumERADER-associated degradationMHCmajor histocompatibility complexHCMHC class I heavy chainRINGreally interesting new geneMARCHmembrane-associated RING-CHTAPtransporter associated with antigen processingTMtransmembrane domainE3ubiquitin ligasemAbmonoclonal antibodyPBSphosphate-buffered salineβ2mβ2-microglobulinhhuman. lumen and membrane. This essential pathway, known as ER-associated degradation (ERAD), prevents the toxic accumulation of misfolded proteins through the regulated degradation of target substrates. Initiation of ERAD involves substrate recognition leading to ubiquitination mediated by ubiquitin ligases (E3). Multiple cellular E3 ligases have been identified that associate with the ER membrane, including Hrd1, Doa10 (known as TEB4 in mammals), and gp78 (1Kostova Z. Tsai Y.C. Weissman A.M. Semin. Cell Dev. Biol. 2007; 18: 770-779Crossref PubMed Scopus (130) Google Scholar, 2Nakatsukasa K. Brodsky J.L. Traffic. 2008; 9: 861-870Crossref PubMed Scopus (235) Google Scholar). These ligases are known to ubiquitinate a multitude of diverse substrates. However, the mechanisms by which substrates are selected remain poorly understood. Although evidence exists for direct binding of some substrates to E3 ligases (3Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (380) Google Scholar, 4Ravid T. Kreft S.G. Hochstrasser M. EMBO J. 2006; 25: 533-543Crossref PubMed Scopus (211) Google Scholar), cofactor molecules in the ER lumen, membrane, and cytosol appear to provide an essential substrate recruitment function (1Kostova Z. Tsai Y.C. Weissman A.M. Semin. Cell Dev. Biol. 2007; 18: 770-779Crossref PubMed Scopus (130) Google Scholar, 2Nakatsukasa K. Brodsky J.L. Traffic. 2008; 9: 861-870Crossref PubMed Scopus (235) Google Scholar, 5Carvalho P. Goder V. Rapoport T.A. Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 6Ballar P. Zhong Y. Nagahama M. Tagaya M. Shen Y. Fang S. J. Biol. Chem. 2007; 282: 33908-33914Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 7Ye Y. Shibata Y. Kikkert M. van Voorden S. Wiertz E. Rapoport T.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14132-14138Crossref PubMed Scopus (282) Google Scholar, 8Denic V. Quan E.M. Weissman J.S. Cell. 2006; 126: 349-359Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 9Ravid T. Hochstrasser M. Nat. Rev. Mol. Cell Biol. 2008; 9: 679-690Crossref PubMed Scopus (609) Google Scholar). Indeed, it is now appreciated that E3 ligases in the ER membrane associate with a complex set of accessory molecules that, collectively, facilitate ERAD. The complexity of these systems confounds the characterization of substrate selection, but the fact that ERAD has been implicated in numerous diseases (10Yoshida H. FEBS J. 2007; 274: 630-658Crossref PubMed Scopus (894) Google Scholar) magnifies the importance of attaining a fuller understanding of substrate recruitment/selection. endoplasmic reticulum ER-associated degradation major histocompatibility complex MHC class I heavy chain really interesting new gene membrane-associated RING-CH transporter associated with antigen processing transmembrane domain ubiquitin ligase monoclonal antibody phosphate-buffered saline β2-microglobulin human. endoplasmic reticulum ER-associated degradation major histocompatibility complex MHC class I heavy chain really interesting new gene membrane-associated RING-CH transporter associated with antigen processing transmembrane domain ubiquitin ligase monoclonal antibody phosphate-buffered saline β2-microglobulin human. Members of the RING finger domain-containing E3 ligase family are known to play a critical role in ERAD (1Kostova Z. Tsai Y.C. Weissman A.M. Semin. Cell Dev. Biol. 2007; 18: 770-779Crossref PubMed Scopus (130) Google Scholar, 2Nakatsukasa K. Brodsky J.L. Traffic. 2008; 9: 861-870Crossref PubMed Scopus (235) Google Scholar). In general, RING E3 ligases have been divided into two broad classes, single- and multi-subunit (11Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3341) Google Scholar). Single-subunit E3 ligases possess discrete domains that mediate substrate binding and ubiquitin-conjugating enzyme recruitment. In contrast, multi-subunit E3 ligases are dependent upon a complex of protein subunits that act together to mediate substrate binding and ubiquitin conjugation. The mK3 protein, encoded by the murine γ-herpesvirus 68, is a member of a family of E3 ligases found in several γ-herpesviruses and poxviruses, as well as in eukaryotes. These molecules are membrane-anchored and possess a cytosol-facing RING domain of the RING-CH subtype (12Ohmura-Hoshino M. Goto E. Matsuki Y. Aoki M. Mito M. Uematsu M. Hotta H. Ishido S. J. Biochem. 2006; 140: 147-154Crossref PubMed Scopus (92) Google Scholar, 13Lehner P.J. Cresswell P. Curr. Opin. Immunol. 2004; 16: 82-89Crossref PubMed Scopus (72) Google Scholar). Like many of its viral homologs, mK3 is a presumed single-subunit E3 ligase. MK3 is employed by the virus to interfere with the host immune response by inhibiting the major histocompatibility complex (MHC) class I antigen presentation pathway (14Stevenson P.G. May J.S. Smith X.G. Marques S. Adler H. Koszinowski U.H. Simas J.P. Efstathiou S. Nat. Immunol. 2002; 3: 733-740Crossref PubMed Scopus (147) Google Scholar). In the presence of mK3, which localizes to the ER membrane, nascent class I heavy chains (HC) are ubiquitinated, leading to their rapid degradation in a proteasome-dependent fashion (15Boname J.M. Stevenson P.G. Immunity. 2001; 15: 627-636Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). This ubiquitination is known to require a cytosolic tail on the class I HC (15Boname J.M. Stevenson P.G. Immunity. 2001; 15: 627-636Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 16Wang X. Connors R. Harris M.R. Hansen T.H. Lybarger L. J. Virol. 2005; 79: 4099-4108Crossref PubMed Scopus (30) Google Scholar). Furthermore, class I HC that are incapable of associating with the class I peptide-loading complex in the ER, consisting of TAP-1/2, tapasin, and additional accessory proteins (17Peaper D.R. Cresswell P. Annu. Rev. Cell Dev. Biol. 2008; 24: 343-368Crossref PubMed Scopus (161) Google Scholar), are resistant to mK3-mediated ubiquitination (18Lybarger L. Wang X. Harris M.R. Virgin 4th, H.W. Hansen T.H. Immunity. 2003; 18: 121-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Interestingly, the stable expression and function of mK3 require TAP-1, TAP-2, and tapasin. In fact, mK3 associates with this complex even in the absence of the class I HC (18Lybarger L. Wang X. Harris M.R. Virgin 4th, H.W. Hansen T.H. Immunity. 2003; 18: 121-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 19Yu Y.Y. Harris M.R. Lybarger L. Kimpler L.A. Myers N.B. Virgin 4th, H.W. Hansen T.H. J. Virol. 2002; 76: 2796-2803Crossref PubMed Scopus (40) Google Scholar). Furthermore, only the class I HC (and not TAP-1, TAP-2, or tapasin) is detectably ubiquitinated and rapidly degraded in the presence of mK3 (20Boname J.M. de Lima B.D. Lehner P.J. Stevenson P.G. Immunity. 2004; 20: 305-317Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Thus, it was initially assumed that mK3 would bind directly to a unique determinant on the peptide-loading complex-associated class I HC; this would be consistent with a single-subunit E3 ligase. However, diverse transmembrane (TM) and cytosolic tails could be appended to the class I HC without loss of mK3-dependent ubiquitination (21Wang X. Lybarger L. Connors R. Harris M.R. Hansen T.H. J. Virol. 2004; 78: 8673-8686Crossref PubMed Scopus (47) Google Scholar). These observations, coupled with the dependence of mK3 on the peptide-loading complex for stable expression, led to an alternative model to explain the specificity of mK3 for MHC class I HC; the association of mK3 with TAP-1/2 and tapasin positions its RING-CH domain such that only the tail of class I HC but not the cytosolic domains of other proteins within the peptide-loading complex can be ubiquitinated (21Wang X. Lybarger L. Connors R. Harris M.R. Hansen T.H. J. Virol. 2004; 78: 8673-8686Crossref PubMed Scopus (47) Google Scholar). In this study, we show that a protein unrelated to class I HC in sequence can, upon association with the peptide-loading complex, be ubiquitinated by mK3. In addition, changing its relative position within the peptide-loading complex can alter the fate of this novel substrate. These data provide direct evidence to support an “adapter-mediated” model of substrate recruitment in which the peptide-loading complex functions as an “adapter complex” for mK3 and serves to recruit the substrate (class I HC) and orient the RING-CH domain of mK3. Additionally, our results highlight the distinction between substrate recruitment by adapters and specific requirements of the E3 ligase for ubiquitination of the substrates. Indeed, the features of class I HC ubiquitination by mK3 (a cytosolic tail with either lysine, serine, threonine, or cysteine residues (22Wang X. Herr R.A. Chua W.J. Lybarger L. Wiertz E.J. Hansen T.H. J. Cell Biol. 2007; 177: 613-624Crossref PubMed Scopus (218) Google Scholar)), held true for our unrelated chimeric substrate. The relationship of mK3 with viral and cellular E3 ligases, including Doa10/TEB4 (23Hassink G. Kikkert M. van Voorden S. Lee S.J. Spaapen R. van Laar T. Coleman C.S. Bartee E. Früh K. Chau V. Wiertz E. Biochem. J. 2005; 388: 647-655Crossref PubMed Scopus (132) Google Scholar), suggests that studies of substrate recruitment/selection by this E3 ligase can provide insights that can inform our understanding of this process by other E3 ligases. Experiments were performed using the 3KO murine embryo fibroblast line (β2m−/−, class I Kb−/−, and class I Db−/−) (18Lybarger L. Wang X. Harris M.R. Virgin 4th, H.W. Hansen T.H. Immunity. 2003; 18: 121-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and a tapasin-deficient murine embryo fibroblast line (Tpn−/− and H-2b) (24Grandea 3rd, A.G. Golovina T.N. Hamilton S.E. Sriram V. Spies T. Brutkiewicz R.R. Harty J.T. Eisenlohr L.C. Van Kaer L. Immunity. 2000; 13: 213-222Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) into which various constructs were introduced. 293T cells (25DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Mol. Cell. Biol. 1987; 7: 379-387Crossref PubMed Scopus (915) Google Scholar) were used for the production of ecotropic retrovirus vectors following transient transfection. All cells were maintained in complete RPMI 1640 medium (Mediatech, Manassas, VA) supplemented with 10% fetal calf serum (HyClone, Logan, UT), 1 mm HEPES (Invitrogen), 2 mml-glutamine, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, and 100 units/ml penicillin/streptomycin (all from Mediatech). Cells were cultured for 24 h in the presence of 100 units of mouse γ-interferon/ml to increase transcription of the peptide-loading complex protein genes. Retrovirus-containing supernatants were generated using the Vpack vector system (Stratagene, La Jolla, CA) for transient production in 293T cells to generate ecotropic virus for infection. Packaging cells were transfected using FuGENE 6 (Roche Diagnostics) according to the supplier's instructions. Virus-containing supernatants were collected 48–72 h post-transfection and added to target cells in the presence of hexadimethrine bromide (Polybrene) (Sigma) at a concentration of 8 μg/ml. Stable transductants were produced by selection with the appropriate antibiotics, at the following concentrations: puromycin, 2.5 μg/ml; Zeocin, 0.2 mg/ml; hygromycin, 0.2 mg/ml; geneticin, 0.5 mg/ml. The vectors used for expression of each cDNA were all murine stem cell virus-derived nonreplicating retroviral vectors. Each vector is bicistronic with the gene of interest upstream of an internal ribosome entry site element that precedes the antibiotic resistance marker. pMIP and pMIB are derivatives of pMSCV-IRES-GFP (pMIG (26Van Parijs L. Refaeli Y. Lord J.D. Nelson B.H. Abbas A.K. Baltimore D. Immunity. 1999; 11: 281-288Abstract Full Text PDF PubMed Scopus (395) Google Scholar)) in which the green fluorescent protein cDNA was replaced with the puromycin and bleomycin (Zeocin) resistance genes, respectively. pMIN and pMIH have been described (16Wang X. Connors R. Harris M.R. Hansen T.H. Lybarger L. J. Virol. 2005; 79: 4099-4108Crossref PubMed Scopus (30) Google Scholar) and encode neomycin and hygromycin resistance, respectively. All cDNAs used in this study were expressed from one of these vectors. The cDNA clones for soluble human β2m, mouse B7.2 (CD86), MHC class I Ld heavy chain, and mouse tapasin were all obtained by reverse transcription-PCR using human- and mouse-derived cells as RNA sources, respectively. Generation of the Ld T134K mutant and the mK3 cDNA have been described (19Yu Y.Y. Harris M.R. Lybarger L. Kimpler L.A. Myers N.B. Virgin 4th, H.W. Hansen T.H. J. Virol. 2002; 76: 2796-2803Crossref PubMed Scopus (40) Google Scholar, 28Yu Y.Y. Turnquist H.R. Myers N.B. Balendiran G.K. Hansen T.H. Solheim J.C. J. Immunol. 1999; 163: 4427-4433PubMed Google Scholar). Transmembrane versions of human β2m all contain the entire open reading frame of hβ2m, including the N-terminal signal peptide fused in frame with various C-terminal domains. Tβ2m.B7.B7 consists of hβ2m fused to residues 208–284 of murine B7.2 (TM and cytosolic domains). Tβ2m.Tpn.Tpn consists of hβ2m fused to residues 381–442 of murine tapasin (TM and cytosolic domains). Tβ2m.B7.Tpn consists of hβ2m fused to residues 208–239 of B7.2 (TM domain) and residues 418–442 of tapasin (cytosolic tail). These chimeric constructs were generated by overlap-extension (fusion) PCR with the respective templates. Truncations of cytosolic domains were generated by PCR in which the reverse oligonucleotide incorporated a stop codon at the desired location. The class I Ld tail-deletion mutant (HC-ΔCyt) retained only six residues (KRRRNT). The Tβ2m.B7.B7 tail-deletion mutant (Tβ2m.B7.ΔCyt) retained only five residues (CHRRP; the two arginine residues are lysines in the native B7.2 sequence). All site-directed mutagenesis was performed using the QuickChange mutagenesis kit (Stratagene) according to the manufacturer's instructions. A sequence comparison of the cytosolic tail sequences present in the various constructs is given in FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6. The correct sequence for all of the constructs was confirmed by DNA sequence analysis.FIGURE 2Tβ2m. B7.B7 is membrane-anchored and supports class I expression and mK3 function. A, 3KO fibroblasts (β2m−/−, class I Kb−/−, and class I Db−/−) were stably transduced with class I HC (Ld), hβ2m, and Tβ2m.B7.B7, as indicated. Cells were then stained for surface expression of hβ2m (top row) or fully conformed class I HC (bottom row), with or without low pH treatment to denature surface class I molecules. Samples were then analyzed by flow cytometry to quantify the expression levels. In each histogram, background staining (secondary antibody alone) is indicated in dark gray; staining of untreated cells is indicated in light gray shaded peaks, and staining of low pH-treated cells is indicated in black. B, class I HC were denatured and immunoprecipitated (IP) from 3KO cells (+ class I HC and hβ2m, or + class I HC and Tβ2m.B7.B7) ± mK3 using mAb 64-3-7. Precipitates were then treated with endoglycosidase H (Endo H), separated by SDS-PAGE, and blotted for ubiquitin (Ub) and class I HC.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Membrane-anchored β2m, but not soluble β2m, is ubiquitinated by mK3. A, immunoblot for steady-state levels of peptide-loading complex proteins in 3KO cells expressing the indicated constructs, ± mK3. Actin blot is included as a loading control. Asterisk indicates the specific band. B, sequential immunoprecipitations (IP) were performed from cells expressing the indicated constructs. Primary anti-TAP-1 precipitates were denatured, and Tβ2m.B7.B7 or hβ2m was recovered by immunoprecipitation, followed by immunoblot for ubiquitin (Ub) and β2m.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Proteasome-dependent degradation of Tβ2m. B7.B7 induced by mK3. A, 3KO cells (+ class I HC and Tβ2m.B7.B7) ± mK3 were pulse-labeled 10 min with 35S-Met/Cys and then chased for the indicated times in medium containing an excess of unlabeled Met/Cys. Lysates from cells harvested at each time point were immunoprecipitated (IP) for either class I HC or Tβ2m.B7.B7. Precipitates were separated by SDS-PAGE, and bands were visualized by autoradiography. Band intensities were quantified from gels from three independent experiments using ImageJ software and plotted as a percentage of the signal at time 0. (*, p < 0.05.) B, 3KO cells (+ class I HC and hβ2m) ± mK3 were treated, quantified, and analyzed as in A. (*, p < 0.05.) C, pulse-chase labeling and immunoprecipitation were performed similar to above but with a single chase point, 1 h. Where indicated, cells were chased in the presence of the proteasome inhibitor MG132.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Sequence requirements for mK3-mediated ubiquitination and degradation of Tβ2m. B7.B7. A, left panel, sequential immunoprecipitations (IP) were performed from cells expressing the indicated constructs. Primary anti-TAP-1 precipitates were denatured, and Tβ2m.B7.ΔCyt or Tβ2m.B7.B7 was recovered by immunoprecipitation, followed by immunoblot for ubiquitin (Ub) and β2m. Right panel, class I HC were denatured and immunoprecipitated from 3KO cells (+ class I HC and Tβ2m.B7.ΔCyt) ± mK3 using mAb 64-3-7. Precipitates were then treated with endoglycosidase H (Endo H), separated by SDS-PAGE, and blotted for ubiquitin and class I HC. B, pulse-chase metabolic labeling and immunoprecipitation were performed with cells expressing Tβ2m.B7.ΔCyt and class I HC, as described in Fig. 4. After labeling, Tβ2m.B7.ΔCyt or class I HC was directly precipitated. Samples were separated by SDS-PAGE and visualized by autoradiography. Data are plotted as the signal remaining at each time point as percentage of the signal at time 0. (*, p < 0.05.) C, left panel, sequential immunoprecipitations were performed from cells expressing Tβ2m.B7.B7 and class I HC-ΔCyt. Primary anti-TAP-1 precipitates were denatured, and Tβ2m.B7.B7 was recovered by immunoprecipitation, followed by immunoblot for ubiquitin and β2m. Right panel, class I HC-ΔCyt were denatured and immunoprecipitated using mAb 64-3-7. Precipitates were then treated with endoglycosidase H, separated by SDS-PAGE, and blotted for ubiquitin and class I HC. D, pulse-chase metabolic labeling and immunoprecipitation were performed as described in Fig. 4. After labeling, Tβ2m.B7.B7 and class I HC-ΔCyt were directly precipitated. Samples were separated by SDS-PAGE and visualized by autoradiography. Bands intensities were quantified from gels from three independent experiments using ImageJ software and plotted as the signal remaining at each time point as percentage of the signal at time 0. (*, p < 0.05.) E, Tβ2m.B7.B7 mutants lacking either lysine residues (K-less), or lysine, serine, and threonine residues (KST-less) in the cytosolic domain were expressed in 3KO (with class I HC) ± mK3 (see Fig. 1 for sequence comparison). Sequential immunoprecipitation was performed; primary anti-TAP-1 precipitates were denatured, and the indicated Tβ2m.B7.B7 mutant molecules or Tβ2m.B7.B7 were recovered by immunoprecipitation, followed by immunoblot for ubiquitin and β2m.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Transmembrane β2m is not ubiquitinated by mK3 when it binds to the peptide-loading complex in the place of tapasin. A, depiction and sequence comparison of the constructs used in these experiments. Potential ubiquitin-acceptor residues are indicated in red. The TM and cytosolic domains are shown. Tβ2m.B7.Tpn contains the B7.2 TM domain and the cytosolic tail of tapasin. Tβ2m.Tpn.Tpn contains the TM and cytosolic domains of tapasin (upper panel). Schematic diagrams are shown of the potential arrangements of Tβ2m.B7.Tpn or Tβ2m.Tpn.Tpn in tapasin-sufficient 3KO cells (lower panel of A). B, Tβ2m.B7.Tpn or Tβ2m.Tpn.Tpn was expressed in 3KO cells (with class I HC) ± mK3. Sequential immunoprecipitation (IP) was performed; primary anti-TAP-1 precipitates were denatured, and the indicated transmembrane β2m chimeric molecules were recovered by immunoprecipitation, followed by immunoblot for ubiquitin and β2m. C, depiction of the possible arrangements of the indicated molecules in tapasin-deficient cells (Tpn−/−). D, immunoblot for steady-state levels of peptide-loading complex proteins were examined in tapasin-deficient fibroblasts following transduction of the indicated constructs, including full-length tapasin. Asterisk indicates the specific band. E, sequential anti-TAP-1/anti-β2m immunoprecipitations were performed from Tpn−/− cells expressing the indicated constructs. In some cases, full-length tapasin was transduced into the cells. Samples were blotted for ubiquitin and β2m.View Large Image Figure ViewerDownload Hi-res image Download (PPT) BBM1 recognizes native and denatured hβ2m (29Brodsky F.M. Bodmer W.F. Parham P. Eur. J. Immunol. 1979; 9: 536-545Crossref PubMed Scopus (249) Google Scholar). Anti-ubiquitin monoclonal antibody (mAb) P4D1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). mAb 64-3-7 recognizes open forms (unassembled) of the Ld class I HC or epitope-tagged class I heavy chains; mAb 30-5-7 is specific for fully conformed (β2m-associated) class I Ld; 5D3 is a hamster anti-mouse tapasin mAb; all have been described previously (18Lybarger L. Wang X. Harris M.R. Virgin 4th, H.W. Hansen T.H. Immunity. 2003; 18: 121-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Rabbit antisera against C-terminal sequences of mK3 (residues 167–187), and mouse TAP-1 have been described (18Lybarger L. Wang X. Harris M.R. Virgin 4th, H.W. Hansen T.H. Immunity. 2003; 18: 121-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). mAb ACTN05 (C4) against actin was purchased from Abcam (Cambridge, MA). Mouse anti-CD86 (B7.2) clone GL1 was purchased from eBioscience (San Diego). All flow cytometric analyses were performed using a FACSCalibur (BD Biosciences), and data were analyzed using CellQuest software (BD Biosciences). Cells were stained on ice with the appropriate dilution of the indicated unconjugated primary antibodies. Phycoerythrin-conjugated goat anti-mouse IgG (Pharmingen) was used to visualize the staining levels of the primary antibodies. For low pH treatment of cells to denature class I molecules, cells were suspended in 0.5% glycine buffer, pH 2.8, for 3 min at 37 °C. Excess complete culture medium was added to neutralize the samples, which were then treated as above for flow cytometric analysis. For co-immunoprecipitation, cells were lysed in phosphate-buffered saline (PBS) containing 1% digitonin (Wako, Richmond, VA), 20 mm iodoacetamide, 1 mm phenylmethylsulfonyl fluoride (both from Sigma), and Protease Inhibitor Cocktail III (Calbiochem). Separately, protein A- Sepharose beads (GE Healthcare) were incubated for 4 h at 4 °C with saturating amounts of antibody. Excess antibody was removed from beads by washing three times with 0.1% digitonin/PBS buffer. Post-nuclear lysates were incubated with immobilized antibody overnight at 4 °C. Beads were washed four times with 0.1% digitonin/PBS buffer, and proteins were eluted by boiling in nonreducing lithium dodecyl sulfate sample buffer (Invitrogen). Sequential immunoprecipitations were performed as described above with the exception of the elution step following the primary immunoprecipitation, which was performed by boiling in 0.5% SDS in 10 mm Tris-Cl, pH 6.8. Samples were diluted with 1% IGEPAL CA-630 (Nonidet P-40) (from Sigma) in PBS for a final concentration of 0.1% SDS. Supernatants were incubated with the second protein A-immobilized antibody overnight at 4 °C. Beads were washed four times with 0.1% IGEPAL CA-630 in PBS, and proteins were eluted by boiling in nonreducing lithium dodecyl sulfate sample buffer. Direct denaturing immunoprecipitations were performed essentially as described above, except that IGEPAL CA-630 was substituted for digitonin. Cells were lysed in buffer containing 1% IGEPAL in PBS and protease inhibitors. Post-nuclear lysates were denatured by the addition of 0.5% SDS and then boiled for 5 min. Samples were diluted with 1% IGEPAL CA-630 buffer for a final concentration of 0.1% SDS prior to incubation with immobilized antibody. Beads were washed four times with 0.1% IGEPAL/PBS, and proteins were eluted by boiling in nonreducing lithium dodecyl sulfate sample buffer. For endoglycosidase-H (endo-H) treatment of precipitates, post-immunoprecipitation samples were eluted by boiling in 10 mm Tris-Cl, pH 6.8, 0.5% SDS. Eluates were mixed with an equal volume of 100 mm sodium acetate, pH 5.4, and either digested (or mock-digested) at 37 °C for >1 h with 1 milliunit of endoglycosidase H (New England Biolabs, Ipswich, MA) that was reconstituted in 50 mm sodium acetate, pH 5.4. For immunoblot of cell lysates, cells were lysed in 1% IGEPAL/PBS. Post-nuclear lysates were mixed with lithium dodecyl sulfate sample buffer and 2-mercaptoethanol (1% final concentration). Protein content was determined using the BCA protein assay from Thermo Scientific (Rockford, IL). Samples were electrophoresed on NuPAGE SDS-polyacrylamide gels (Invitrogen). Separated proteins were then transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked for 1 h with either 5% dried milk, 0.1% Tween 20 (Sigma), 0.1% SDS in PBS, or 0.1% Tween 20, 0.1% SDS. The latter formulation (no milk) was used for the ubiquitin blots. After washing three times with 0.1% Tween 20, 0.1% SDS in PBS, membranes were incubated" @default.
• W2000555535 created "2016-06-24" @default.
• W2000555535 creator A5064568180 @default.
• W2000555535 creator A5086081568 @default.
• W2000555535 creator A5087099599 @default.
• W2000555535 date "2009-06-01" @default.
• W2000555535 modified "2023-09-27" @default.
• W2000555535 title "Adapter-mediated Substrate Selection for Endoplasmic Reticulum-associated Degradation" @default.
• W2000555535 cites W1481795283 @default.
• W2000555535 cites W1483402518 @default.
• W2000555535 cites W1501553911 @default.
• W2000555535 cites W1507598785 @default.
• W2000555535 cites W1594939553 @default.
• W2000555535 cites W1765185969 @default.
• W2000555535 cites W1964269825 @default.
• W2000555535 cites W1967986514 @default.
• W2000555535 cites W1976435906 @default.
• W2000555535 cites W1981849277 @default.
• W2000555535 cites W1984347348 @default.
• W2000555535 cites W1985495705 @default.
• W2000555535 cites W1989525395 @default.
• W2000555535 cites W1998535540 @default.
• W2000555535 cites W2000447975 @default.
• W2000555535 cites W2010617137 @default.
• W2000555535 cites W2010862596 @default.
• W2000555535 cites W2017776769 @default.
• W2000555535 cites W2018925832 @default.
• W2000555535 cites W2021658855 @default.
• W2000555535 cites W2029119458 @default.
• W2000555535 cites W2030413639 @default.
• W2000555535 cites W2032818498 @default.
• W2000555535 cites W2033313049 @default.
• W2000555535 cites W2033352476 @default.
• W2000555535 cites W2034133901 @default.
• W2000555535 cites W2034414781 @default.
• W2000555535 cites W2036925007 @default.
• W2000555535 cites W2037488340 @default.
• W2000555535 cites W2037657846 @default.
• W2000555535 cites W2040798958 @default.
• W2000555535 cites W2045298605 @default.
• W2000555535 cites W2051881845 @default.
• W2000555535 cites W2053118119 @default.
• W2000555535 cites W2058209707 @default.
• W2000555535 cites W2060736883 @default.
• W2000555535 cites W2061156149 @default.
• W2000555535 cites W2069904188 @default.
• W2000555535 cites W2073337768 @default.
• W2000555535 cites W2084416493 @default.
• W2000555535 cites W2091022868 @default.
• W2000555535 cites W2093930948 @default.
• W2000555535 cites W2095392756 @default.
• W2000555535 cites W2113164415 @default.
• W2000555535 cites W2121077625 @default.
• W2000555535 cites W2122166146 @default.
• W2000555535 cites W2123322767 @default.
• W2000555535 cites W2129291354 @default.
• W2000555535 cites W2139577310 @default.
• W2000555535 cites W2140219059 @default.
• W2000555535 cites W2140921627 @default.
• W2000555535 cites W2142424369 @default.
• W2000555535 cites W2145174769 @default.
• W2000555535 cites W2147291971 @default.
• W2000555535 cites W2149368193 @default.
• W2000555535 cites W2149616074 @default.
• W2000555535 cites W2150123812 @default.
• W2000555535 cites W2164155929 @default.
• W2000555535 cites W2166472526 @default.
• W2000555535 cites W2167352490 @default.
• W2000555535 cites W2169221124 @default.
• W2000555535 cites W2170924043 @default.
• W2000555535 doi "https://doi.org/10.1074/jbc.m808258200" @default.
• W2000555535 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2719388" @default.
• W2000555535 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19366690" @default.
• W2000555535 hasPublicationYear "2009" @default.
• W2000555535 type Work @default.
• W2000555535 sameAs 2000555535 @default.
• W2000555535 citedByCount "6" @default.
• W2000555535 countsByYear W20005555352012 @default.
• W2000555535 countsByYear W20005555352014 @default.
• W2000555535 crossrefType "journal-article" @default.
• W2000555535 hasAuthorship W2000555535A5064568180 @default.
• W2000555535 hasAuthorship W2000555535A5086081568 @default.
• W2000555535 hasAuthorship W2000555535A5087099599 @default.
• W2000555535 hasBestOaLocation W20005555351 @default.
• W2000555535 hasConcept C158617107 @default.
• W2000555535 hasConcept C177284502 @default.
• W2000555535 hasConcept C185592680 @default.
• W2000555535 hasConcept C18903297 @default.
• W2000555535 hasConcept C2777289219 @default.
• W2000555535 hasConcept C2779679103 @default.
• W2000555535 hasConcept C41008148 @default.
• W2000555535 hasConcept C76155785 @default.
• W2000555535 hasConcept C86803240 @default.
• W2000555535 hasConcept C9390403 @default.
• W2000555535 hasConcept C95444343 @default.
• W2000555535 hasConceptScore W2000555535C158617107 @default.
• W2000555535 hasConceptScore W2000555535C177284502 @default.
• W2000555535 hasConceptScore W2000555535C185592680 @default.

• next