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• W2000694202 abstract "Any protein synthesized in the secretory pathway has the potential to misfold and would need to be recognized and ubiquitylated for degradation. This is astounding, since only a few ERAD-specific E3 ligases have been identified. To begin to understand substrate recognition, we wished to map the ubiquitylation sites on the NS-1 nonsecreted immunoglobulin light chain, which is an ERAD substrate. Ubiquitin is usually attached to lysine residues and less frequently to the N terminus of proteins. In addition, several viral E3s have been identified that attach ubiquitin to cysteine or serine/threonine residues. Mutation of lysines, serines, and threonines in the NS-1 variable region was necessary to significantly reduce ubiquitylation and stabilize the protein. The Hrd1 E3 ligase was required to modify all three amino acids. Our studies argue that ubiquitylation of ER proteins relies on very different mechanisms of recognition and modification than those used to regulate biological processes. Any protein synthesized in the secretory pathway has the potential to misfold and would need to be recognized and ubiquitylated for degradation. This is astounding, since only a few ERAD-specific E3 ligases have been identified. To begin to understand substrate recognition, we wished to map the ubiquitylation sites on the NS-1 nonsecreted immunoglobulin light chain, which is an ERAD substrate. Ubiquitin is usually attached to lysine residues and less frequently to the N terminus of proteins. In addition, several viral E3s have been identified that attach ubiquitin to cysteine or serine/threonine residues. Mutation of lysines, serines, and threonines in the NS-1 variable region was necessary to significantly reduce ubiquitylation and stabilize the protein. The Hrd1 E3 ligase was required to modify all three amino acids. Our studies argue that ubiquitylation of ER proteins relies on very different mechanisms of recognition and modification than those used to regulate biological processes. Promiscuity in amino acid modification allows Hrd1 to recognize numerous ER proteins Hrd1 required for Ub conjugation not only on lysines but also on serines/threonines Hrd1-mediated ubiquitin chain elongation is via conventional isopeptide linkages Mutation of K, S, and T residues is required to inhibit Ub and stabilize NS-1 LC The folding and assembly of nascent proteins in the mammalian ER is carefully monitored by a process referred to as “ER quality control” (Ellgaard and Helenius, 2003Ellgaard L. Helenius A. Quality control in the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2003; 4: 181-191Crossref PubMed Scopus (1593) Google Scholar). Proteins that pass this inspection can exit the ER for residence in other organelles of the secretory pathway, secretion, or expression at the cell surface. However, proteins that fail to mature properly in the ER are identified, retrotranslocated to the cytosol, and targeted for degradation by the 26S proteasome via an incompletely understood process termed ER-associated degradation (ERAD) (Werner et al., 1996Werner E.D. Brodsky J.L. McCracken A.A. Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate.Proc. Natl. Acad. Sci. USA. 1996; 93: 13797-13801Crossref PubMed Scopus (379) Google Scholar, Vembar and Brodsky, 2008Vembar S.S. Brodsky J.L. One step at a time: endoplasmic reticulum-associated degradation.Nat. Rev. Mol. Cell Biol. 2008; 9: 944-957Crossref PubMed Scopus (967) Google Scholar). Like the degradation of cytosolic proteins by the 26S proteasome, this pathway is dependent on ubiquitylation of the unfolded substrates. The ERAD pathway was first described in yeast (Werner et al., 1996Werner E.D. Brodsky J.L. McCracken A.A. Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate.Proc. Natl. Acad. Sci. USA. 1996; 93: 13797-13801Crossref PubMed Scopus (379) Google Scholar) and is conserved in mammalian cells (Wiertz et al., 1996Wiertz E.J. Jones T.R. Sun L. Bogyo M. Geuze H.J. Ploegh H.L. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol.Cell. 1996; 84: 769-779Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar, Plemper et al., 1997Plemper R.K. Bohmler S. Bordallo J. Sommer T. Wolf D.H. Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation.Nature. 1997; 388: 891-895Crossref PubMed Scopus (462) Google Scholar). The role of ER chaperones (Brodsky et al., 1999Brodsky J.L. Werner E.D. Dubas M.E. Goeckeler J.L. Kruse K.B. McCracken A.A. The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct.J. Biol. Chem. 1999; 274: 3453-3460Crossref PubMed Scopus (223) Google Scholar, Taxis et al., 2003Taxis C. Hitt R. Park S.H. Deak P.M. Kostova Z. Wolf D.H. Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD.J. Biol. Chem. 2003; 278: 35903-35913Crossref PubMed Scopus (160) Google Scholar) and ubiquitylation (Hiller et al., 1996Hiller M.M. Finger A. Schweiger M. Wolf D.H. ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway.Science. 1996; 273: 1725-1728Crossref PubMed Scopus (598) Google Scholar, Meusser et al., 2005Meusser B. Hirsch C. Jarosch E. Sommer T. ERAD: the long road to destruction.Nat. Cell Biol. 2005; 7: 766-772Crossref PubMed Scopus (932) Google Scholar) in disposing of the misfolded proteins is well documented, whereas the proteins that play a role in the extraction and ubiquitylation of the ERAD substrates have more recently been identified. A number of proteins have been identified that assist in the dislocation and degradation of misfolded ER proteins. In S. cerevisiae, these include the multipass transmembrane protein Der1p (Knop et al., 1996Knop M. Finger A. Braun T. Hellmuth K. Wolf D.H. Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.EMBO J. 1996; 15: 753-763Crossref PubMed Scopus (309) Google Scholar), which may form part of the channel. Two cytosolically oriented, integral membrane E3 ubiquitin ligases, Hrd1p (Bays et al., 2001Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation.Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (368) Google Scholar) and Doa10p (Swanson et al., 2001Swanson R. Locher M. Hochstrasser M. A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation.Genes Dev. 2001; 15: 2660-2674Crossref PubMed Scopus (354) Google Scholar), are essential for ubiquitylation of many different ERAD substrates. Lumenally oriented Hrd3 forms a complex with Hrd1 (Wilhovsky et al., 2000Wilhovsky S. Gardner R. Hampton R. HRD gene dependence of endoplasmic reticulum-associated degradation.Mol. Biol. Cell. 2000; 11: 1697-1708Crossref PubMed Scopus (94) Google Scholar), and Usa1 links the Hrd1p/Hrd3p complex to Der1p (Carvalho et al., 2006Carvalho P. Goder V. Rapoport T.A. Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). Finally, the Cdc48p AAA ATPase complex supplies the energy to extract the substrates from the ER (Rabinovich et al., 2002Rabinovich E. Kerem A. Frohlich K.U. Diamant N. Bar-Nun S. AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation.Mol. Cell. Biol. 2002; 22: 626-634Crossref PubMed Scopus (456) Google Scholar). Different compliments of these proteins are required depending on the substrate. Mammalian equivalents of these yeast proteins have been identified, including three Der1p homologs, Derlin-1-3 (Ye et al., 2004Ye Y. Shibata Y. Yun C. Ron D. Rapoport T.A. A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol.Nature. 2004; 429: 841-847Crossref PubMed Scopus (764) Google Scholar, Lilley and Ploegh, 2004Lilley B.N. Ploegh H.L. A membrane protein required for dislocation of misfolded proteins from the ER.Nature. 2004; 429: 834-840Crossref PubMed Scopus (559) Google Scholar, Oda et al., 2006Oda Y. Okada T. Yoshida H. Kaufman R.J. Nagata K. Mori K. Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation.J. Cell Biol. 2006; 172: 383-393Crossref PubMed Scopus (274) Google Scholar). Sel1L is an ortholog of Hrd3p (Mueller et al., 2006Mueller B. Lilley B.N. Ploegh H.L. SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER.J. Cell Biol. 2006; 175: 261-270Crossref PubMed Scopus (149) Google Scholar) and plays a role in targeting glycoproteins that are associated with EDEM to the retrotranslocon (Cormier et al., 2009Cormier J.H. Tamura T. Sunryd J.C. Hebert D.N. EDEM1 recognition and delivery of misfolded proteins to the SEL1L-containing ERAD complex.Mol. Cell. 2009; 34: 627-633Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Herp is an ortholog of Usa1 and contains a ubiquitin-like domain (Kokame et al., 2000Kokame K. Agarwala K.L. Kato H. Miyata T. Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress.J. Biol. Chem. 2000; 275: 32846-32853Crossref PubMed Scopus (247) Google Scholar), which might account for its ability to bind both to the 26S proteasome and ubiquitylated substrates (Okuda-Shimizu and Hendershot, 2007Okuda-Shimizu Y. Hendershot L.M. Characterization of an ERAD pathway for nonglycosylated BiP substrates, which requires Herp.Mol. Cell. 2007; 28: 544-554Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Herp also associates with the E3 ligase Hrd1 and the p97 AAA ATPase (Schulze et al., 2005Schulze A. Standera S. Buerger E. Kikkert M. van Voorden S. Wiertz E. Koning F. Kloetzel P.M. Seeger M. The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway.J. Mol. Biol. 2005; 354: 1021-1027Crossref PubMed Scopus (159) Google Scholar). At least two E3 mammalian ubiquitin ligases have been identified that show broad substrate specificity: gp78 (Fang et al., 2001Fang S. Ferrone M. Yang C. Jensen J.P. Tiwari S. Weissman A.M. The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum.Proc. Natl. Acad. Sci. USA. 2001; 98: 14422-14427Crossref PubMed Scopus (337) Google Scholar) and Hrd1 (Kikkert et al., 2004Kikkert M. Doolman R. Dai M. Avner R. Hassink G. van Voorden S. Thanedar S. Roitelman J. Chau V. Wiertz E. Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum.J. Biol. Chem. 2004; 279: 3525-3534Crossref PubMed Scopus (267) Google Scholar), with several additional E3 ligases, Rma1 (Matsuda et al., 2001Matsuda N. Suzuki T. Tanaka K. Nakano A. Rma1, a novel type of RING finger protein conserved from Arabidopsis to human, is a membrane-bound ubiquitin ligase.J. Cell Sci. 2001; 114: 1949-1957Crossref PubMed Google Scholar, Wang et al., 2008Wang L. Dong H. Soroka C.J. Wei N. Boyer J.L. Hochstrasser M. Degradation of the bile salt export pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II.Hepatology. 2008; 48: 1558-1569Crossref PubMed Scopus (64) Google Scholar, Delaunay et al., 2008Delaunay A. Bromberg K.D. Hayashi Y. Mirabella M. Burch D. Kirkwood B. Serra C. Malicdan M.C. Mizisin A.P. Morosetti R. et al.The ER-bound RING finger protein 5 (RNF5/RMA1) causes degenerative myopathy in transgenic mice and is deregulated in inclusion body myositis.PLoS ONE. 2008; 3: e1609https://doi.org/10.1371/journal.pone.0001609Crossref PubMed Scopus (52) Google Scholar), TEB4 (Hassink et al., 2005Hassink 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. TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum.Biochem. J. 2005; 388: 647-655Crossref PubMed Scopus (125) Google Scholar ; Zavacki et al., 2009Zavacki A.M. Arrojo E Drigo R. Freitas B.C. Chung M. Harney J.W. Egri P. Wittmann G. Fekete C. Gereben B. Bianco A.C. The E3 ubiquitin ligase TEB4 mediates degradation of type 2 iodothyronine deiodinase.Mol. Cell. Biol. 2009; 29: 5339-5347Crossref PubMed Scopus (66) Google Scholar), and parkin (Hassink et al., 2005Hassink 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. TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum.Biochem. J. 2005; 388: 647-655Crossref PubMed Scopus (125) Google Scholar, Wang et al., 2008Wang L. Dong H. Soroka C.J. Wei N. Boyer J.L. Hochstrasser M. Degradation of the bile salt export pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II.Hepatology. 2008; 48: 1558-1569Crossref PubMed Scopus (64) Google Scholar, Kitada et al., 1998Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism.Nature. 1998; 392: 605-608Crossref PubMed Scopus (3887) Google Scholar), showing much more limited substrate specificity. A particularly perplexing, unresolved issue is how so few E3 ligases can potentially be responsible for the disposal of any protein synthesized in the ER that fails to mature properly. To begin to understand how ERAD substrates might be recognized, we wished to identify the site(s) of ubiquitylation on a soluble ERAD substrate. For this study, we chose the nonsecreted NS-1 immunoglobulin κ LC (Skowronek et al., 1998Skowronek M.H. Hendershot L.M. Haas I.G. The variable domain of non-assembled Ig light chains determines both their half-life and binding to BiP.Proc. Natl. Acad. Sci. USA. 1998; 95: 1574-1578Crossref PubMed Scopus (82) Google Scholar, Okuda-Shimizu and Hendershot, 2007Okuda-Shimizu Y. Hendershot L.M. Characterization of an ERAD pathway for nonglycosylated BiP substrates, which requires Herp.Mol. Cell. 2007; 28: 544-554Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Ubiquitylation of proteins occurs in a multistep transfer between a series of ubiquitin ligases (Pickart and Eddins, 2004Pickart C.M. Eddins M.J. Ubiquitin: structures, functions, mechanisms.Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (925) Google Scholar, Scheffner et al., 1995Scheffner M. Nuber U. Huibregtse J.M. Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade.Nature. 1995; 373: 81-83Crossref PubMed Scopus (685) Google Scholar). The first step is the covalent attachment of the C-terminal glycine of ubiquitin to a cysteine on the E1 enzyme via a thioester bond. The next step is the transfer of ubiquitin from the E1 to a cysteine residue on one of many different E2s. The association of an E2 with an E3 provides the specificity of the reaction, as there are even more E3s, which interact specifically with a very limited group or in some cases even a single substrate (Pickart, 2001Pickart C.M. Mechanisms underlying ubiquitination.Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2755) Google Scholar). The E2/E3 pair most commonly transfers ubiquitin to the ɛ-amino group of a lysine residue on the substrate via an isopeptide bond (Pickart, 2001Pickart C.M. Mechanisms underlying ubiquitination.Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2755) Google Scholar). However, there are a number of instances where ubiquitin is placed on the N-terminal amino group of the substrate protein (Ciechanover and Ben-Saadon, 2004Ciechanover A. Ben-Saadon R. N-terminal ubiquitination: more protein substrates join in.Trends Cell Biol. 2004; 14: 103-106Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Notably, several viral E3s induce ubiquitylation of nonamino groups including the sulfhydral group on cysteine (Cadwell and Coscoy, 2005Cadwell K. Coscoy L. Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase.Science. 2005; 309: 127-130Crossref PubMed Scopus (303) Google Scholar) or hydroxyl groups serine/threonines (Wang et al., 2007Wang X. Herr R.A. Chua W.J. Lybarger L. Wiertz E.J. Hansen T.H. Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3.J. Cell Biol. 2007; 177: 613-624Crossref PubMed Scopus (203) Google Scholar) on their target proteins. Once the ubiquitin chain is elongated to about four or more molecules, the substrate can be recognized by the proteasome and degraded. We attempted to map the site(s) of ubiquitylation on the NS-1 LC using extensive mutagenesis coupled with enzymatic cleavage and chemical treatment. We found that it was apparently ubiquitylated predominantly on serine/threonine residues, but if these amino acids were mutated it was then modified on lysines. Mutation of all three types of amino acids was required to significantly reduce ubiquitylation and stabilize the NS-1 LC. Modification of all three amino acids was dependent on the Hrd1 E3 ligase. The nonsecreted NS-1 κ LC was previously shown to be an ERAD substrate that is degraded by the 26S proteasome (Knittler and Haas, 1992Knittler M.R. Haas I.G. Interaction of BiP with newly synthesized immunoglobulin light chain molecules: cycles of sequential binding and release.EMBO J. 1992; 11: 1573-1581Crossref PubMed Scopus (143) Google Scholar, Skowronek et al., 1998Skowronek M.H. Hendershot L.M. Haas I.G. The variable domain of non-assembled Ig light chains determines both their half-life and binding to BiP.Proc. Natl. Acad. Sci. USA. 1998; 95: 1574-1578Crossref PubMed Scopus (82) Google Scholar). Our previous data demonstrated that this LC is ubiquitylated, which occurs on a folding intermediate in which the VL domain is reduced but the CL domain remains oxidized (Okuda-Shimizu and Hendershot, 2007Okuda-Shimizu Y. Hendershot L.M. Characterization of an ERAD pathway for nonglycosylated BiP substrates, which requires Herp.Mol. Cell. 2007; 28: 544-554Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). We first confirmed the ubiquitylation of this substrate by treating the LC with SDS. The LC was still ubiquitylated, demonstrating that the attachment was covalent (see Figure S1A available online). We also used a rabbit antiserum to detect ubiquitin (Figure S1B), so that the secondary antibody would not interact directly with the NS-1 LC. Again, ubiquitin was detected on the immunoprecipitated LC, ruling out the possibility that the signal at the top of the blot represented aggregated LC instead of ubiquitylated LC. Finally, we used two linkage-specific antisera to probe the LC and found that the anti-K48 linkage antibody reacted with precipitated material but not the anti-K63 linkage antibody (Figure S1C). These three pieces of data, coupled with our previous 2D data showing that the ubiqutin signal emanates only from the partially oxidized (ox1) form of LC (Okuda-Shimizu and Hendershot, 2007Okuda-Shimizu Y. Hendershot L.M. Characterization of an ERAD pathway for nonglycosylated BiP substrates, which requires Herp.Mol. Cell. 2007; 28: 544-554Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), assured us that the NS-1 LC was indeed ubiquitylated. Thus, we began our studies by making a number of mutants in which the six lysine residues present in the VL domain were changed to arginine either singly or in various combinations, including one in which all six lysines were mutated (Figure 1 and Figure S2A). Mutant proteins lacking any (data not shown) or all of the lysines in the VL domain were still ubiquitylated (Figure 1) and were degraded by the 26S proteasome with kinetics similar to that of the wild-type protein, demonstrating that ubiquitylation of the lysines in the VL domain was not required for the degradation of this ERAD substrate. Although the CL domain remained oxidized and was unlikely to be ubiquitylated, we proceeded to mutate the seven lysines in this domain alone (K7-13R) or together with the lysines in the VL domain (K1-13R). Examination of these mutants revealed that even the lysine-less LC was still ubiquitylated and readily degraded by the proteasome (Figure 1). The K1-13R mutant was consistently degraded somewhat faster than the wild-type protein, which might reflect additional destabilization of the LC by these mutations. Thus, either lysine ubiquitylation does not occur on this ERAD substrate or it makes only a minor contribution to the ubiquitin signal and does not contribute to its degradation. As a number of cellular proteins are ubiquitylated at the N terminus, we examined this possibility using two approaches. First, we engineered a Factor Xa cleavage site four residues after the N terminus of the mature sequence of the NS-1 LC (Xa4) (Figure S2B). Ubiquitylated wild-type and Xa4 LCs were isolated and incubated with or without Factor Xa (Figure 2A ). Factor Xa recognizes the sequence Ile-Glu-Gly-Arg and cleaves immediately after the arginine (Nagai and Thogersen, 1987Nagai K. Thogersen H.C. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherchia coli.Methods Enzymol. 1987; 153: 461-481Crossref PubMed Scopus (342) Google Scholar). As expected, Factor Xa did not remove ubiquitin from wild-type LC that does not have a Xa cleavage site. When the Xa4 LC was similarly treated, there was no significant decrease in the ubiquitin signal, arguing that the N terminus either was not ubiquitylated or did not contribute significantly to the signal. As an alternative method to determine if N-terminal ubiquitylation contributed to the degradation of the NS-1 LC, we mutated the first two amino acids of the mature N terminus from Asn-Ile to either Ser-Asp or Ala-Asp. Cytosolic proteins possessing these residues are resistant to N-terminal ubiquitylation due to acetylation of these amino acids (Ciechanover and Ben-Saadon, 2004Ciechanover A. Ben-Saadon R. N-terminal ubiquitination: more protein substrates join in.Trends Cell Biol. 2004; 14: 103-106Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, Polevoda and Sherman, 2003Polevoda B. Sherman F. N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins.J. Mol. Biol. 2003; 325: 595-622Crossref PubMed Scopus (335) Google Scholar), although it is important to note that this has not been tested for a protein synthesized in the ER. In both cases, the protein was still ubiquitylated (Figure 2B) and turned over with kinetics very similar to that observed for the wild-type protein (Figure 2C). Together these two approaches argue that the NS-1 LC is not ubiquitylated at the N terminus either. Previous reports demonstrated that the MIR1 E3 ligase of the Kaposi's sarcoma-associated herpes virus ubiquitylates the cytosolic tail of the MHC I heavy chain on a single cysteine residue, which is sufficient to target this protein to the proteasome for degradation (Cadwell and Coscoy, 2005Cadwell K. Coscoy L. Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase.Science. 2005; 309: 127-130Crossref PubMed Scopus (303) Google Scholar). While this activity was performed by a viral E3, not a cellular E3, it does demonstrate that this linkage can occur and be recognized on an ERAD substrate, in addition to the well-characterized ubiquitylation of E1s and E2s on cysteine residues (Pickart and Eddins, 2004Pickart C.M. Eddins M.J. Ubiquitin: structures, functions, mechanisms.Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (925) Google Scholar). There are two cysteines available in the reduced VL domain of the ox1 form of LC that would be potential sites for ubiquitylation. These were mutated either alone (C23,88A) or in combination with the six lysines in the VL domain (K1-6R, C23,88A) (Figure S2C). Because the thioester bond can be broken with the β-mercaptoethanol present in reducing buffers, we analyzed the mutants under both reducing and nonreducing conditions. We found that the wild-type and mutant LCs were similarly ubiquitylated under both conditions (Figures 3A and 3B ). The cysteine mutants were degraded even more rapidly than the wild-type protein (Figure 3C), which may reflect the fact that these mutants are only present in the partially oxidized form that is the substrate for degradation (Okuda-Shimizu and Hendershot, 2007Okuda-Shimizu Y. Hendershot L.M. Characterization of an ERAD pathway for nonglycosylated BiP substrates, which requires Herp.Mol. Cell. 2007; 28: 544-554Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), as opposed to the wild-type NS-1 LC in which the fully oxidized (ox2) form must be converted to the partially oxidized (ox1) form for degradation. Given the unexpected findings obtained thus far, we wished to verify that ubiquitylation was restricted to the reduced VL domain before proceeding. To do so, we inserted a Factor Xa site at position 50 in the mature sequence (Xa50) of the VL domain and a second one just at the end of this domain at position 109 (Xa109) (Figure S2D). LCs were isolated from untreated and MG132-treated cells, and ubiquitylation was monitored before and after incubating with Factor Xa (Figure 4A ). In the case of the Xa50 mutant, Factor Xa treatment produced a band at ∼18 kDa. This was the expected size of the remaining LC, which could be recognized by the anti-κ antibody that is specific for the CL domain (Figure 4B). We found that removing the first 50 amino acids reduced the amount of ubiquitylation (Figure 4A) associated with the LC, arguing that at least one modified residue exists between amino acids 4 and 50. Cleavage of the Xa109 mutant produced an even smaller LC fragment of the expected size (Figure 4B) and resulted in almost complete removal of the ubiquitin signal from the remaining LC fragment (Figure 4A). In fact, the signal is nearly the same as that obtained with lysing buffer and immunoprecipitating antibody alone (Figure 4A, buffer). These data demonstrate that the NS-1 LC is ubiquitylated on at least two residues in the VL domain and that this domain possesses the majority, if not all, of the ubiquitylation sites. The mK3 E3 from the γ-herpes virus modifies serines and threonines on the cytosolic tail of the MHC I heavy chain and targets it for proteasomal degradation (Wang et al., 2007Wang X. Herr R.A. Chua W.J. Lybarger L. Wiertz E.J. Hansen T.H. Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3.J. Cell Biol. 2007; 177: 613-624Crossref PubMed Scopus (203) Google Scholar). Again, these data at least provide proof of principal for this type of modification on an ERAD substrate. However, unlike the cytosolic tail of the MHC I heavy chain, which contained only one threonine and four serines, the VL domain of the NS-1 LC has a total of 24 serine/threonine residues, making it an unwieldy target for PCR-induced mutagenesis. Therefore, because the ester bond found in this type of linkage is sensitive to high pH (Wang et al., 2007Wang X. Herr R.A. Chua W.J. Lybarger L. Wiertz E.J. Hansen T.H. Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3.J. Cell Biol. 2007; 177: 613-624Crossref PubMed Scopus (203) Google Scholar), we began by isolating the ubiquitylated LC and incubating it with and without NaOH (Figure 5A ). This treatment readily released ubiquitin from the NS-1 LC but only slightly reduced the background signal associated with the vector control (Figure 5A, lanes 2 and 3). Free ubiquitin cannot be detected under these conditions or with this antibody. Thus, we repeated the experiment on NS-1 LC isolated from the P3U.1 cells, but in this case the membrane was autoclaved before probing with an antibody that recognizes both free and bound ubiquitin. We detected a ladder of bands that reacted with the anti-ubiquitin antibody starting as small as three-ubiquitin chains but not with anti-κ LC (Figure S3A). No free ubiquitin was detected, arguing that this substrate was not monoubiquitylated and that the elongation of ubiquitin chains is likely to occur primarily through more conventional isopeptide bonds, in keeping with our data showing that K48 linkages could be detected (Figure S1C). The NaOH-eluted material was reprecipitated with either anti-ubiquitin or anti-κ LC and blotted for ubiquitin (Figure S3B). The strong signal obtained when the sample was both immunoprecipitated and blotted with anti-ubiquitin made it difficult to convincingly demonstrate that ubiquitin was released by this treatment, although the ubiquitin signal was clearly lost from LC. Thus, COS cells were transfected with His-tagged ubiquitin and the κ LC. In this case, nickel agarose was clearly able to reprecipitate the released His-tagged ubiquitin (Figure S3C). In toto, these data strongly suggest that this ERAD substrate is modified on a serine/threonine residue(s) in the VL domain. Given the largely unprecedented nature of these results, we synthesized a VL domain in which all 24 serines and threonines were mutated to alanine (ST−) and one in which they were mutated in combination with substituting the lysines in this domain with arginines (STK−). Cells were transfected with these two constructs, the wild-type NS-1 or the lysine-less VL domain mutant, and the effects on ubiq" @default.
• W2000694202 created "2016-06-24" @default.
• W2000694202 creator A5005203312 @default.
• W2000694202 creator A5068769982 @default.
• W2000694202 creator A5069199599 @default.
• W2000694202 date "2010-12-01" @default.
• W2000694202 modified "2023-10-13" @default.
• W2000694202 title "Ubiquitylation of an ERAD Substrate Occurs on Multiple Types of Amino Acids" @default.
• W2000694202 cites W1553768169 @default.
• W2000694202 cites W1563724009 @default.
• W2000694202 cites W1631620978 @default.
• W2000694202 cites W1668688100 @default.
• W2000694202 cites W1832030339 @default.
• W2000694202 cites W1877873625 @default.
• W2000694202 cites W1895779241 @default.
• W2000694202 cites W1971579210 @default.
• W2000694202 cites W1977498240 @default.
• W2000694202 cites W1981551685 @default.
• W2000694202 cites W1981625315 @default.
• W2000694202 cites W1986342078 @default.
• W2000694202 cites W1986675822 @default.
• W2000694202 cites W1987428238 @default.
• W2000694202 cites W2008044453 @default.
• W2000694202 cites W2017562400 @default.
• W2000694202 cites W2021918518 @default.
• W2000694202 cites W2023397454 @default.
• W2000694202 cites W2023662773 @default.
• W2000694202 cites W2029119458 @default.
• W2000694202 cites W2035349696 @default.
• W2000694202 cites W2045151558 @default.
• W2000694202 cites W2046933512 @default.
• W2000694202 cites W2049656093 @default.
• W2000694202 cites W2051094352 @default.
• W2000694202 cites W2051881845 @default.
• W2000694202 cites W2053462962 @default.
• W2000694202 cites W2057668553 @default.
• W2000694202 cites W2058209707 @default.
• W2000694202 cites W2064609154 @default.
• W2000694202 cites W2066537326 @default.
• W2000694202 cites W2079420530 @default.
• W2000694202 cites W2081104776 @default.
• W2000694202 cites W2083690210 @default.
• W2000694202 cites W2099386409 @default.
• W2000694202 cites W2105628355 @default.
• W2000694202 cites W2114366341 @default.
• W2000694202 cites W2117313136 @default.
• W2000694202 cites W2119363024 @default.
• W2000694202 cites W2121576917 @default.
• W2000694202 cites W2121812741 @default.
• W2000694202 cites W2123018879 @default.
• W2000694202 cites W2139577310 @default.
• W2000694202 cites W2146183177 @default.
• W2000694202 cites W2146969978 @default.
• W2000694202 cites W2149368193 @default.
• W2000694202 cites W2149789119 @default.
• W2000694202 cites W2150925238 @default.
• W2000694202 cites W2153693056 @default.
• W2000694202 cites W2157668086 @default.
• W2000694202 cites W2166440816 @default.
• W2000694202 cites W2171862076 @default.
• W2000694202 cites W2220626911 @default.
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