Ubiquitylation of an ERAD substrate occurs on multiple types of amino acids.

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.

[1]  J. Boyer,et al.  Degradation of the bile salt export pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II , 2008, Hepatology.

[2]  Tom A. Rapoport,et al.  Distinct Ubiquitin-Ligase Complexes Define Convergent Pathways for the Degradation of ER Proteins , 2006, Cell.

[3]  J. Brodsky,et al.  The Requirement for Molecular Chaperones during Endoplasmic Reticulum-associated Protein Degradation Demonstrates That Protein Export and Import Are Mechanistically Distinct* , 1999, The Journal of Biological Chemistry.

[4]  Stephen N. Jones,et al.  The ER-Bound RING Finger Protein 5 (RNF5/RMA1) Causes Degenerative Myopathy in Transgenic Mice and Is Deregulated in Inclusion Body Myositis , 2008, PloS one.

[5]  R. Gardner,et al.  HRD gene dependence of endoplasmic reticulum-associated degradation. , 2000, Molecular biology of the cell.

[6]  R. Hitt,et al.  Use of Modular Substrates Demonstrates Mechanistic Diversity and Reveals Differences in Chaperone Requirement of ERAD* , 2003, Journal of Biological Chemistry.

[7]  Anna M. Keller,et al.  Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its N-terminal fragment , 2007, The Journal of cell biology.

[8]  K. Fröhlich,et al.  AAA-ATPase p97/Cdc48p, a Cytosolic Chaperone Required for Endoplasmic Reticulum-Associated Protein Degradation , 2002, Molecular and Cellular Biology.

[9]  S. Minoshima,et al.  Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism , 1998, Nature.

[10]  L. Hendershot,et al.  The variable domain of nonassembled Ig light chains determines both their half-life and binding to the chaperone BiP. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[11]  T. Rapoport,et al.  A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol , 2004, Nature.

[12]  J. Roth,et al.  Multiple Lysine Mutations in the C-Terminal Domain of p53 Interfere with MDM2-Dependent Protein Degradation and Ubiquitination , 2000, Molecular and Cellular Biology.

[13]  Martin Scheffner,et al.  Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade , 1995, Nature.

[14]  H. Ploegh,et al.  A membrane protein required for dislocation of misfolded proteins from the ER , 2004, Nature.

[15]  A. Ciechanover,et al.  N-terminal ubiquitination: more protein substrates join in. , 2004, Trends in cell biology.

[16]  Toshiyuki Miyata,et al.  Herp, a New Ubiquitin-like Membrane Protein Induced by Endoplasmic Reticulum Stress* , 2000, The Journal of Biological Chemistry.

[17]  A. Nakano,et al.  Rma1, a novel type of RING finger protein conserved from Arabidopsis to human, is a membrane-bound ubiquitin ligase. , 2001, Journal of cell science.

[18]  C. Pickart,et al.  Ubiquitin: structures, functions, mechanisms. , 2004, Biochimica et biophysica acta.

[19]  R. Plemper,et al.  Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation , 1997, Nature.

[20]  C. Pickart,et al.  Mechanisms underlying ubiquitination. , 2001, Annual review of biochemistry.

[21]  H. Ploegh,et al.  SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER , 2006, The Journal of cell biology.

[22]  R. Kaufman,et al.  Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation , 2006, The Journal of cell biology.

[23]  J. Harney,et al.  The E3 Ubiquitin Ligase TEB4 Mediates Degradation of Type 2 Iodothyronine Deiodinase , 2009, Molecular and Cellular Biology.

[24]  Jeffrey L. Brodsky,et al.  One step at a time: endoplasmic reticulum-associated degradation , 2008, Nature Reviews Molecular Cell Biology.

[25]  J. Bonifacino,et al.  Serine Residues in the Cytosolic Tail of the T-cell Antigen Receptor α-Chain Mediate Ubiquitination and Endoplasmic Reticulum-associated Degradation of the Unassembled Protein* , 2010, The Journal of Biological Chemistry.

[26]  E. Wiertz,et al.  Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degradation substrates , 2009, The Journal of cell biology.

[27]  M. Ferrone,et al.  The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[28]  L. Hendershot,et al.  BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. , 1999, Molecular biology of the cell.

[29]  P. Kloetzel,et al.  The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway. , 2005, Journal of molecular biology.

[30]  M. Knittler,et al.  Interaction of BiP with newly synthesized immunoglobulin light chain molecules: cycles of sequential binding and release. , 1992, The EMBO journal.

[31]  E. Wiertz,et al.  Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3 , 2007, The Journal of cell biology.

[32]  M. Bogyo,et al.  The Human Cytomegalovirus US11 Gene Product Dislocates MHC Class I Heavy Chains from the Endoplasmic Reticulum to the Cytosol , 1996, Cell.

[33]  M. Knop,et al.  Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. , 1996, The EMBO journal.

[34]  M. Hochstrasser,et al.  A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation. , 2001, Genes & development.

[35]  D. Wolf,et al.  ER Degradation of a Misfolded Luminal Protein by the Cytosolic Ubiquitin-Proteasome Pathway , 1996, Science.

[36]  I. Wada,et al.  Enhancement of Endoplasmic Reticulum (ER) Degradation of Misfolded Null Hong Kong α1-Antitrypsin by Human ER Mannosidase I* , 2003, Journal of Biological Chemistry.

[37]  L. Hendershot,et al.  Characterization of an ERAD pathway for nonglycosylated BiP substrates, which require Herp. , 2007, Molecular cell.

[38]  T. Rapoport,et al.  JCB Article , 2001 .

[39]  J. Brodsky,et al.  Proteasome-dependent endoplasmic reticulum-associated protein degradation: An unconventional route to a familiar fate , 1996 .

[40]  T. Sommer,et al.  ERAD: the long road to destruction , 2005, Nature Cell Biology.

[41]  Ari Helenius,et al.  Quality control in the endoplasmic reticulum , 2003, Nature Reviews Molecular Cell Biology.

[42]  C. Joazeiro,et al.  Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation , 2000, Nature Cell Biology.

[43]  A. Amerik,et al.  Mechanism and function of deubiquitinating enzymes. , 2004, Biochimica et biophysica acta.

[44]  K. Nagai,et al.  Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. , 1987, Methods in enzymology.

[45]  A. Le,et al.  Intracellular degradation of the transport-impaired human PiZ alpha 1-antitrypsin variant. Biochemical mapping of the degradative event among compartments of the secretory pathway. , 1990, The Journal of biological chemistry.

[46]  F. Sherman,et al.  N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins. , 2003, Journal of molecular biology.

[47]  K. Früh,et al.  TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum. , 2005, The Biochemical journal.

[48]  D. Hebert,et al.  EDEM1 recognition and delivery of misfolded proteins to the SEL1L-containing ERAD complex. , 2009, Molecular cell.

[49]  K. Cadwell,et al.  Ubiquitination on Nonlysine Residues by a Viral E3 Ubiquitin Ligase , 2005, Science.

[50]  Sumire V. Kobayashi,et al.  Cytosolic Degradation of T-cell Receptor α Chains by the Proteasome* , 1997, The Journal of Biological Chemistry.

[51]  Wei Li,et al.  A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate , 2007, Nature.

[52]  A. Weissman,et al.  The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[53]  V. Chau,et al.  Human HRD1 Is an E3 Ubiquitin Ligase Involved in Degradation of Proteins from the Endoplasmic Reticulum* , 2004, Journal of Biological Chemistry.