title: Ubiquitylation of an ERAD substrate occurs on multiple types of amino acids.
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.
authors.affiliation: Department of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Shimizu
authors.forename: Yuki
authors.initials: Y
authors.lastname: Okuda-Shimizu
authors.forename: Linda M
authors.initials: LM
authors.lastname: Hendershot
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007145:Immunoglobulin kappa-Chains / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D012972:Sodium Hydroxide / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination
chemical_list: D007145:Immunoglobulin kappa-Chains; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D013912:Threonine; D012694:Serine; D012972:Sodium Hydroxide; C104621:HRD1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D008239:Lysine; D003545:Cysteine