A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway

The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor α (TCRα) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCRα occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCRα in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCRα is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCRα peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCRα remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms. 1. Introduction Approximately one-third of newly synthesized proteins are targeted for the degradation by the proteasome within minutes of their synthesis as a result of premature termination or misfolding. ER-associated degradation (ERAD) is a specialized pathway that targets misfolded nascent proteins in the endoplasmic reticulum (ER) for degradation via the proteasome [1]. In the ER, misfolded proteins are recognized by several quality control mechanisms that escort them to a channel for retrotranslocation to the cytosol. Soon after retrotranslocation, these proteins are ubiquitinated and extracted from the ER membrane with the help of the Cdc48p/p97 complex. Ubiquitination acts as a signal to target proteins to the proteasome and thus is an essential process in the degradation of newly synthesized defective proteins. Ubiquitination is a post-translational modification that regulates many cellular events such as protein degradation, endocytosis, and the cell cycle. The process of ubiquitination involves the concerted action of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase. The last