Fig. 3

The four main steps for ERAD. I. Recognition occurs during protein synthesis. Here a misfolded region (red stars) are recognized by either cytoplasmic, ER luminal and/or transmembrane recognition factors depending on the site of lesion. II. Polyubiquitination starts when chaperones and co-chaperones direct the misfolded substrate to ubiquitination machinery. An ubiquitin activating enzyme (E1) transfers ubiquitin (Ub) (grey circles) to cysteine residue in an active site of an ubiquitin conjugating enzyme (E2) using ATP as energy. Ubiquitin ligase then transfers Ub to a lysine residue on the substrate protein. The latter process occurs on either the ER or cytoplasmic side of the membrane. III. Retrotranslocation ensues when the substrate protein is escorted to the dislocation machinery made up of a protein scaffold such as SEL1L adaptor subunit of ERAD E3 ubiquitin ligase (SEL1L), synoviolin 1 (SYVN1), cytochrome c oxidase assembly factor 7 (COA7) (not shown), derlin 1,2,3 (DERL1,2,3), selenoprotein S (SELENOS), homocysteine inducible ER protein with ubiquitin like domain 1 (HERPUD1), and valosin-containing protein (VCP). The substrate protein is removed either by passing through a retrotranslocon or by complete elimination of the protein. This is mainly done by the cytoplasmic ATPases associated with diverse cellular activities (AAA⁺ ATPase) p97 (commonly known as VCP), which interacts with Ub on the substrate and de-ubiquitinates the mutant protein and sends it off to the 26S proteasome. IV. Degradation is the final step where polyubiquitinated substrates are escorted to the 26S proteasome for degradation of faulty proteins. N-glycans are cleaved off by peptide N-glycanase associated with the ERAD machinery and Ub moieties are removed by de-ubuitinating enzymes found in the cytoplasm or in the proteasome cap to release small peptides shown as blue triangles (Milhem 2015)