Pathogenic Hijacking of ER-Associated Degradation: Is ERAD Flexible? Daisuke Morito, Kazuhiro Nagata  Molecular Cell  Volume 59, Issue 3, Pages 335-344 (August 2015) DOI: 10.1016/j.molcel.2015.06.010 Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Invasion Pathways of Viruses and Protein Toxins (A and B) A polyomavirus first attaches to the host cell surface via an association with glycolipid ganglioside and is then internalized by caveolae-mediated endocytosis and directly transported to the ER. The virus appears to be transported using ERAD components, although the mechanism remains largely unknown. (C and D) Protein toxins invade the cell through endocytosis after associating with specific cell surface receptors and finally reach the ER lumen through the Golgi apparatus. Toxins are then transported from the ER to the cytosol through combined usage of the Sec61 complex and ERAD components. Molecular Cell 2015 59, 335-344DOI: (10.1016/j.molcel.2015.06.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Oligosaccharide-Chain Processing Associates with Protein Maturation and Degradation The polypeptide is first modified with the G3M9 form of an oligosaccharide chain, which contains two N-acetylglucosamines (GlcNAc), nine mannoses, and three glucoses. The G3M9 form is processed into the G1M9 form by glucosidase I (Glu I) and II (Glu II). The G1M9 form is then specifically recognized by the molecular chaperones calnexin and calreticulin. Either calnexin or calreticulin promotes folding of the polypeptide until the oligosaccharide chain is further processed into the G0 form by Glu II. When the polypeptide remains unfolded, UDP-glucose:glycoprotein glucosyltransferase (UGGT) again attaches glucose to the G0M9 form and thus restores the G1M9 form. Hence, calnexin and calreticulin can again recognize it and promote folding. ER mannosidase I (ER Man I) and ERAD-enhancing mannosidase-like protein (EDEM) 2 process the M9 form into the M8 form. EDEM3 further processes this into the M5–7 forms. The M5–8 forms are recognized by EDEM1 and Osteosarcoma 9 (OS9) and recruited to the ERAD machinery. By contrast, successfully folded polypeptides are properly secreted via the secretory pathway. Molecular Cell 2015 59, 335-344DOI: (10.1016/j.molcel.2015.06.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Rough Scheme of Polypeptide Relay from De Novo Synthesis to ERAD (A) The polypeptide is first synthesized by an ER membrane-associated ribosome and is co-translationally transported into the ER lumen through the Sec61 translocon. Most polypeptides are modified in the ER with oligosaccharide chains and disulfide bonds by specific enzymes such as ER oxidoreductin 1 (ERO1) and PDI during their translation and folding. Immunoglobulin heavy-chain-binding protein (BiP) is a molecular chaperone that promotes polypeptide folding. (B) When a polypeptide fails to be folded properly, it is isolated by lectins and ERAD components, such as EDEM1, OS9, ERdj5, BiP, and SEL1L, and recruited to the so-called ERAD complex, which involves HRD1, Derlin-1, Ubc6e, Ubc7, and the p97 complex. (C) The targeted polypeptide is somehow transported into the cytosol through the ERAD complex. The polypeptide is poly-ubiquitylated during its dislocation and further extracted into the cytosol by the p97 complex. HRD1, which acts as a ubiquitin ligase, and Derlin-1 are candidates for the dislocation channel. (D) The oligosaccharide portion is processed by peptide:N-glycanase (PNGase), and then the protein portion is degraded into short peptides by the proteasome. Molecular Cell 2015 59, 335-344DOI: (10.1016/j.molcel.2015.06.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Maturation and Degradation of MHC Class I Molecules (A) The proteasome degrades endogenous proteins into short peptides. (B) Peptides are transported into the ER through the transporter associated with antigen processing (TAP) complex. Calreticulin, ERp57, and tapasin mediate assembly of MHC class I molecules, which consist of MHC class I heavy chain (HC), β2 macroglobulin (β2 m), and the transported peptide antigen. (C) Maturated MHC class I molecules are transported to the cell surface. (D) Thereafter, the complex mediates antigen presentation. (E) However, virus US11 protein extracts the HC molecule from the maturation pathway and targets it to ERAD through a direct interaction with Derlin-1. Thus, US11 can prevent MHC class I molecule-mediated antigen presentation. Cs, cytosol; ExC, extracellular space. Molecular Cell 2015 59, 335-344DOI: (10.1016/j.molcel.2015.06.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Colorful Modalities of Pathogenic Exploitation Suggest Plasticity and Potential Flexibility of Physiological ERAD Machinery (A) SEL1L, HRD1, and Derlins are considered to compose a core complex in physiological ERAD. OS9 and BiP mediate substrate recognition and recruitment. p97 and its cofactors, Npl4 and Ufd1, provide a driving force for dislocation. Usage of the Sec61 complex is currently very controversial. (B) HCMV-encoded US2 physically associates with MHC class I heavy chain (HC) and promotes ERAD in conjunction with BiP (Hegde et al., 2006), the Sec61 translocon (Wiertz et al., 1996b), and p97 (Soetandyo and Ye, 2010). However, US2 does not require SEL1L (Mueller et al., 2006), Derlin-1 (Lilley and Ploegh, 2004), Ufd1, or Npl4 (Soetandyo and Ye, 2010). Instead of HRD1, US2 utilizes the ER membrane-spanning ubiquitin ligase TRC8 (Stagg et al., 2009). US2 further exploits PDI (Lee et al., 2010) and PNGase (Blom et al., 2004). The involvement of signal peptide peptidase is currently controversial (Boname et al., 2014; Lee et al., 2010; Loureiro et al., 2006). (C) HCMV-encoded US11 directly associates with MHC class I HC and targets it to the ERAD pathway in cooperation with BiP (Hegde et al., 2006), SEL1L (Mueller et al., 2006), Derlin-1 (Lilley and Ploegh, 2004; Ye et al., 2004), VCP-interacting membrane protein (VIMP) (Ye et al., 2004), p97, Ufd1, and Npl4 (Ye et al., 2001), whereas US11 uses the ubiquitin ligase TMEM129 (van de Weijer et al., 2014; van den Boomen et al., 2014) instead of HRD1. (D) MHV68-encoded viral ubiquitin ligase mK3 leads to ERAD of MHC class I HC in cooperation with endogenous tapasin, a TAP (Lybarger et al., 2003), Derlin-1, and p97 (Wang et al., 2006). The involvement of SEL1L, BiP, and OS9 is unknown. (E) HIV-encoded virus protein U (Vpu) leads to ERAD of CD4 with p97, Npl4, Ufd1 (Magadán et al., 2010), and the cytosolic complex ubiquitin ligase β-TrCP (Margottin et al., 1998). A yeast study suggested that the endogenous ERAD factors Hrd1p, Hrd3p (an ortholog of mammalian SEL1L), and Ubc7p are not involved in this system (Meusser and Sommer, 2004). No ER luminal or membrane-associated components, which support retrograde transport of CD4, except for Vpu itself have been identified. (F) Cholera toxin (CTx) is targeted to Derlin-1, HRD1, and/or the HRD1 homologous ubiquitin ligase gp78 (Bernardi et al., 2008, 2010). This complex also includes ERdj5 and SEL1L (Williams et al., 2013). Unfolding by PDI is essential prior to retrotranslocation (Inoue et al., 2011). In addition, CTx possibly exploits the Sec61 translocon (Schmitz et al., 2000), TorsinA (Nery et al., 2011), p97 (Abujarour et al., 2005; Kothe et al., 2005), and hsp90 (Taylor et al., 2010). (G) Retrotranslocation of ricin A chain is mediated by a set of common factors including HRD1, SEL1L, and Derlin-2 (Eshraghi et al., 2014; Redmann et al., 2011), whereas the ubiquitin ligase activity of HRD1 is likely not required (Li et al., 2010). The driving force is likely provided by the ATPase subunit of the proteasome, not by p97 (Li et al., 2010). Calreticulin and EDEM1 are also involved in this pathway (Day et al., 2001; Slominska-Wojewodzka et al., 2006). (H) Prior to membrane penetration, simian virus 40 (SV40) is remodeled by PDI and ERp57 into the penetration-competent state (Schelhaas et al., 2007). In addition to SEL1L and Derlin-1 (Schelhaas et al., 2007), DnaJB12, DNAJB14, SGTA, hsc70 (Walczak et al., 2014), BiP, RMA1, BAP29, and BAP31 (Geiger et al., 2011) contribute to viral membrane penetration. Other ERAD-related factors including HRD1, Herp, Sec61β, TRAM1, EDEM1, and p97 are dispensable for this process (Geiger et al., 2011). The molecular mechanism by which the abovementioned components support viral penetration remains completely unknown. Molecular Cell 2015 59, 335-344DOI: (10.1016/j.molcel.2015.06.010) Copyright © 2015 Elsevier Inc. Terms and Conditions