3.A.16 The Endoplasmic Reticular Retrotranslocon (ER-RT) Family
Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are degraded in the cytoplasm of eukaryotic cells by proteosomes after translocation across the membrane by a retrotranslocon. The process involves recognition of a substrate in the ER lumen, translocation through the ER membrane, and binding to the cytosolic p97 ATPase (Cdc48 in yeast) which pulls the misfolded protein through the membrane at the expense of ATP hydrolysis. In mammals, the p97-interacting membrane protein is Derlin-1 (Der1 in yeast). Derlin-1, an integral membrane protein with 5 or 6 TMSs, interacts with the substrate proteins as they move through the membrane. Inactivation of Derlin-1 in C. elegans causes ER stress. Derlin-1 interacts with a virally-encoded ER protein that targets MHC class I heavy chains for export from the ER as well as VIMP (selenoprotein S; VPC-interacting protein; a 1 TMS ER protein), which recruits P97 and its cofactor proteins, Ufd1 (ubiquitin fusion degradation 1 protein) and Npl4. Homologues of Derlin-1 are found in all types of eukaryotes and many contain multiple paralogues. Distant homologues may be present in bacteria. These homologues include the DNA internalization-related competence protein, ComEC of Enterococcus faecalis (AAO82165), homologous to the B. subtilis ComEC protein (TC #3.A.11.1.1).The ER-RT family is also called the ER-associated degradation (ERAD) transport apparatus (Bolte et al., 2011). TMS hydrophobicity is an energetic barrier during the retrotranslocation of transmembrane ERAD substrates (Guerriero et al. 2017).
The ER retrotranslocon interacts with the US11 protein of human cytomegalovirus (HCMV) to target newly synthesized major histocompatibility complex (MHC) class I heavy chains for retro-translocation. This allows the virus to selectively destroy cellular proteins required for immune defense of the host. Thus, the retrotranslocon is important for the establishment of viral infections. It plays a role in other human diseases as well.
Derlins (Der1, 2, and 3) are functional components of ERAD for misfolded lumenal proteins, and may act by
forming a channel that allows the retrotranslocation of misfolded
glycoproteins into the cytosol where they are ubiquitinated and degraded
by the proteasome. They may mediate the interaction between VCP and
misfolded glycoproteins (Lilley and Ploegh 2005, Oda et al. 2006). They may also be involved in ER stress-induced
pre-emptive quality control, a mechanism that selectively attenuates the
translocation of newly synthesized proteins into the endoplasmic
reticulum and reroutes them to the cytosol for proteasomal degradation (Kadowaki et al. 2015). The involvement of derlins in protein translocation across the ER membrane has been confirmed after some controversy (Neal et al. 2018).
A complex involving Derlin-1 and p97 mediates the retrotranslocation and endoplasmic reticulum (ER)-associated degradation of misfolded proteins in yeast and is used by certain viruses to promote host cell protein degradation (Romisch, 2005). Derlin-1 and p97 form complexes with non-ubiquitylated CFTR in human airway epithelial cells. Derlin-1 interacted with CFTR, whereas p97 associated with ubiquitylated CFTR. Exogenous expression of Derlin-1 led to its co-localization with CFTR in the ER where it reduced wild type (WT) CFTR expression and efficiently degraded the disease-associated CFTR folding mutants (>90%). Thus, Derlin-1 recognizes misfolded, non-ubiquitylated CFTR to initiate its dislocation and degradation early in the course of CFTR biogenesis, perhaps by detecting structural instability within the first transmembrane domain (Sun et al., 2006).
Cholera toxin (CT) intoxicates cells by using its receptor-binding B subunit (CTB) to traffic from the plasma membrane to the endoplasmic reticulum (ER). In this compartment, the catalytic A1 subunit (CTA1) is unfolded by protein disulfide isomerase (PDI) and retro-translocated to the cytosol where it triggers a signaling cascade leading to secretory diarrhea. Using a semipermeabilized-cell retro-translocation assay, Bernardi et al., (2007) demonstrated that a dominant-negative Derlin-1-YFP fusion protein attenuates the ER-to-cytosol transport of CTA1. Derlin-1 interacts with CTB and the ER chaperone PDI as assessed by coimmunoprecipitation experiments. An in vitro membrane-binding assay showed that CTB stimulated the unfolded CTA1 chain to bind to the ER membrane. Moreover, intoxication of intact cells with CTB stabilized the degradation of a Derlin-1-dependent substrate, suggesting that CT uses the Derlin-1 pathway. Thus, Derlin-1 facilitates the retro-translocation of CT. CTB may play a role in this process by targeting the holotoxin to Derlin-1, enabling the Derlin-1-bound PDI to unfold the A1 subunit and prepare it for transport (Bernardi et al., 2008).
Misfolded polytopic membrane proteins can be extracted from the ER, and the process involves the ER retrotranslocon. Chaperones play a role, and there is requirement for Ufd2p, a ubiquitin chain extension enzyme, during membrane protein quality control (Nakatsukasa et al., 2008).
The ERAD-machinery is well studied in Saccharomyces cerevisiae, where three different modes of ERAD complexes are utilized depending on the substrate (Carvalho et al. 2006; Bolte et al. 2011). Whereas the ERAD-L system is responsible for retro-translocation of soluble proteins and membrane proteins with misfolded lumenal regions, ERAD-M and ERAD-C mediate retro-translocation of membrane proteins possessing misfolded sections in transmembrane domains (ERAD-M) or in cytosolic domains (ERAD-C) of membrane proteins, respectively. For soluble ERAD-L substrates, a complete translocation from the ER lumen into the cytosol occurs.
Aberrant proteins are recognised within the ER lumen as ERAD-L substrates. Following recognition by a soluble receptor protein (such as Yos9p in the case of glycoproteins), this complex is bound by the membrane receptor protein Hrd3p - a protein with a large luminal domain comprising multiple TPR motifs. In the next step, the substrate is presumably inserted into a translocation channel, the identity of which was elusive in 2011. Prominent candidates for forming such a channel are the Sec61p complex, the ubiquitin-ligase Hrd1p and the membrane protein Der1p.
Once inserted into the channel, substrate translocation involves ubiquitination by E1, E2 and E3 enzymes on the cytosolic side. Ubiquitinated substrates are subsequently bound by the ATPase Cdc48p, which provides the energy for pulling the proteins out of the ERAD-L translocation channel (Fig. 1A in Bolte et al., 2011). A central player for substrate ubiquitination in the ERAD-L pathway is the E3 enzyme Hrd1p, a RING-finger ubiquitin ligase with six transmembrane helices. It has been shown to interact with the membrane receptor Hrd3p and the membrane protein Usa1p - an adaptor for Hrd1p and Der1p interaction. The catalytic RING-H2 domain of Hrd1p is located on the cytoplasmic side of the ER and catalyses the transfer of ubiquitin to lysine residues of ERAD-L substrates. Prior activation of ubiquitin is mediated by the ubiquitin-activating protein Uba1p , followed by subsequent transfer to the ubiquitin conjugating enzymes, Ubc1p and Ubc7p [37, 38]. The ubiquitin ligase Hrd1p binds both the ERAD-substrate and the Ubc protein and catalyses the transfer of ubiquitin to the substrate . This ubiquitination process is essential for proteasomal degradation but, additionally, it is vital for the retro-translocation process itself [40, 41]. Ubiquitinated ERAD-L substrates are specifically bound to and extracted by the cytosolic ATPase, called Cdc48p in yeast or p97 in mammals, thereby completing the process of retro-translocation. Cdc48p/p97 belongs to the AAA ATPase family. Together with the ERAD-specific co-factors Ufd1p and Npl4p, Cdc48p/p97 functions in the context of ERAD [42, 45, 49]. The recruitment of the ATPase to the ER membrane is supported by the membrane protein Ubx2p, enabling Cdc48p/p97 to exert mechanical force for membrane release of ERAD-L substrates 50, 51.
During endoplasmic reticulum-associated degradation (ERAD), misfolded lumenal and membrane proteins in the ER are recognized by the transmembrane Hrd1 ubiquitin ligase complex and retrotranslocated to the cytosol for ubiquitination and degradation. Substrates are believed to be delivered to the proteasome only after the ATPase Cdc48p/p97 acts. Nakatsukasa et al. 2013 provided evidence that inactivation of Cdc48p/p97 stalls retrotranslocation and triggers formation of a complex that contains the 26S proteasome, Cdc48p/p97, ubiquitinated substrates, select components of the Hrd1 complex, and the lumenal recognition factor, Yos9p. Possibly the actions of Cdc48p/p97 and the proteasome are tightly coupled during ERAD, and the Hrd1 complex links substrate recognition and degradation on opposite sides of the ER membrane.
The reaction catalyzed by the ER-RT is:
misfolded protein (ER lumen) → misfolded protein (cytosol)