1. Introduction
1.8 Endoplasmic reticulum quality control
Proteins that need to be secreted or inserted into membranes are cotranslationally transported into the Endoplasmic Reticulum (ER) where their folding and assembly is monitored by the Endoplasmic reticulum quality control (ERQC). The signal recognition particle (SRP) binds signal sequences in newly synthesized polypeptides in the cytosol and
thereby arrests translation (Walter and Blobel 1981). The SRP is recognized by a receptor in the ER membrane which brings it to the translocon, a complex consisting of Sec61, and (Deshaies, Sanders et al. 1991). After its release the ribosome starts again translation of the polypeptide through the translocon pore into the ER lumen where it is bound by chaperones to assist folding. Subsequently the signal sequence of the polypeptide is cleaved by signal peptidases (Jackson and Blobel 1977) and the preformed oligosaccharide consisting of three glucose, nine mannose and two N‐acetylglucosamin residues (Glc3Man9GlcNAc2) is added to an asparagine. This oligosaccharide is directly trimmed by glucosidase I and II to GlcMan9GlcNAc2 so that the lectin‐like chaperones calnexin (CNX) and calreticulin (CRT) can interact with the polypeptide to prevent aggregation and promote proper folding (Rodan, Simons et al. 1996). The CNX/CRT interacting oxidoreductase ERp57 catalyzes the formation of disulphide bonds between the cysteines of the protein which is released upon removal of the last glucose by glucosidase II. The UDP‐glucose/glycoprotein glucosyl transferase (UGGT) controls the folding state of the protein and adds glucose again to repeat the assisted folding.
Correctly folded proteins are transported to their destination (Araki and Nagata 2011).
1.8.1. ERAD
When proteins ultimately can’t be folded correctly they have to be removed from the ER to prevent their accumulation. This process is called ER‐associated degradation (ERAD) and at least in yeast it can be divided into ERAD‐C (cytosol), ERAD‐M (membrane) and ERAD‐L (lumen) depending on the localisation of the substrate and therefore on the involved enzymes (Carvalho, Goder et al. 2006). In mammals these pathways are defined by complexes containing the ubiquitin E3 ligases Ring finger protein (RNF) 5 (or RMA1), glycoprotein (gp) 78 or HMG‐CoA reductase degradation (Hrd) 1 which share some cofactors and partially target
the same substrates (Morito, Hirao et al. 2008). During repeated folding cycles the probability of the glycosylated proteins to be exposed to ER‐mannosidase I increases. Trimmed oligosaccharides are recognized by ER degradation‐enhancing ‐mannosidase like (EDEM) proteins which can cut off even more mannosidase residues. They hand over the substrate proteins to the chaperone Binding immunoglobulin Protein (BiP, GRP78) which retains the substrate until retrotranslocation into the cytosol in an unfolded state. The reduced glycosylation increases hydrophobicity of the proteins which is recognized by the lectins OS‐9 and XTB‐3B. Abnormal non‐glycosylated proteins are also directly recognized by BiP and co‐
chaperones of the DnaJ family without the help of EDEM proteins. BiP, XTB‐3B and OS‐9 interact via SEL1L with the transmembrane HRD1 ubiquitin E3 ligase complex. Derlin‐1, which is associated with Hrd1 or gp78, interacts with Sec61 and promotes the transport of the substrate through the translocon into the cytosol. On the cytoplasmic site of the ligase complex the associated E1 UBA1 and the E2 UBC6e (Ube2j1) start the enzyme cascade resulting in the ubiquitination of the substrate mostly at serines or threonines. UBXD8 and UBXD2 are membrane bound cofactors which recruit the Valosin‐containing protein (VCP, p97) to the ligase complex. VCP is an ATPase involved in the release of the translocated ERAD substrates in order to make them accessible to degradation by the proteasome. Although VCP can also interact with Hrd1 and gp78 directly, it needs its cofactors Ufd1 and Npl4 to deliver the ubiquitinated substrates for processing. Additionally the deubiquitinating enzymes Ataxin‐
3, VCIP135 and YOD1 associate with VCP to modify ubiquitination of either substrates or parts of the ERAD machinery (reviewed in (Araki and Nagata 2011, Christianson and Ye 2014). When the substrates are translocated into the cytosol they are handed over to ubiquitin receptors like Rad23 or Dsk2 which bind to the proteasome to deliver the substrate for degradation (Medicherla, Kostova et al. 2004). There are some characteristic proteins like the T‐cell receptor ‐chain (TCR), ‐1 antitrypsin (1AT) or the cystic fibrosis transmembrane conductance regulator (CFTR) which can be used as model substrates to study ERAD‐related processes (reviewed in (Baek, Cheng et al. 2013)).
1.8.2. Unfolded‐protein response
In case of accumulation of proteins that don’t fold properly because of mutations or stress conditions and thus can’t be exported from the ER, the unfolded‐protein response (UPR) takes place to reduce ER stress. Three principal branches of the UPR have been defined by the ER‐
resident signalling components as inositol requiring enzyme (IRE) 1, doublestranded RNA‐
activated protein kinase (PKR)‐like ER kinase (PERK) and activating transcription factor (ATF) 6 pathway. They employ different mechanisms for reducing the amount of unfolded proteins by translational control or production of helpful proteins in order to restore protein homeostasis and normal ER function (reviewed in (Walter and Ron 2011)).
ATF6 is a transmembrane protein with a large ER‐luminal domain bound to the chaperone BiP.
Upon accumulation of unfolded proteins ATF6 is released from BiP and transported from the ER to the Golgi. ATF6 is cut by the Golgi‐resident site‐1 protease (S1P) and subsequently by S2P next to the membrane which releases the N‐terminal part of ATF6 into the cytosol. This N‐terminus acts as a transcription factor for BiP, glucose‐regulated protein (GRP) 94 and other ER‐resident chaperones (Walter and Ron 2011).
During ER stress the transmembrane kinase PERK oligomerizes, phosphorylates itself and inhibits the translation initiation factor eIF2 by phosphorylation. Translation of most proteins is stopped to reduce the burden of protein folding in the ER. However, some mRNAs are particularly translated when eIF2 is inactive, e.g. the transcription factor ATF4 which targets the transcription factor C/EBP homologous protein (CHOP) and growth arrest and DNA
damage‐inducible (GADD) 34. CHOP induces expression of genes involved in apoptosis, whereas GADD34 is a subunit of the protein phosphatase PP1C which dephosphorylates eIF2
thus restoring its normal activity (Walter and Ron 2011).
IRE1 is a transmembrane protein with kinase and endoribonuclease activity. Unfolded proteins lead to oligomerization and autophosphorylation of IRE1. This activates its nuclease activity towards the mRNA of the X‐box binding protein (XBP) 1 excising one intron. The translated protein XBP‐1s acts as a transcription factor for chaperones and other ERAD proteins as well as for proteins involved in lipid synthesis (Walter and Ron 2011).