QUALITY CONTROL IN THE ENDOPLASMIC RETICULUM

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1 QUALITY CONTROL IN THE ENDOPLASMIC RETICULUM Lars Ellgaard and Ari Helenius The endoplasmic reticulum (ER) has a quality-control system for proof-reading newly synthesized proteins, so that only native conformers reach their final destinations. Non-native conformers and incompletely assembled oligomers are retained, and, if misfolded persistently, they are degraded. As a large fraction of ER-synthesized proteins fail to fold and mature properly, ER quality control is important for the fidelity of cellular functions. Here, we discuss recent progress in understanding the conformation-specific sorting of proteins at the level of ER retention and export. Institute of Biochemistry, Swiss Federal Institute of Technology (ETH) Zürich, Hönggerberg, CH 8093 Zürich, Switzerland. Correspondence to A.H. ari.helenius@bc.biol.ethz.ch doi: /nrm1052 In the assembly of a complex machine, such as a car, every essential component must conform to carefully defined specifications, and is therefore subject to stringent quality control (QC). In the cell something similar occurs there are QC systems for practically every step that leads to the synthesis of DNA, RNA and protein molecules 1 5. As a result, the number of accumulated errors in macromolecules that are ultimately deployed by cells is extremely low. For proteins, proof-reading occurs at the level of transcription, translation, folding and assembly. To pass the final QC checkpoints, a protein must typically have reached a correctly folded conformation. This is generally the so-called native conformation that corresponds to the most energetically favourable state. In the case of proteins with several subunits, proper oligomeric assembly is usually necessary. If the folding and maturation process fails, a protein molecule is not transported to its final destination in the cell, and is eventually degraded. To distinguish between native and non-native protein conformations, the cell uses various sensor molecules. By definition, the sensors include the molecular chaperones, because these interact specifically with incompletely folded proteins. Molecular chaperones often have the dual role of assisting the folding process and dispatching any improperly folded proteins for destruction. The conformationsensing system also includes enzymes that selectively and covalently tag misfolded proteins for recognition by the folding and degradation machinery. The best-known tags are ubiquitin, a small protein that is attached to lysine side chains as a degradation signal 6, and glucose, which is added to the N-linked glycans of glycoproteins as a retention signal in the endoplasmic reticulum (ER) 7,8. In this review, we discuss the QC process that functions on newly synthesized proteins in the ER of eukaryotic cells. The stringent distinction between proteins that can or cannot be transported along the secretory pathway secures the fidelity and functionality of proteins that are expressed in the extracellular space, the plasma membrane and in the compartments that are involved in secretion and endocytosis. ER QC also regulates the degradation of those proteins that are not correctly folded 9. Disposal occurs by a process called ER-associated degradation (ERAD); misfolded proteins are retro-translocated from the ER into the cytosol where they are ubiquitylated and subsequently degraded by proteasomes. As ERAD has recently been reviewed in detail in this journal 10 and elsewhere 11,12, we place our emphasis on aspects of transport regulation. Protein folding and quality control in the ER The ER provides an environment that is optimized for protein folding and maturation. Like the lumen of other organelles of the secretory pathway (BOX 1), the ER lumen is extracytosolic and is therefore topologically equivalent to the extracellular space. Consequently, the milieu of the ER differs from that of the cytosol with NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 4 MARCH

2 Box 1 Secretory-pathway organelles that are involved in quality control ERAD Translocon complex Ribosome a Nascent protein chain ER b COPI vesicle c ER exit site COPII vesicle ERGIC Lysosome/ vacuole Golgi complex The endoplasmic reticulum (ER) is the site of synthesis and maturation of proteins entering the secretory pathway (see figure, part a). It contains molecular chaperones and folding factors that assist protein folding and retain non-native conformers. Terminally misfolded proteins and unassembled oligomers are retro-translocated to the cytosol and are degraded by the proteasome a process referred to as ER-associated degradation (ERAD). Once they are correctly folded, native conformers enter ER exit sites (see figure, part b). Vesicles that are coated with the coatomer protein (COP)II coat bud off and traffic through the ER Golgi intermediate compartment (ERGIC) to the cis-face of the Golgi complex. In certain cases, the retrieval of misfolded proteins from the Golgi complex by COPI vesicles has been observed (see figure, part c). The Golgi complex does not contain molecular chaperones and does not seem to support protein folding. Once they have passed through the cis-golgi, proteins proceed through the trans-golgi network (TGN) to the plasma membrane or beyond. In special cases, which have been observed mainly in Saccharomyces cerevisiae, non-native proteins can be diverted from the TGN to the lysosome/vacuole for degradation (see figure, part d). TGN respect to ions, redox conditions and the complement of molecular chaperones. In the ER, several cotranslational and post-translational modifications take place that do not occur in the cytosol, such as disulphidebond formation, signal-peptide cleavage, N-linked glycosylation and glycophosphatidylinositol (GPI)-anchor addition. These covalent changes are important for correct protein folding. The rules for folding in the ER are different from those that are applied in the cytosol. Usually, proteins that have evolved to fold in the ER do not fold correctly if targeted to the cytosol and vice versa. In mammalian cells, proteins are translocated into the ER cotranslationally, and folding typically occurs in three phases. First, cotranslational and cotranslocational protein folding occurs in the context of the translocon complex a proteinaceous channel through which the nascent chain enters the ER lumen or the ER membrane. Second, post-translational folding takes place after the completed chain has been released from the ribosome and the translocon complex. Finally, oligomeric assembly takes place. This usually occurs when the subunits have reached a conformation that is close to the final folded state. Chaperones and folding enzymes (BOX 2) d Cytosol Plasma membrane reside in the ER lumen in high concentrations, and participate in all three stages of folding. The presence of a strict QC system in the ER is essential for several reasons. By preventing the premature exit of folding intermediates and incompletely assembled proteins from the ER, it extends the exposure of the substrates to the folding machinery in the ER lumen and thereby improves the chance of correct maturation; downstream organelles in the secretory pathway do not generally support protein folding 13. Furthermore, ER QC ensures that proteins are not dispatched to terminal compartments when they are still incompletely folded and therefore potentially damaging to the cell. For example, it is essential that non-functional or partially functional ion channels, transporters and receptors do not reach the plasma membrane, where their presence could be toxic 14,15. Finally, cells use ER QC to regulate the transportation and activation of specific proteins posttranslationally. These proteins are involved in processes such as gene regulation and nutrient storage, and some have extracellular carrier functions for ligands that have to be loaded in the ER before the proteins become secretioncompetent Primary quality control The system that regulates the transport of proteins from the ER to the Golgi apparatus is complicated and sophisticated. It works at a general level ( primary QC ) that is applied to all proteins, regardless of their origin and individual characteristics, and at a specific level that is reserved for selected categories of proteins ( secondary QC ) 19. Primary QC is based on common structural and biophysical features that distinguish native from non-native protein conformations. This means that, although many cargo molecules acquire functionality while they are in the ER, transport competence is not dependent on protein function. Important features for recognition include the exposure of hydrophobic regions, unpaired cysteine residues and the tendency to aggregate. The molecular chaperones and folding sensors that are used in primary QC are abundant in the ER. They include BiP, calnexin, calreticulin, glucose-regulated protein (GRP)94 and the thiol-disulphide oxidoreductases protein disulphide isomerase (PDI) and ERp57 (BOX 2). Frequently, even minor deviations from the native conformation, because of incomplete folding or misfolding, lead to a protein being bound by one or more of these factors and therefore to its retention in the ER In fact, there are various serious diseases that derive from endogenous proteins that contain mutations and defects that affect folding and lead to protein accumulation in the ER As no specific signals or amino-acid sequence motifs are needed for primary QC, it applies not only to endogenous wild-type proteins, but also to chimeric and recombinantly expressed heterologous proteins. A correlation with protein stability. Primary QC is a retention-based system in which the incompletely folded conformers and unassembled oligomers are recognized 182 MARCH 2003 VOLUME 4

3 Box 2 The main chaperone families of the endoplasmic reticulum The endoplasmic reticulum (ER) contains chaperones that belong to several of the classical chaperone families, such as heat shock protein (Hsp)40, Hsp70 and Hsp90, with the two main exceptions being the Hsp104 and Hsp60/Hsp10 proteins. The ER also contains chaperones and folding enzymes that are unique, such as calnexin and calreticulin and the family of thiol-disulphide oxidoreductases. Hsp70s The main protein of this family is BiP, which takes part in many aspects of ER quality control (QC). It binds to various nascent and newly synthesized proteins and assists their folding. In addition, it is involved in the processes of ER-associated degradation and the unfolded protein response. The functions of the second Hsp70 family member, glucose-regulated protein (GRP)170, are relatively unexplored. Hsp40s Five ER proteins of the Hsp40 family (ERdj1 5) are known. They contain a luminally exposed J-domain and can stimulate BiP ATPase activity in vitro. Hsp90 The only known Hsp90 family member is GRP94. Despite being abundant in the ER, it is not essential for cell viability and seems to limit its interactions to a small set of substrates. Peptidyl-prolyl isomerases Peptidyl-prolyl isomerases (PPIases) from both of the two main PPIase families the cyclophilins and the FK506-binding proteins are found in the ER. Catalysis of cis/trans isomerization of peptidyl-prolyl bonds in vivo remains to be shown conclusively for these proteins. Calnexin and calreticulin These two lectin chaperones interact with and assist the folding of proteins that carry monoglucosylated N-linked glycans. Thiol-disulphide oxidoreductases This large family of enzymes, of which protein disulphide isomerase is the best known, catalyses the oxidation, isomerization and reduction of disulphide bonds. CONFORMATIONAL STABILITY The conformational stability of a protein is defined as the free energy change, G, for the conversion of its unfolded (denatured) form to its folded (native) form. and selectively retained. Folded conformers are not detected and are therefore free to leave. Which factors influence the efficiency of secretion for a given protein? When Wittrup and colleagues 26,27 measured the secretion efficiency of a series of folding mutants of bovine pancreatic trypsin inhibitor (BPTI) in Saccharomyces cerevisiae, they found that there was a clear correlation with the in vitro measured thermal stability of the mutant proteins (that is, an increased thermal stability resulted in an increased secretion efficiency). This correlation was observed even when the melting temperatures for all of the mutants were well above the temperature used for protein expression. Therefore, an important parameter that determines the efficiency of secretion is the CONFORMATIONAL STABILITY of the folded protein, that is, the free energy of folding. The generality of this observation has been confirmed in several systems. The introduction of point mutations in insulin and T-cell-receptor chains, which were designed to make these proteins more stable than the respective wild-type proteins, were shown to result in higher levels of secretion 28,29. When mutants of BPTI were screened and those that had an increased secretion efficiency selected, all of the mutants showed a higher thermal stability than the wild-type protein 30. Together with studies using the yeast surface display system (for an example, see REF. 31), these observations confirm that the lower the free-energy barrier between native and non-native structures, the larger the fraction of protein that is retained in the ER and then degraded. These findings indicate that QC in the ER might depend on dynamic and kinetic properties, that is, on structural fluctuations and not merely on the timeaveraged structure. Even when completely folded, both wild-type and mutant proteins might unfold transiently and expose chaperone-binding, misfolded conformations when they are in the ER. The lower the overall stability of a protein, the more frequently this occurs. The longer the period of time a protein spends in non-native conformations, the lower its chances of leaving the ER. The above ideas are summarized in FIG. 1. The conformations of a protein are shown to interchange between incompletely folded free forms (I), incompletely folded chaperone-bound forms (I C) and the native conformation (N). Although a few exceptions are known 32,33, exit from the ER to the Golgi complex is, in this model, limited to proteins that have reached the native conformation, whereas misfolded forms are retro-translocated and degraded in the cytosol. The higher the stability of the protein s native form, the more efficient its export and the smaller the degraded fraction. Inherent to this model is the idea that, although the ER environment promotes and supports the folding of proteins, it might also induce unfolding. That this is possible was illustrated by experiments with the tso45 folding mutant of the vesicular stomatitis virus (VSV) G-protein: the VSV G-protein is partially unfolded by a rise in temperature, but only when it is located in the ER 13,34. Furthermore, the unfolding power of the ER is used by cholera toxin to gain access to the cytosol by retro-translocation 35,36. In addition, at least partial unfolding by chaperones is probably needed before the retro-translocation of misfolded proteins from the ER to the cytosol, through the translocon channel, for degradation 10,36. Molecular properties that determine ER retention. Although the stability argument provides an explanation for the observation that proteins differ widely in their secretion efficiency, the molecular cues that direct folding sensors to non-native conformers remain largely unclear. However, some information, albeit incomplete, is available for BiP and also for the calnexin/calreticulin system, which is discussed below. In vitro, BiP binds heptapeptides that have aliphatic amino-acid side chains in alternating positions 37,38. Although such sequence determinants are present in most proteins, only a limited set are actually used by BiP as binding sites during protein folding 22. Part of the reason might be that many segments are buried rapidly as a protein folds. It might also be that, although exposed, the sequences are sterically inaccessible to the peptidebinding site in BiP 39. In the same way that the efficiency of proteolytic cleavage at exposed sites on the surface of native and partly folded proteins correlates with the flexibility of the polypeptide segment 40, the binding of NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 4 MARCH

4 ERAD Translocon complex ER Cytosol Chaperone Figure 1 The effects of the endoplasmic reticulum environment on folding equilibria. The equilibrium between the native (N) and incompletely folded (I) forms of a protein (green) in the endoplasmic reticulum (ER) is affected by the ER environment, in particular by the presence of molecular chaperones and folding enzymes. Incompletely folded conformers are bound by chaperones (I C), which transiently stabilize them and protect them from aggregation. Chaperones also directly promote folding and, in many cases, this process occurs by sequential or even simultaneous interactions with different chaperones. In addition, chaperones have a role in transferring misfolded proteins for degradation by the ER-associated degradation (ERAD) pathway. It seems reasonable to assume that the chaperone system in the ER can shift the equilibrium away from the native conformation if it is not sufficiently stable. So, high conformational stability is likely to protect proteins from recapture by the ER quality control system and to favour ER export. Modified with permission from REF. 26. the American Society for Biochemistry and Molecular Biology (1998). LECTIN A protein that binds carbohydrates. F-BOX PROTEIN A protein component of a ubiquitin-ligase complex that contains an F-box domain, which is responsible for the interaction with a specific substrate protein. UBIQUITIN LIGASE A protein or protein complex that mediates the ubiquitylation of a substrate protein through interactions with other components of the ubiquitylation machinery. CFTR (Cystic fibrosis transmembrane conductance regulator). A plasma membrane Cl channel. CFTR F508 A folding-defective and principal disease-causing allele of the cystic fibrosis transmembrane conductance regulator (CFTR). I C I N Golgi hydrophobic peptide sequences to chaperones might depend not only on exposure, but also on the dynamic properties of the target sequence 22,39. The stamp of disapproval. An example of a particularly well-characterized primary QC system is the so-called calnexin/calreticulin cycle (FIG. 2). This system is responsible for promoting the folding of glycoproteins, retaining non-native glycoproteins in the ER until they are correctly folded and, in some cases, targeting misfolded glycoproteins for degradation 8, Calnexin and calreticulin are homologous proteins that are resident in the ER, and both are LECTINS that interact with monoglucosylated, trimmed intermediates of the N-linked core glycans on newly synthesized glycoproteins 7,44,46,47. Calnexin is a transmembrane protein and calreticulin is a soluble lumenal protein. Their interaction with glycans occurs through a binding site in their globular lectin domain, which is structurally related to legume lectins 48. The specificity of calnexin and calreticulin for binding monoglucosylated glycan (Glc 1 Man GlcNAc 7- ) (where Glc is glucose, Man is mannose, 9 2 and GlcNAc is N-acetylglucosamine) leads to the transient association of one or both of these chaperones with almost all of the glycoproteins that are synthesized in the ER 21. Whether there are further contacts, beyond the glycan, between substrate glycoproteins and the lectin chaperones is a matter of ongoing investigation Both calnexin and calreticulin form complexes with ERp57 (REFS 52,53) a thiol-disulphide oxidoreductase that is known to form transient disulphide bonds with calnexin- and calreticulin-bound glycoproteins 54. NMR and biochemical studies have shown that ERp57 binds to the tip of an arm-like domain that extends ~110 and ~140 Å from the lectin domains of calreticulin and calnexin, respectively A protected space is formed for the bound substrate between ERp57, the arm-like domain and the lectin domain (FIG. 2). Other ER chaperones and folding enzymes, such as BiP and PDI, are also often involved in the folding of glycoproteins. Two functionally independent ER enzymes mediate the on- and off-cycle in this chaperone system (FIG. 2). Glucosidase II is responsible for dissociating the substrate glycoprotein from calnexin or calreticulin by hydrolysing the glucose from the monoglucosylated core glycan. UDP-glucose:glycoprotein glucosyltransferase (GT), on the other hand, is reponsible for reglucosylating the substrate so that it can reassociate with calnexin or calreticulin. It is well known from in vivo and in vitro studies that re-glucosylation by GT happens only if the glycoprotein is incompletely folded 8. In this way, GT works as a folding sensor. A protein can only exit the cycle when GT fails to re-glucosylate it. The glucose acts as a selective tag for incompletely folded proteins it is, in a sense, a stamp of disapproval that tells the system that the protein is not yet ready to be deployed. The cycles of glucosylation and de-glucosylation continue until the glycoprotein has either reached its native conformation or is targeted for degradation. For the degradation of glycoproteins, trimming of a single mannose by the ER α1,2-mannosidase I in the middle branch of the oligosaccharide leads to an association with ER degradation-enhancing 1,2-mannosidase-like protein (EDEM) a newly discovered lectin 58,59. This interaction probably diverts the glycoprotein from the calnexin/calreticulin cycle and promotes its degradation. The recent discovery that the F-BOX PROTEIN Fbx2 of the SCF Fbx2 ubiquitin ligase complex functions as a UBIQUITIN LIGASE specifically for proteins that carry N-linked high-mannose oligosaccharides provides another link between the processes of ER QC and ERAD of glycoproteins 60. So, how does GT distinguish native from incompletely folded proteins? In vitro studies have shown that it preferentially re-glucosylates glycoproteins in partially folded, molten globule-like conformations, and that an important feature for recognition is the exposure of hydrophobic amino-acid clusters 61.It ignores glycoproteins in native and random-coil conformations, as well as short glycopeptides and isolated glycans 8,62. GT is able to recognize limited, highly localized folding defects and, in doing so, it only re-glucosylates those glycan chains that are present in the misfolded regions 63 (C. Ritter, K. Quirin and A.H., unpublished observations). This means that the enzyme focuses specifically on regions of the substrate glycoproteins that are partially misfolded and contain glycans, and highlights the fact that GT is a sophisticated and sensitive detector of misfolding in the ER lumen and in ER exit sites. The placement of N-linked glycans in the sequence of 184 MARCH 2003 VOLUME 4

5 Cytosol ER ERp57 S S G M ER exit site glycoproteins might have evolved to direct the attention of the QC system to particular regions of the folding protein. Conversely, N-linked glycans might be missing from other regions where an increased mobility is needed. Glucosidases I and II Calreticulin Glucosidase II M M G GG G M Ribosome Translocon complex Nascent protein chain N-linked glycan UDP G Figure 2 The calnexin/calreticulin cycle. Calnexin and calreticulin assist the folding of glycoproteins in the endoplasmic reticulum (ER) (for simplicity, only calreticulin is depicted). After transfer of the core oligosaccharide (Glc 3 Man 9 GlcNAc 2, where Glc is glucose (red circles), Man is mannose (blue circles) and GlcNAc is N-acetylglucosamine) to the nascent chain of the protein, two glucoses are removed by glucosidases I and II. This generates a monoglucosylated (Glc 1 Man 9 GlcNAc 2 ) glycoprotein that can interact with calnexin and calreticulin. Both chaperones associate with the thiol-disulphide oxidoreductase ERp57 through an extended arm-like domain. During the catalysis of disulphide-bond formation, ERp57 forms interchain disulphide bonds (S S) with bound glycoproteins. Cleavage of the remaining glucose by glucosidase II terminates the interaction with calnexin and calreticulin. On their release, correctly folded glycoproteins can exit the ER. By contrast, non-native glycoproteins are substrates for the UDP-glucose:glycoprotein glucosyltransferase, which places a single glucose back on the glycan and thereby promotes a renewed association with calnexin and calreticulin. If the protein is permanently misfolded, the mannose residue in the middle branch of the oligosaccharide is removed by ER α1,2-mannosidase I. This leads to recognition by the ER degradation-enhancing 1,2-mannosidase-like protein (EDEM), which probably targets glycoproteins for ER-associated degradation (ERAD). Please note that, for simplicity, only some of the sugar moieties of the oligosaccharide core structure are depicted fully in the figure. M UDP UDP-glucose:glycoprotein glucosyltransferase α1,2-mannosidase I EDEM ERAD Translocon complex Chemical and pharmacological chaperones. Given a system in which retention is based on conformational criteria and on differences in the stability of native and non-native conformations, it should be possible to improve maturation and secretion by stabilizing proteins in the early secretory pathway. This approach could potentially pave the road for therapeutic intervention in ER-storage diseases in which folding-defective proteins fail to be secreted. Indeed, several studies using tissue-culture cells have shown that such an effect can be obtained, for example, by lowering the temperature or by using small cell-permeable molecules such as glycerol, dimethylsulphoxide and trimethylamine N-oxide. These chemical chaperones exert a nonspecific, folding-promoting effect presumably by stabilizing native or native-like conformers or by reducing aggregation. A well-known case involves the primary disease-causing allele of the cystic fibrosis transmembrane conductance regulator (CFTR) the CFTR 508 folding-defective mutant. In this case, the use of different chemical chaperones leads to increased cell-surface expression of the structurally destabilized, but functional, protein 64. Similar, but more specific, effects can be achieved with pharmacological chaperones. This term refers to ligands that target a specific protein (for a review, see REF. 65). Recent elegant work, on G-protein-coupled receptors (such as the δ opioid receptor 66 and V2 vasopressin receptor mutants 67 ), tyrosinase 68, P-glycoprotein mutants 69 and antibodies that carry mutations in the heavy chain complementarity-determining region 70, has demonstrated the potential of these molecules. The specificity of the approach is shown by a correlation between ligand-binding affinity and ligand-mediated rescue 66,70. The beneficial effects of pharmacological chaperones probably stem from the structural stabilization of native-like conformers that are able to interact with ligands. Therefore, they might help to shift the equilibrium from the incompletely folded (I) forms of the protein to the native (N) form (FIG.1) enough to increase secretory efficiency. Pharmacological chaperones mimic the effect that certain physiological ligands have on protein secretion. For example, the retinol binding protein (RBP) is retained in the ER in the unliganded form. Only on binding retinol does export of RBP take place 18.As retinol binding results in only a minor conformational change in RBP, as detected by X-ray crystallography 71, this effect is probably best explained by ligand-induced stabilization of the RBP structure. Secondary quality control To be secreted, many proteins must fulfil criteria beyond those that are imposed by the primary QC system. The term secondary QC refers to various selective mechanisms that regulate the export of individual protein species or protein families 19. Each of the factors involved has its own specific recognition mechanism and many of these factors interact with the folded cargo proteins or late folding intermediates 19,24,72. Among the colourful mixture of secondary QC proteins, Herrmann and colleagues 72 have classified those proteins that are needed NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 4 MARCH

6 TYPE I MEMBRANE PROTEIN A transmembrane protein that is orientated with its carboxyl terminus in the cytosol PROTEINS A family of abundant proteins that bind to phosphoserine- and phosphothreonine-containing motifs in a sequence-specific manner. INVARIANT CHAIN A specific chaperone and escort protein for MHC class II molecules. TYPE II MEMBRANE PROTEIN A transmembrane protein that is orientated with its amino terminus in the cytosol. to fold and assemble specific proteins as outfitters, those needed to accompany proteins out of the ER as escorts and those needed to provide signals for intracellular transport as guides. The group of outfitters includes specialized chaperones and enzymes such as Nina A a peptidyl-prolyl cis/trans isomerase that ensures the transport competence of specific rhodopsins in Drosophila melanogaster 73. A well-known escort is the receptor-associated protein (RAP), which binds to members of the low density lipoprotein-receptor (LDLR) family in the ER and escorts them to the Golgi complex to protect them from premature ligand-binding in the early secretory pathway 74. Among the guides, the lectin that is known as ER Golgi intermediate compartment (ERGIC)-53 cycles between the ER and the Golgi complex and seems to act as a transport receptor for certain proteins that carry high-mannose N-linked glycans 75,76. For a more complete listing of secondary QC factors, see REF. 19. Secondary QC processes are often cell-type specific. In addition, they are frequently involved in the regulation of ER retention and export. For example, cellular cholesterol levels are controlled by the regulated ER export of the sterol regulatory element binding protein (SREBP) through its cholesterol-dependent interaction with the escort factor SCAP (SREBP cleavage-activating protein) 77. In this case, cholesterol binding by SCAP brings about a conformational change in the molecule that leads to ER retention of the SCAP SREBP complex 16. This retention is facilitated through the interaction of the sterol-sensing domain of SCAP with either of two homologous transmembrane ER proteins (insig-1 and insig-2) 78,79. At present, the ER-retention mechanism for the ternary SCAP SREBP insig complex is not known. Protein sorting and ER quality control ER-retention signals. The ER is continuously feeding cargo-containing vesicles into the secretory pathway and receiving retrograde membrane traffic from the Golgi complex in return (BOX 1). A cohort of folded, assembled, native secretory and membrane proteins, as well as membrane lipids, are exported continuously from the ER. To avoid this fate, most resident ER proteins, including chaperones and folding enzymes, contain ER-retention and -retrieval signals (for reviews, see REFS 80,81). Of these signals, the wellcharacterized tetrapeptide KDEL sequence (where K is lysine, D is aspartate, E is glutamate and L is leucine) is present at the carboxyl terminus of many soluble ER proteins. This motif interacts with the KDEL receptor and thereby ensures that the protein is retrieved from the Golgi complex by coatomer protein (COP)I-coated vesicles (BOX 1) Similar to the KDEL sequence, KKXX or KXKXX signals (where X is any amino acid) that are present at the carboxyl terminus of TYPE I MEMBRANE PROTEINS are known to mediate retrieval by direct interaction with the COPI coat In addition, exposed cysteine residues can also function as retention signals in the lumen of the ER 20. Retention and retrieval signals that are present in proteins destined for export are also important in QC, as they help to retain proteins in the ER until correct folding or assembly has taken place. The best characterized of these signals is the RKR motif (where R is arginine), which was first described in subunits of ATP-sensitive potassium channels 14. In most cases, this signal helps to retain individual subunits and incomplete oligomers in the ER until they are masked by the correct oligomeric assembly 89. Another intriguing example of transport regulation involves the recently described role of a PROTEIN in promoting ER export of proteins that contain dibasic COPI-interacting retention signals 90. The binding of to short sequence motifs which contain a phosphorylated serine residue and are present, for example, in different potassium channels and in the INVARIANT CHAIN was shown to exclude simultaneous binding to the COPI-protein β-cop and to result in ER export. So, mutually exclusive binding of and β-cop seems to underlie this phosphorylation-dependent regulation of ER export and retention. In the case of the TYPE II TRANSMEMBRANE PROTEIN ATF6 (activating transcription factor 6), transport from the ER is regulated by ER stress. Under stress conditions that lead to the accumulation of misfolded proteins in the ER, the transcription factor ATF6 is transported to the Golgi complex where intramembrane proteolysis releases the transcription-factor domain, which is exposed to the cytosol 91. The transcription-factor domain then traffics to the nucleus in which it activates ER-chaperone-gene transcription. The ER export of ATF6 is controlled by BiP 92. Using a mechanism that probably involves the masking of signals for forward transport to the Golgi, binding of BiP retains ATF6 in the ER under normal cellular conditions. On the accumulation of misfolded protein in the ER, it is conceivable that such conformers compete for binding of BiP. This then results in BiP release from the lumenal domain of ATF6 and, as a consequence, the protein becomes transport competent. ER exit sites. Exit of proteins from the ER occurs at socalled transitional elements or ER exit sites 93,94 (FIG. 3). These form buds or small membrane clusters that are contiguous with the ER membrane and are coated with the COPII coat 95. Importantly, incompletely folded cargo proteins and ER chaperones are generally excluded from exit sites 13,96,97. If the tso45 VSV G-protein is rendered misfolded by a temperature shift when present in exit sites, it is re-glucosylated by GT and, instead of being transported to the Golgi complex, is returned to the ER 13. Therefore, the exit sites function as an intrinsic part of the ER QC system. The reasons why misfolded proteins are not included in export vesicles could be: that they are retained by interactions with molecules elsewhere in the ER; that they are somehow prevented from entering the exit sites by restrictions imposed by the exit sites themselves; or that cargo receptors that are present in the exit sites fail to recognize them. Here, the underlying models are 186 MARCH 2003 VOLUME 4

7 a b c ER ER exit sites Golgi complex Figure 3 Trafficking of a cargo glycoprotein from the endoplasmic reticulum through exit sites to the Golgi complex. a A light-microscopy image of the endoplasmic reticulum (ER). Tissue-culture cells were transfected with a green fluorescent protein-labelled version of the temperature sensitive tso45 variant of the vesicular stomatitis virus (VSV) G-protein and were incubated at 39.5 C. At this temperature, the VSV G-protein is partially unfolded and is therefore retained in the ER (white meshwork) by the quality-control system. b A light-microscopy image of the ER and ER exit sites. On shifting the cells from 39.5 C to 10 C for 1 h, the VSV G-protein that is discussed in part a refolds, but it remains in the ER and in ER exit sites, which are visible as small dots (the labels highlight two examples of ER exit sites) 13. c A light-microscopy image of the Golgi complex. On shifting the temperature from 39.5 C to 20 C, followed by incubation for 2 h, the VSV G-protein refolds and traffics to the Golgi complex, which is visible as an intensely stained, bright area (see label). A weak reticular ER stain is visible from VSV G-protein that is still present in the ER. This figure was kindly provided by A. Mezzacasa, ETH Zürich, Switzerland. those of bulk flow, in which selectivity is based on retaining interactions 98, and of cargo capture, in which folded cargo is selectively recognized in the exit sites by receptors that are thought to be transmembrane proteins, which associate directly or indirectly with the COPII coat 99. Although evidence has been presented in favour of both models, neither adequately explains the full range of sorting phenomena that are observed during the ER-to-Golgi trafficking of proteins. As is often the case in biology, elements taken from numerous models might together provide the full picture. Protein mobility in the ER. Some misfolded proteins fail to be transported to the Golgi complex because they aggregate in the ER 2. In addition to non-native proteins, such aggregates typically contain molecular chaperones and thiol-disulphide oxidoreductases, and they are often stabilized by non-native, interchain disulphide bonds 100,101. Protein aggregation in the ER is most often irreversible and in many cases connected directly to a disease state. However, recent work showed that aggregates of a genetically engineered variant of the FKBP12 protein (FK506 binding protein), which was recombinantly expressed in the ER, could be dissolved on the addition of a small synthetic ligand 102. When fusion proteins of FKBP12 linked to therapeutically important hormones (such as insulin) through a cleavage site for furin (which is a Golgi-resident protease) were expressed in tissue-culture cells or mice, regulated ligandinduced secretion of the hormone could be achieved 102. This approach shows that the modulation of the processes that are involved in protein sorting and ER QC can potentially lead to the development of new therapeutic strategies. Sheer size might be enough to explain why aggregates in the ER fail to be exported. However, the upper size limit for particles that can be exported is quite high; for example, viruses and virus-like particles that are 50 nm in diameter can bud into the ER, and procollagen fibres with a length of 300 nm can be transported to the Golgi complex Nevertheless, it is possible that some of the aggregates that are formed by misfolded proteins exceed these limits. Fluorescence recovery after photobleaching (FRAP) analysis of green fluoresent protein (GFP)-labelled cargo molecules has shown that certain aggregated, non-native proteins are effectively immobilized in the rough ER So, another reason why aggregates of misfolded proteins fail to be transported to the Golgi is probably a lack of mobility in the ER. They might not be able to move to ER exit sites. It has been proposed that molecular chaperones and other resident proteins that are present in the ER lumen at high concentrations form a viscous, interconnected protein network that might restrict mobility, not only of large aggregates but also of smaller complexes that contain incompletely folded proteins However, recent reports, also based on FRAP analysis, show that several transport-incompetent cargo proteins are actually quite mobile in the ER. These transportincompetent cargo proteins include GFP-labelled tso45 VSV G-protein 106, the CFTR 508 mutant 113,a mutant of aquaporin 2 (REF. 107) and the heavy chain of unassembled major histocompatibility complex (MHC) class I molecules 108. When compared to the folded forms of the same proteins, little difference in lateral mobility was observed. The estimated diffusion constants indicate that the proteins retained in the ER are in the form of monomers or small to mid-size aggregates. So, although aggregation can be a factor that might help to exclude certain misfolded proteins from exit sites, the FRAP data imply that many misfolded proteins are mobile in the ER. More work is needed to address the important question of why such proteins are not exported through exit sites like their folded counterparts. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 4 MARCH

8 Quality control in the Golgi complex. Although most of the reported QC events occur in the ER, the Golgi complex can also participate 32,33. In certain cases, retrieval of misfolded proteins from the Golgi complex to the ER occurs. For unassembled T-cell antigen receptor α-chains, this retrieval pathway involves COPI-coated vesicles and is dependent on BiP and the KDEL-receptor 114. Similarly, when the misfolded tso45 variant of the VSV G-protein was overexpressed in Chinese hamster ovary (CHO) cells, the protein was seen to cycle between the ER and the cis-golgi in a complex with BiP 115.However, the KDEL-receptor has the ability to retrieve KDELcontaining proteins and complexes not only from the cis-golgi, but from the entire Golgi complex 116.The retrieval pathway from the Golgi complex probably represents a back-up system for when the ER-retention capacity for a protein is saturated. In other cases, the recycling of misfolded proteins might be required for ERAD 33, The Golgi complex of mammalian cells seems to lack resident chaperones and folding enzymes, but the localization of calreticulin, GT and glucosidase II to the ERGIC might indicate a potential for glycoprotein folding in this compartment 120,121. Particularly in S. cerevisiae, the Golgi complex seems to be an important site for QC.Vps10 a Golgi protein that is responsible for sorting vacuolar hydrolases has been implicated as a folding sensor for the vacuolar transport of misfolded proteins 122,123. Recently, a multispanning membrane protein of the Golgi Tul1, which is a putative ubiquitin ligase was identified as an enzyme that recognizes polar residues in the transmembrane region of membrane cargo proteins 124. It is possible that Tul1 singles out membrane proteins that are either not folded correctly in the transmembrane region or are not completely oligomerized for vacuolar targeting. The efficiency of protein maturation Although some proteins fold quite efficiently, many fold inefficiently. One possible explanation for the poor success rate of protein maturation might be that, during evolution, proteins have been optimized for function, but not necessarily for folding and assembly. Therefore, this would mean that these latter processes are intrinsically error-prone. Often polypeptides get caught in nonnative conformations from which they cannot escape within a reasonable time, even in the presence of chaperones. For certain ER proteins, such as CFTR and the δ opioid receptor, 40% or more of newly synthesized wild-type protein molecules fail to mature and are degraded 125,126. Although the physiological temperature might be optimal for protein function, it might be too high for efficient folding. Consequently, an increase in folding efficiency is often found when cells are grown at reduced temperatures (for a review, see REF. 2). For hetero-oligomeric proteins, a poor success rate of maturation is often observed. Orphan subunits and partially assembled oligomers are retained in the ER and eventually degraded. For example, this has been observed in cultured muscle cells, in which only ~30% of acetylcholine receptor α-subunits were incorporated into the native hetero-pentameric receptor molecules 127. However, non-stoichiometric production of protein subunits can also have a distinct purpose. In the case of immunoglobulin assembly in plasma cells, the light chains are expressed in excess of the heavy chains to ensure high rates of heavy-chain incorporation into the oligomeric complex, because the heavy chain is aggregation prone and potentially toxic in the free form 128. Likewise, the level of protein expression often exceeds the availability of specific ligands that are essential for secretion. This is the case for MHC class I molecules only the fraction that is loaded with high affinity peptides is transported to the cell surface 129 and for RBP, which needs to associate with retinol 18. Recent reports, which used pulse-labelling and proteasome inhibitors to block the degradation of newly synthesized proteins, indicated that 30 75% of the proteins that are synthesized are degraded within 20 min, and that some of the proteins that are being degraded are growing nascent chains How can the cell afford this large-scale degradation of newly synthesized proteins? Yewdell and co-workers 130 have argued that the apparent wastefulness actually has an important function, because the ongoing degradation of newly synthesized and nascent proteins provides a continuous supply of peptides that are presented on the cell surface bound to MHC class I molecules. It is known that most of the peptides that are presented in this way are, in fact, from proteins that have just been synthesized 134. Therefore, when a cell is infected by a virus and starts to produce viral protein, this infection can be rapidly detected by T-cells. Although the formation of misfolded proteins might have a useful role in immune defence, the downside is the various diseases in which aggregated proteins accumulate in the cells and tissues in non-native conformations. For example, the formation of amyloid plaques is clearly due to a failure in QC and/or in the proteindegradation system 135. It is apparent that these conformations are either not recognized efficiently as being misfolded or, once they are formed, they cannot be degraded. Conclusions and perspectives The ER is unique among cellular compartments in the sense that most newly synthesized proteins are exported from the ER and find their final location in places that are devoid of chaperones or other factors that could help them refold if they were to become damaged. Therefore, the requirements for protein stability might be set higher in the ER than in the cytosol. The stability of proteins that are produced in the ER is enhanced by the formation of disulphide bonds and by the addition of protein-bound glycans. Although these modifications make proteins more stable, they impose more complexities on the folding process. The number of enzymes that are involved solely in the oxidation and isomerization of disulphide bonds is large, and so is the number of enzymes that are needed to synthesize and trim N-linked glycans. It is also probable that the ER exposes folded proteins to further prodding by chaperones to ensure their stability. 188 MARCH 2003 VOLUME 4

9 As we learn more about the folding and maturation of individual proteins in the ER, and about the mechanisms that are used by individual chaperones, it will be important to consider the overall principles of these systems. How are degradation decisions made in this compartment? How do chaperones and folding enzymes work together? Which are the crucial folding sensors, and how do they recognize their substrates? What is the mechanism for selective cargo loading in ER exit sites? Whether at the level of structural fluctuations in the folded and unfolded proteins or the mobility of the proteins and assemblies in the ER lumen and membrane, we will also need to focus more on dynamic aspects. The questions above are important, and the answers might allow us to enhance the expression of recombinant proteins that are of crucial industrial and pharmaceutical significance. They might also help us to further understand the many disease states that have their basis in aberrant protein folding in the ER, and they will certainly provide us with a better understanding of the post-translational aspects of protein expression and its regulation in general. 1. Kurland, C. G. Translational accuracy and the fitness of bacteria. Annu. Rev. Genet. 26, (1992). 2. Hurtley, S. M. & Helenius, A. Protein oligomerization in the endoplasmic reticulum. Annu. Rev. Cell Biol. 5, (1989). 3. Wickner, S., Maurizi, M. R. & Gottesman, S. Posttranslational quality control: folding, refolding, and degrading proteins. Science 286, (1999). 4. Ibba, M. & Söll, D. Quality control mechanisms during translation. Science 286, (1999). 5. Lindahl, T. & Wood, R. D. Quality control by DNA repair. Science 286, (1999). 6. Glickman, M. H. & Ciechanover, A. The ubiquitin proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, (2002). 7. Hammond, C., Braakman, I. & Helenius, A. Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc. Natl Acad. Sci. USA 91, (1994). 8. Parodi, A. J. Protein glucosylation and its role in protein folding. Annu. Rev. Biochem. 69, (2000). 9. Klausner, R. D. & Sitia, R. Protein degradation in the endoplasmic reticulum. Cell 62, (1990). 10. Tsai, B., Ye, Y. & Rapoport, T. A. Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nature Rev. Mol. Cell Biol. 3, (2002). This review summarizes retro-translocation across the ER membrane to the cytosol, for example, as it occurs in ERAD. 11. Brodsky, J. L. & McCracken, A. A. ER protein quality control and proteasome-mediated protein degradation. Semin. Cell Dev. Biol. 10, (1999). 12. Jarosch, E., Geiss-Friedlander, R., Meusser, B., Walter, J. & Sommer, T. Protein dislocation from the endoplasmic reticulum pulling out the suspect. Traffic 3, (2002). 13. Mezzacasa, A. & Helenius, A. The transitional ER defines a boundary for quality control in the secretion of tso45 VSV glycoprotein. Traffic 3, (2002). 14. Zerangue, N., Schwappach, B., Jan, Y. N. & Jan, L. Y. A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron 22, (1999). This paper was the first to show the importance of the RKR motif for ER retention. 15. Bichet, D. et al. The I-II loop of the Ca 2+ channel α1-subunit contains an endoplasmic reticulum retention signal antagonized by the β-subunit. Neuron 25, (2000). 16. Brown, A., Sun, L., Feramisco, J., Brown, M. & Goldstein, J. Cholesterol addition to ER membranes alters conformation of SCAP, the SREBP escort protein that regulates cholesterol metabolism. Mol. Cell 10, (2002). This report shows that SCAP undergoes a conformational change on sterol binding, which, in turn, results in the retention of the SCAP/SREBP complex in the ER. 17. Galili, G. et al. Wheat storage proteins: assembly, transport and deposition in protein bodies. Plant Physiol. Biochem. 34, (1996). 18. Melhus, H., Laurent, B., Rask, L. & Peterson, P. A. Liganddependent secretion of rat retinol-binding protein expressed in HeLa cells. J. Biol. Chem. 267, (1992). 19. Ellgaard, L., Molinari, M. & Helenius, A. Setting the standards: quality control in the secretory pathway. Science 286, (1999). 20. Fra, A. M., Fagioli, C., Finazzi, D., Sitia, R. & Alberini, C. M. Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation. EMBO J. 12, (1993). 21. Helenius, A., Trombetta, E. S., Hebert, D. N. & Simons, J. F. Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol. 7, (1997). 22. Hellman, R., Vanhove, M., Lejeune, A., Stevens, F. J. & Hendershot, L. M. The in vivo association of BiP with newly synthesized proteins is dependent on the rate and stability of folding and not simply on the presence of sequences that can bind to BiP. J. Cell Biol. 144, (1999). 23. Amara, J. F., Cheng, S. H. & Smith, A. E. Intracellular protein trafficking defects in human disease. Trends Cell Biol. 2, (1992). 24. Aridor, M. & Balch, W. E. Integration of endoplasmic reticulum signaling in health and disease. Nature Med. 5, (1999). 25. Rutishauser, J. & Spiess, M. Endoplasmic reticulum storage diseases. Swiss Med. Wkly 132, (2002). 26. Kowalski, J. M., Parekh, R. N., Mao, J. & Wittrup, K. D. Protein folding stability can determine the efficiency of escape from endoplasmic reticulum quality control. J. Biol. Chem. 273, (1998). This paper describes the correlation between protein stability and secretion efficiency as determined for mutants of BPTI. 27. Kowalski, J. M., Parekh, R. N. & Wittrup, K. D. Secretion efficiency in Saccharomyces cerevisiae of bovine pancreatic trypsin inhibitor mutants lacking disulfide bonds is correlated with thermodynamic stability. Biochemistry 37, (1998). 28. Kjeldsen, T. et al. Engineering-enhanced protein secretory expression in yeast with application to insulin. J. Biol. Chem. 277, (2002). 29. Shusta, E. V., Kieke, M. C., Parke, E., Kranz, D. M. & Wittrup, K. D. Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency. J. Mol. Biol. 292, (1999). 30. Hagihara, Y. & Kim, P. S. Toward development of a screen to identify randomly encoded, foldable sequences. Proc. Natl Acad. Sci. USA 99, (2002). 31. Shusta, E. V., Holler, P. D., Kieke, M. C., Kranz, D. M. & Wittrup, K. D. Directed evolution of a stable scaffold for T-cell receptor engineering. Nature Biotechnol. 18, (2000). 32. Minami, Y., Weissman, A. M., Samelson, L. E. & Klausner, R. D. Building a multichain receptor: synthesis, degradation, and assembly of the T-cell antigen receptor. Proc. Natl Acad. Sci. USA 84, (1987). 33. VanSlyke, J. K., Deschenes, S. M. & Musil, L. S. Intracellular transport, assembly, and degradation of wildtype and disease-linked mutant gap junction proteins. Mol. Biol. Cell 11, (2000). 34. de Silva, A. M., Balch, W. E. & Helenius, A. Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro. J. Cell Biol. 111, (1990). 35. Tsai, B., Rodighiero, C., Lencer, W. I. & Rapoport, T. A. Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin. Cell 104, Tsai, B. & Rapoport, T. A. Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1. J. Cell Biol. 159, (2002). 37. Flynn, G. C., Pohl, J., Flocco, M. T. & Rothman, J. E. Peptide-binding specificity of the molecular chaperone BiP. Nature 353, (1991). 38. Blond-Elguindi, S. et al. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell 75, (1993). 39. Knarr, G., Kies, U., Bell, S., Mayer, M. & Buchner, J. Interaction of the chaperone BiP with an antibody domain: implications for the chaperone cycle. J. Mol. Biol. 318, (2002). 40. Hubbard, S. J. The structural aspects of limited proteolysis of native proteins. Biochim. Biophys. Acta 1382, (1998). 41. Ou, W. J., Cameron, P. H., Thomas, D. Y. & Bergeron, J. J. Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature 364, (1993). 42. Hammond, C. & Helenius, A. Quality control in the secretory pathway. Curr. Opin. Cell Biol. 7, (1995). 43. McCracken, A. A. & Brodsky, J. L. Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP. J. Cell Biol. 132, (1996). 44. Ware, F. E. et al. The molecular chaperone calnexin binds Glc 1 Man 9 GlcNAc 2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. J. Biol. Chem. 270, (1995). 45. Liu, Y., Choudhury, P., Cabral, C. M. & Sifers, R. N. Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome. J. Biol. Chem. 274, (1999). 46. Hebert, D. N., Foellmer, B. & Helenius, A. Glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum. Cell 81, (1995). 47. Spiro, R. G., Zhu, Q., Bhoyroo, V. & Soling, H. D. Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi. J. Biol. Chem. 271, (1996). 48. Schrag, J. D. et al. The structure of calnexin, an ER chaperone involved in quality control of protein folding. Mol. Cell 8, The unusual crystal structure of the calnexin ectodomain shows a molecule that comprises a globular lectin domain and a long extended arm-like domain. The structure provides important clues to the function of the protein. 49. Ihara, Y., Cohen-Doyle, M. F., Saito, Y. & Williams, D. B. Calnexin discriminates between protein conformational states and functions as a molecular chaperone in vitro. Mol. Cell 4, (1999). 50. Saito, Y., Ihara, Y., Leach, M. R., Cohen-Doyle, M. F. & Williams, D. B. Calreticulin functions in vitro as a molecular chaperone for both glycosylated and non-glycosylated proteins. EMBO J. 18, (1999). 51. Danilczyk, U. G. & Williams, D. B. The lectin chaperone calnexin utilizes polypeptide-based interactions to associate with many of its substrates in vivo. J. Biol. Chem. 276, Oliver, J. D., van der Wal, F. J., Bulleid, N. J. & High, S. Interaction of the thiol-dependent reductase ERp57 with nascent glycoproteins. Science 275, (1997). 53. Oliver, J. D., Roderick, H. L., Llewellyn, D. H. & High, S. ERp57 functions as a subunit of specific complexes formed with the ER lectins calreticulin and calnexin. Mol. Biol. Cell 10, (1999). 54. Molinari, M. & Helenius, A. Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells. Nature 402, (1999). NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 4 MARCH