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Protein quality control in the early secretory pathway

2008; Springer Nature; Volume: 27; Issue: 2 Linguagem: Inglês

10.1038/sj.emboj.7601974

1460-2075

Tiziana Anelli, Roberto Sitia,

Lipid Membrane Structure and Behavior

Focus Quality Control23 January 2008free access Protein quality control in the early secretory pathway Tiziana Anelli Tiziana Anelli Department of Functional Genomics and Molecular Biology, Università Vita-Salute San Raffaele Scientific Institute, DiBiT-HSR, Milano, Italy Search for more papers by this author Roberto Sitia Corresponding Author Roberto Sitia Department of Functional Genomics and Molecular Biology, Università Vita-Salute San Raffaele Scientific Institute, DiBiT-HSR, Milano, Italy Search for more papers by this author Tiziana Anelli Tiziana Anelli Department of Functional Genomics and Molecular Biology, Università Vita-Salute San Raffaele Scientific Institute, DiBiT-HSR, Milano, Italy Search for more papers by this author Roberto Sitia Corresponding Author Roberto Sitia Department of Functional Genomics and Molecular Biology, Università Vita-Salute San Raffaele Scientific Institute, DiBiT-HSR, Milano, Italy Search for more papers by this author Author Information Tiziana Anelli1 and Roberto Sitia 1 1Department of Functional Genomics and Molecular Biology, Università Vita-Salute San Raffaele Scientific Institute, DiBiT-HSR, Milano, Italy *Corresponding author. Department of Functional Genomics and Molecular Biology, Università Vita-Salute San Raffaele Scientific Institute, DiBiT-HSR, via Olgettina 58, Milan 20132, Italy. Tel.: +39 02 2643 4763; Fax: +39 02 2643 4723; E-mail: [email protected] The EMBO Journal (2008)27:315-327https://doi.org/10.1038/sj.emboj.7601974 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Eukaryotic cells are able to discriminate between native and non-native polypeptides, selectively transporting the former to their final destinations. Secretory proteins are scrutinized at the endoplasmic reticulum (ER)–Golgi interface. Recent findings reveal novel features of the underlying molecular mechanisms, with several chaperone networks cooperating in assisting the maturation of complex proteins and being selectively induced to match changing synthetic demands. ‘Public’ and ‘private’ chaperones, some of which enriched in specializes subregions, operate for most or selected substrates, respectively. Moreover, sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion. Introduction As the per-capita income increases in Western societies, the quality of the products that appear in the market is becoming more important than their quantity. Depending on high quality and innovative design, industries employ abundant personnel and devices to ensure a stringent control of their products, the quality of which must fulfill strict pre-determined standards. This key activity is usually referred to as ‘quality control’ (QC). At the same time, the need for innovation makes it very hard to dictate a fixed set of standards and rules. Moreover, selling more (high quality) products remains a common goal of any commercial activity. Italian parents often say to their hasty offspring ‘Presto e bene raro avviene’ (Fast and good is a rare combination). How can our factories contradict this rather wise saying? And, of more interest for The EMBO Journal readership, how do cells cope with somewhat similar problems? In this essay, we analyze the mechanisms that cells employ to couple abundant synthesis and high quality for secretory proteins. After synthesis, proteins must rapidly fold to perform their biological activities. Folding takes place in three main sub-cellular compartments, cytosol, endoplasmic reticulum (ER) and mitochondria. Each organelle is equipped with a specific set of chaperones and folding enzymes optimized to work in the local conditions. In all cases, the final outcome must be a native molecule devoid of errors. Moreover, structural maturation must be completed within a rather short time frame. In the crowded environment of the cell, unfolded proteins are a danger as they may aggregate and become toxic. In viable cells, extensive aggregation is prevented by several proteolytic systems that rapidly dispose of aberrant or damaged polypeptides (see Goldberg, Liberek, Haas, this issue). A considerable fraction of the proteome consists of molecules that are destined to the extracellular space (Chen et al, 2005): these are either secreted by the cell or inserted in membranes, to act as ligands and receptors, respectively. Proteins destined to the extracellular space are synthesized on ER-bound ribosomes, and are cotranslationally translocated into the ER lumen where they attain their native conformation, before being transported to the Golgi and downstream compartments (Figure 1). Secreted and membrane proteins are the main devices of intercellular communications. The fidelity of ligand–receptor interactions requires that both molecules attain the very conformations that allow signals to be properly transmitted and understood. Protein folding in the secretory pathway must therefore be controlled in the tightest way. Figure 1.The early secretory pathway. Proteins destined to the extracellular space or to organelles of the secretory route are synthesized by ER-bound ribosomes and cotranslationally translocated (entry) into the ER. Here they attain their native structure (folding), under strict QC scrutiny. Only properly folded and assembled proteins can reach the Golgi, where they are further modified, to be transported to the extracellular space or to lysosomes. Gray arrows indicate the direction of vesicles moving among different compartments; dark arrows indicate the pathways followed by cargoes in the early secretory pathway; red lines show homeostatic control pathways (+ stimulatory, − inhibitory). Misfolded proteins are recognized, retained and eventually routed to degradation by ERAD or autophagy (which are likely reciprocally regulated, as indicated by the blue arrows). Some misfolded soluble ERAD substrates are transported to ERGIC or cis-Golgi before retrotranslocation and degradation. Too high load for the folding machinery or the accumulation of misfolded proteins activate resident ER stress sensors, which elicit the UPR. ER stress can selectively inhibit protein entry into the ER, and increase the capacity of folding and degradation (via ERAD and autophagy). The UPR induces also molecules acting downstream of the ER. Download figure Download PowerPoint Protein QC in the secretory compartment In the late 1980s, work on oligomeric viral proteins (Kreis and Lodish, 1986; Boulay et al, 1988; Gething and Sambrook, 1989), the T-cell receptor (Bonifacino et al, 1989; Sancho et al, 1989) and immunoglobulins (Bole et al, 1986; Sitia et al, 1987; Hendershot and Kearney, 1988) revealed that assembly is a requisite for transport to the Golgi apparatus and onwards along the secretory route. Klausner (1989) referred to this phenomenon as ‘Architectural editing’; the term ‘ER quality control’ (Hurtley and Helenius, 1989; Hurtley et al, 1989) eventually stuck to indicating the processes of conformation-dependent molecular sorting of secretory proteins. Until then, the lysosome was considered the site where secretory molecules are degraded. Since proteins retained in the ER cannot reach downstream lysosomes, the question arose as to how aberrant proteins are degraded (Klausner and Sitia, 1990). In the mid 1990s, studies on CFTR and MHC class I (Michalek et al, 1993; Ward et al, 1995; Wiertz et al, 1996) revealed that proteins that fail to fold or assemble are eventually retrotranslocated (or dislocated) across the ER membrane for degradation by cytosolic proteasomes. The players, mechanisms and physiopathologic implications of this process (ER-associated degradation, ERAD) remain a hot topic in molecular cell biology (Yoshida, 2007). During their lifetime, cells must integrate all the different reactions schematized in Figure 1, and adapt them to face possible changes in the quality and quantity of secretory proteins they produce during differentiation. As colocalized signals can dictate assembly, retention and degradation of membrane and soluble cargo proteins (Bonifacino et al, 1990; Fra et al, 1993), a competition between ER export and degradation can explain homeostatic control. An interesting mathematical model has been recently introduced, which considers protein folding in the ER (ERAF, ER-assisted folding), ERAD and ER export as single biochemical parameters (see Wiseman et al, 2007 and references therein). Despite the limits imposed by the simplification, this approach leads to some interesting and testable predictions: export efficiency of a particular cargo protein depends on the activity of the ERAF, ERAD and export systems, which in turn are influenced by the proteome expressed by the cell. This partially simplified model could be further expanded and tested to integrate new emerging evidence. Recent data highlight a spatial subdivision of the early secretory compartment that seems particularly suited for the biogenesis of complex, multimeric proteins. Both parallel and sequential QC pathways coexist in cells, some common to all polypeptides, others specific for particularly demanding proteins. This diversity likely evolved to cope with the myriads of polypeptides that our cells produce, often in exuberant amounts. Protein QC (Box 1) is intimately linked to the processes of folding (Ellgaard and Helenius, 2003; Sitia and Braakman, 2003). Both rely on chaperones and devoted resident enzymes. QC serves different roles: (i) it prevents the deployment of aberrant protein conformers, ensuring that only native proteins proceed along the secretory pathway; (ii) it retains precursors in an environment suitable for their maturation; (iii) it increases their local concentration to favor assembly and polymerization; (iv) it reduces the risks of proteotoxicity by inhibiting aggregation and degrading terminally misfolded proteins; (v) it maintains homeostasis in the early secretory pathway; (vi) it is involved in the developmental regulation of protein secretion (IgM, adiponectin, see below) and (vii) it is important for storing proteins for regulated secretion. In certain plants, in fact, ER retention/accumulation is utilized to store abundant proteins during seed formation (Larkins et al, 1993; Jolliffe et al, 2005; Vicente-Carbajosa and Carbonero, 2005). Table 1. Box 1 The logics of QC 1. Preventing the deployment of aberrant protein conformers 2. Retaining precursor proteins in an environment suitable for their maturation 3. Favoring correct assembly by increasing subunit concentration 4. Reducing the risks of proteotoxicity by inhibiting aggregation and degrading terminally misfolded proteins 5. Maintaining homeostasis in the early secretory pathway 6. Developmental regulation of protein secretion (IgM, adiponectin) 7. Storing proteins for regulated secretion (plants, adipocytes) QC, quality control. Protein folding in the ER Upon cotranslational translocation, nascent secretory proteins enter the crowded environment of the ER lumen and soon begin folding into more stable, lower energy, conformation(s) (Dobson, 2004). While the basic principles governing folding are common to other cellular compartments (Anfinsen and Scheraga, 1975; Dobson, 2004), the ER is unique in sustaining a set of covalent modifications, which include removal of the signal sequences, disulfide bond formation, N-glycosylation and GPI addition. A plethora of enzymes and assistants are found in the early secretory pathway, which catalyze each step (Box 2). How is their synthesis regulated so as to have the right balance in different cell types? How are they functionally interconnected? How are the different steps executed in the right order? How are unfolded proteins recognized (Box 3)? Table 2. Box 2 Workers in the secretory protein factory (an incomplete list) (A) ‘Public’ chaperones and enzymes Class Name Localization Function Chaperones BiP/GRP78 ER Folding assistant/unfoldingRegulation of IRE1, PERK and ATF6 in ER signalingTranslocon gating and regulation GRP94 ER Folding assistant ORP150 ER Folding assistant, hypoxia HERP ER membrane ERAD SEL1L ER membrane ERAD Co-chaperones Sil1/BAPERdjs ERER ATP exchange factorBiP cofactors Lectins CNX ER membrane Folding CRT ER soluble Folding ERGIC-53 ERGIC Transport F5, F8, CatZ, CatC, IgM polymers VIPL ER Transport VIP-36 Cis-Golgi Transport EDEM1, 2, 3 ER subregion ERAD OS9 ER membrane ERAD Erlectin/XTP3-B ER membrane ERAD Enzymes redox Ero1α ER+ERGIC Oxidase Ero1β ER Oxidase PDI ER Oxidase, isomerase, reductaseSubunit of prolyl 4-hydroxylaseSubunit of microsomal triacylglycerol transfer protein ERp57 ER Isomerase, oxidase? ERp72 ER Unclear ERp44 ERGIC-cis-Golgi Thiol-mediated retention/IP3R1 regulation Proline isomerases PPIasesCyclophilins ER ER, mitochondria, nucleus, cytosol Proline isomerization Proline isomerization Sugar processing Glucosidase I ER Glucosidase II ER ER Man I ER ER Man II ER UGGT ER Folding sensor Man IA, IB, IC Golgi (B) ‘Private’ tissue- or substrate-specific factors Name Tissue expression Substrates/function Hsp47 Fibroblasts Collagen biosynthesis/chaperone PDIp Exocrine pancreas Zymogens/oxidoreductase PDILT Sertoli cells in testis Calmegin, Δ-somatostatin/ chaperone Egasyn Ubiquitous β-Glucuronidase/chaperone Invariant chain APC MHC class II assembly and transport Tapasin Ubiquitous MHC class I assembly SCAP-RAP Boca/Mesd Ubiquitous LDL receptor assembly and transport APC, antigen-presenting cell; CNX, calnexin; CRT, calreticulin; ER Man, ER α1,2-mannosidase; ER, endoplasmic reticulum; ERAD, ER-associated degradation; ERGIC, ER–Golgi intermediate compartment; LDL, low-density lipoprotein; MHC, major histocompatibility complex; UGGT, UDP-glucose glycoprotein glucosyltransferase. Table 3. Box 3 Monitoring non-native structure 1. Exposure of hydrophobic patches 2. Presence of immature glycans 3. Exposure of reactive thiols Owing to the fact that N-glycosylation is unique to secretory proteins, the folding and QC of glycoproteins have been analyzed in great detail and can be used as a prototypic example of labor organization in the ER protein factory. As in all assembly lines, transport must follow the execution of a given step. The time allocated to the latter, however, must be precisely controlled in order to allow efficiency and prevent jams along the line. The sequential modifications of the oligosaccharides provide an elegant solution to dictate and time the manufacture of cargo glycoproteins. N-glycosylation involves binding of a preformed oligosaccharide (Glc3Man9GlcNAc2) to asparagine side chains in the sequence NXS/T, where X is any amino acid other than proline (Khalkhall and Marshall, 1975). The sugar moieties are then progressively trimmed by resident enzymes of the secretory pathway. Soon after synthesis, glucosidases I and II sequentially remove the three glucose moieties from the A branch of the oligosaccharide moieties (Figure 2). UDP-glucose glycoprotein glucosyltransferase (UGGT) adds back a glucose residue to N-glycans positioned near regions of disorders (Taylor et al, 2004). Therefore, UGGT acts as a folding sensor and produces monoglucosylated proteins (Glc1Man9GlcNAc2) that can interact with calnexin (CNX) or calreticulin (CRT), two ER chaperones with lectin activity (Waisman et al, 1985; Ahluwalia et al, 1992; reviewed in Williams, 2006). CNX and CRT retain misfolded substrates in the ER, prevent their aggregation and promote oxidative folding via interactions with ERp57 (Ellgaard et al, 2001; Schrag et al, 2001; Frickel et al, 2002; Russell et al, 2004). By removing the terminal glucose, glucosidase II dissociates the substrate from CNX/CRT for a novel round of inspection by UGGT. Figure 2.The CNX/CRT cycle. After transfer of the preformed core oligosaccharide (Glc3Man9GlcNAc2) onto nascent proteins, glucosidase I and II sequentially remove the two terminal glucoses from the A branch. The monoglucosylated Glc1Man9GlcNAc2 unfolded protein can now interact with the lectin chaperones CNX and CRT. In association with the oxidoreductase ERp57, CNX and CRT prevent aggregation and facilitate glycoprotein folding. Removal of the glucose by glucosidase II (Man9GlcNAc2) interrupts the interaction of the protein with CNX/CRT. If the protein has attained its native structure, it can now proceed along the secretory pathway by bulk flow or by interaction with specific lectin transporters such as ERGIC-53 or VIPL. If unfolding persists, the glycoprotein is recognized by UGGT1, which places a single glucose back onto the A branch, causing the protein to enter the CNX/CRT cycle again. Mannose trimming causes exit from the CNX/CRT cycle. Misfolded proteins can be recognized by specific lectins (EDEMs, OS9, etc) and targeted to degradation. Download figure Download PowerPoint How do terminally misfolded proteins escape the cycle? Glycan processing again comes into action, because removing the terminal mannose moieties inhibits glucose re-addition. Mannose trimming hence acts as a timer, discriminating between junior proteins (which should be given the time to fold) and senior ones, which should be either secreted or sent to degradation. Many proteins with mannosidase activity reside in the early secretory apparatus (e.g., ER α1,2-mannosidase I (ER Man I), EDEM1 and 3, Golgi Man IA, IB, IC, ER Man II). Man I inhibitors (e.g., kifunensine), which prevent removal of the terminal B-branch mannose, stabilize ERAD glycoprotein substrates, but do not prevent secretion of native species. Overexpression of ER Man I and EDEMs accelerates degradation (Hosokawa et al, 2006 and references therein). ER–Golgi intermediate compartment-53 (ERGIC-53) (a protein transporter with lectin activity, cycling between the ER and the ERGIC, see below) and possibly other L-type lectins (e.g., VIPL, VIP36; Kamiya et al, 2007) bind high-mannose cargoes, facilitating their forward transport. Further mannose trimming in the ER may favor degradation, possibly also because reducing the hydrodynamic volume of substrate glycoproteins could facilitate their retrotranslocation. It will be of great interest to determine the precise binding specificities and fate of the various intermediates in glycan processing (Helenius and Aebi, 2001). Another well-characterized folding pathway is based on Binding Protein (BiP, also called GRP78), an abundant chaperone of the hsp70 family, which serves also a key regulatory role in ER signalling (Bertolotti et al, 2000). BiP was first isolated as a protein associating with unassembled Ig-H chains (Haas and Wabl, 1983). It consists of an N-terminal ATPase domain and a C-terminal domain with affinity for hydrophobic patches (Flynn et al, 1991; Blond-Elguindi et al, 1993). The affinity for substrates depends on ATP binding at the N-terminal domain. When ATP is hydrolyzed to ADP, a conformational change occurs, which determines substrate release. Thus, substrates can undergo cycles of BiP binding and release, depending on ATP hydrolysis (Gething, 1999). Owing to the weak BiP ATPase activity, hsp40-like co-chaperones containing J domains (ERdj) play a key regulatory role. Five ERdj proteins have been isolated so far (Shen et al, 2002; Cunnea et al, 2003; Kroczynska et al, 2004; Shen and Hendershot, 2005). One of them, ERdj5, also displays oxidoreductase acitivity, possibly linking BiP-dependent folding/unfolding and disulfide bond formation, isomerization or reduction (Nagata et al, personal communication). Very rarely glycoproteins are found to bind simultaneously to BiP and CNX or CRT. Therefore, it seems that a given glycoprotein enters first either the BiP or the CNX/CRT pathway (Figure 3). The initial choice is dictated by the localization of the N-glycans: the closer these are to the N-terminus of the nascent protein, the higher the tendency to use CNX as a chaperone system (Molinari and Helenius, 2000). If the first attempts to fold fail, the protein can shift to the alternative pathway. Altogether, these data imply that sites of conjunction exist in which the substrate can jump from one pathway to another. In principle, however, it should be possible for large multi-domain proteins to engage with both. It would be of interest to determine whether ER sub-regions exist that are enriched in either pathway. Figure 3.QC in the early secretory pathway. Chaperones and folding assistants can be grouped in different classes according to their specificity and subcellular localization. The majority of secretory proteins utilize public chaperones: some initially go with BiP, PDI and their partners, others enter the CNX/CRT cycle, the choice depending on the location of the N-glycans. Certain proteins that are produced in large amounts, or are intrinsically difficult to fold, are assisted by specific (private) chaperones and enzymes (see also Box 2). In addition, QC can occur in sequential steps. After a proximal QC, certain substrates (generally multimeric proteins) seem to undergo also distal QC checkpoints in ERGIC and cis-Golgi. This model could mediate cargo concentration and selective export of oligomerized species, thus coupling fidelity and efficiency in the secretory protein factory. While proximal QC can rely on simple retention, the distal checkpoints likely imply substrate retrieval to the ER, either for further attempts to fold, or for retrotranslocation and degradation. Download figure Download PowerPoint Supplementary Table 1S shows the phenotypes of cells, mice and patients in which individual chaperones, enzymes or sensor molecules are insufficient or absent altogether. BiP−/−, ERp57−/−, CNX−/− and CRT−/− mice show embryonic or perinatal lethality, but their phenotypes vary considerably: CRT−/− mice have severe problems in cardiac development, while large myelinated fibers in peripheral nerves are the main targets in CNX−/− animals (Mesaeli et al, 1999; Denzel et al, 2002; Garbi et al, 2006). BiP is essential also for survival of cells in culture, in agreement with its role in regulating translocation and ER signalling. In contrast, CRT−/− and CNX−/− cells are viable, and their phenotypes surprisingly mild, suggesting redundancy in substrate recognition by the two chaperones. Oxidative folding In terms of ionic composition and redox potential, the ER is similar to the extracellular space, providing an ideal folding place/test bench for proteins destined to the external world. Although certainly important, the higher ratio between oxidized and reduced glutathione (GSSG/GSH) in the ER, compared with the cytosol (Hwang et al, 1992), is not enough to guarantee efficient oxidative folding. Indeed, for many proteins to fold correctly, disulfide bond isomerization, and sometimes also reduction (Jansens et al, 2002), is needed. A hyper-oxidizing environment in the ER lumen may hence inhibit folding of proteins with multiple disulfides, and promote aggregation (Molteni et al, 2004). Therefore, oxidative folding relies primarily on protein–protein interchange relays. The main pathway involves disulfide transfer from PDI or PDI-like proteins to nascent cargoes. PDI consists of four thioredoxin (trx) domains: the two lateral domains (a and a′) are endowed with oxidoreductase activity, while the two central ones, b and b′, provide a hydrophobic surface suited to bind and present nascent proteins to the active sites in a and a′. This overall structure is likely important for the redox-dependent chaperone function of PDI (Wilkinson and Gilbert, 2004; Forster et al, 2006a), particularly with terminally misfolded proteins, which must be reduced before dislocation to the cytosol for proteasomal degradation (Gillece et al, 1999; Fagioli et al, 2001; Tsai et al, 2001, 2002; Molinari et al, 2002). After transferring a disulfide bond to nascent proteins, PDI is re-oxidized by members of the Ero1 flavoprotein family (Frand and Kaiser, 1998; Pollard et al, 1998; Cabibbo et al, 2000; Pagani et al, 2000; Mezghrani et al, 2001). In vitro, yeast Ero1p can use molecular oxygen as terminal electron acceptor, in a reaction that produces hydrogen peroxide in stoichiometric amounts to the disulfides formed (Tu and Weissman, 2002; Gross et al, 2006). Studies are ongoing in several laboratories to determine whether H2O2 is generated in living cells as a byproduct of oxidative folding, because this could serve signalling purposes. However, at least in yeast, disulfide bond formation can proceed in anaerobic conditions (Gross et al, 2006), suggesting that alternative electron acceptors exist. Over the last years, many other ER-resident PDI-like oxidoreductases have been characterized in mammalian cells. The precise role(s) of these molecules, as well as the mechanisms controlling their redox state and activity, remain to be clarified (Ellgaard and Ruddock, 2005). Some of them, for example, PDIp (Desilva et al, 1996) and PDILT (van Lith et al, 2005; van Lith et al, 2007), are selectively expressed in pancreas and testis, respectively, and hence belong to the growing group of substrate- or tissue-specific (‘private’) chaperones, including Hsp47, etc. (Box 2B). Disulfide bond formation is crucial in the folding and QC of secretory proteins. Since they increase the stability of the native conformation, their absence, or even worse, their mispairing, generally produces severely misfolded species. Furthermore, an exposed cysteine in the proper amino acid context is sufficient to cause retention and degradation of otherwise transport competent intermediates (Fra et al, 1993; Guenzi et al, 1994), likely because the reactivity of thiol groups favors the formation of mixed disulfides with PDI, ERp44 and other resident proteins (Reddy et al, 1996; Anelli et al, 2003, 2007). The thiol-dependent retention mechanisms, originally described in the developmental control of IgM (Sitia et al, 1990), have recently been shown to control also adiponectin secretion (Wang et al, 2007). Bulk flow, retention, retrieval and selective export Since the discovery of the KDEL motif as a means to localize soluble proteins in the ER (Munro and Pelham, 1987), the problem arose as to how these extremely abundant residents rarely saturate the KDEL receptors. A possible answer lies in the discovery of supra-molecular complexes comprising different chaperones (BiP, GRP94, ERdj3 and UGGT, but no CRT; Meunier et al, 2002; Gilchrist et al, 2006). Because of their different diffusibility, these complexes could be excluded from forward moving vesicles, and form a matrix to retain folding intermediates in a suitable environment for their maturation. The presence of UGGT in these complexes may be important for shifting misfolded substrates to the CNX–CRT pathway. Unless retained by interactions with resident proteins, a protein could exit from the ER by bulk flow (Wieland et al, 1987). However, many proteins are actively transported out of the ER by interaction with specialized export machineries (see Gurkan et al, 2006 for a review). Export from the ER occurs at ER exit sites (ERES; Mezzacasa and Helenius, 2002), where budding of COPII-coated vesicle takes place. It is now evident that transportable cargoes contribute to the formation of ERES- and COPII-coated vesicles (Forster et al, 2006b). Moreover, in exocrine pancreatic cells, the ER–Golgi interface is where different secretory proteins reach their highest intracellular concentration (Martinez-Menarguez et al, 1999; Oprins et al, 2001), which could have important consequences for the biogenesis of oligomeric proteins (see below). Specific transporter molecules mediate the exit from the ER of certain glycoproteins, concentrating them into forward transport vesicles (see Hauri et al, 2002; Lee et al, 2004 for reviews). In mammalian cells, one of the best characterized is ERGIC-53, a hexameric transmembrane lectin (Schindler et al, 1993) that derives its name from being particularly abundant in the ERGIC. ERGIC-53 is described to capture high-mannose glycoproteins in the ER, and release them in the ERGIC in a Ca2+- and pH-dependent manner (Appenzeller-Herzog et al, 2004). Mutations in ERGIC-53 (also known as LMAN1) or in MCFD2, a gene encoding a small soluble protein that associates with ERGIC-53 (Zhang et al, 2003, 2005; Baines and Zhang, 2007), are responsible for most cases of combined deficiency of coagulation factors V and VIII (F5F8D), a recessive bleeding disorder characterized by decreased serum levels of both clotting factors (Nichols et al, 1998; Neerman-Arbez et al, 1999). The rather limited phenotype of patients who lack functional ERGIC-53 suggests that other lectins serve redundant functions in controlling glycoprotein traffic (e.g., VIP36 (Fiedler et al, 1994), VIPL (Neve et al, 2003), ERGL (Yerushalmi et al, 2001; see Hauri et al, 2002 for a review)). The specificity of these lectins has recently been analyzed (Kamiya et al, 2007): VIPL and VIP36 interact preferentially with glycans carrying, on their A branch, three mannoses but no terminal glucoses, (see also Fullekrug et al, 1999). Unexpectedly, ERGIC-53 displays low-affinity and broad-specificity interactions with high-mannose oligosaccharides also when monoglucosylated at the A branch. Moreover, while VIPL and ERGIC-53 bind better at pH 7, (as found in the ER), VIP36 has an optimum at pH 6.5 (as in the Golgi). From these data, it has been suggested that VIPL binds de-glucosylated cargoes exiting the CNX/CRT