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Home , Calnexin, Calreticulin, Lectin

Protein folding in living cells is a complex, error-prone , process. Numerous mechanisms are in place to ensure that newly synthesized reach their folded and functional form. One of the strategies is the covalent attachment of to asparagine residues of nascent polypeptide chains. Such N-linked glyco- the folding of sylation occurs for the majority of soluble and mem- brane-bound proteins that are synthesized in the (ER). The addition of oligo- saccharides can be beneficial during maturation by making folding intermediates more soluble. In ad- dition, it allows the newly synthesized polypeptides to bind to calnexin and calreticulin, unique pres- ent in the ER, which serve as molecular chaperones. Several reviews have been published on cabrexin and calreticulin in recent years that provided descriptions of the discovery and molecular characterization of these resident ER chaperoneslA. Here, we outline what Calnexin and calreticulin are molecular chaperones in the is currently known about calnexin, calreticulin and ac- endoplasmic reticulum (ER). They are lectins that interact with cessory in the process of folding. newly synthesized glycoproteins that have undergone partial Calnexin and calreticulin are lectins trimming of their core N-linked oligosaccharides. Together with the Calnexin is a nonglycosylated type I membrane (65 kDa, 573 amino acids) with its substrate- enzymes responsible for removal and a glucosyltransferase binding domain in the lumen of the ER. It has an 89-residue cytoplasmic tail that is phosphorylated that re-glucosylates already-trimmed glycoproteins, they provide a and carries a C-terminal RKPRRE sequence that serves novel mechanism for promoting folding, oligometic assembly and as an ER-localization signalz. Calreticulin, the soluble homologue (46 kDa, 400 amino acids), has both a quality control in the ER. KDEL retrieval sequence and a highly negatively charged region responsible for Ca2+ binding and ER retention at its C-terminus4,5. The primary amino acid sequences of calreticulm and the lumenal domain of calnexln show extensive stretches of homology. by the oligosaccharyltransferase associated with the Although reported to be localized in several cell translocation machinery” (Fig. 1). They are attached compartments, calreticulin is clearly more abundant to the side chain of asparagine residues in the in the lumen of the ER, where it is one of the major consensus sequence Asn-X-Ser/Thr. Transfer of the proteins. A variety of functions have been ascribed to generally occurs co-translationally as calreticulin, among them regulation, Ca2+ se- soon as the acceptor site enters the lumen of the ER. questration and RNA bindin&. It is now clear that, The original core contain a string of three like calnexin, it is a molecular that binds glucose residues. Their removal begins on the grow- transiently to many glycoproteins in the ERGy. Both ing nascent chain. Glucosidase I removes the termi- seem to function as monomers, although they may nal oll,2-linked glucose, followed by elimination of be part of a larger dynamic network of proteins that the two remaining al,3-linked by glucosi- includes other ER chaperones and folding enzymes*. dase II (Fig. 1). Compared with other oligosaccharide- Initial experiments in tissue culture cells treated modification reactions in the secretory pathway, glu- with inhibitors showed that calnexin cose trimming is exceptionally efficient; nearly all selectively associated with folding intermediates of N-linked chains are completely de-glucosylated. The glycoproteinsiO. Further studies using glucosidase Golgi complex of some cell types contains an endo- inhibitors demonstrated that calnexin bound to par- mannosidase that eliminates any residual glucose tially glucose-trimmed glycoproteins” . Subsequently, residues that may have been missed in the ER18. it was shown that calnexin1214 and calreticulin6,7 The monoglucosylated glycans that bind to calnexin The authors are in specifically associated with monoglucosylated glyco- and calreticulin arise in the ER as an intermediate dur- the Dept of proteins. Biochemical analysis using isolated oligo- ing the stepwise removal of glucoses. However, the same Cell Biology, saccharides has confirmed that they are a new type structure is also formed by re-glucosylation of fully Yale University of and that they bind monoglucosylated core glucose-trimmed oligosaccharides. This occurs through School of glycans (Glc,Man,-,GlcNAc2), whereas they do not the action of a lumenal , the UDP-Glc:glyco- Medicine, bind glycans with two or three or no glucose resi- protein glucosyltransferase1y*20 (Fig. 1). This enzyme 333 Cedar Street, duesi5,16. Both the affinity for the oligosaccharides counteracts the function of glucosidase II by adding New Haven, and the number of binding sites are unknown. glucose residues back to the oligosaccharides, thus re- CT 06520-8002, generating monoglucosylated glycans2i. The existence USA. Generating the oligosaccharide ligands of de- and re-glucosylation reactions introduces the possi- E-mail: N-linked glycans are added to growing polypeptide bility of a cycle that involves association with, and dis- ari.helenius@ chains as 14resIdue oIigosaccharides (Glc,Mar+GlcNAc,) sociation from, calnexin and calreticulin (see below). yale.edu

trends in CELL BIOLOGY (Vol. 7) May 1997 Copyright 0 1997 Elsevier Science Ltd. All rights reserved. 0962-8924/97/$17.00 193 PII: 50962-8924(97)01032-5 Glucosyl- transferase E Glucosidase II -Asn- -Asn- -Asn- T-Asn-

A Glucose 0 0 N-acetylglucosamine

FIGURE 1

N-glycosylation and glucose trimming in the endoplasmic reticulum (ER). The core oligosaccharide (Glc,Man,GlcNAc,) is synthesized in the ER membrane and transferred to the consensus N-glycosylation sites (Am-X-Ser/lhr) on nascent polypeptide chains. Immediately after transfer, the glucose residues are removed by the sequential action of glucosidases I and II. Clucosidase I removes the terminal al-24inked glucose, whereas glucosidase II removes the two remaining al-3-linked residues17. Fully glucose-trimmed high-mannose glycans can be re-glucosylated by UDP-glucose:glycoprotein glucosyltransferase if present on incompletely folded or assembled proteinslg.

The enzymes that generate the monoglucosylated A model for transient glycoprotein binding to glycoproteins have been known for many years, but calnexin and calreticulin their have been cloned only recently (Table 1). Binding of glycoproteins to calnexin and calreticu- Glucosidase I is a type-11 membrane glycoprotein lin is a transient step during early maturation. Depend- (92 kDa). Glucosidase II, by contrast, is soluble and ing on the distribution of glycans in the polypeptide does not share any primary with chain, associationcan begin with the growing nascent glucosidase I. In mammalian cells, it is a heterodimer chain30. It continues posttranslationally for variable in which the cx subunit (104 kDa) contains the active periods of time-ranging from a few minutes to hours. site, and the B subunit (58 kDa) carries retrieval and With permanently misfolded proteins, or unassembled retention sequences for ER localization. subunits of oligomers, the association usually con- UDP-Glc:glycoprotein glucosyltransferase is a sol- tinues until the protein is degraded9J*J2J3,31-33.Final uble enzyme of 170 kDa. It and glucosidase II are the releasefrom calnexin and calreticulin occurs when only soluble enzymes involved in the processing of the polypeptides have reached a fully folded confor- N-linked in the secretory pathway. Its mation or when they have undergone oligomeric C-terminal 500 amino acids share homology between assembly (for reviews, seeRefs 2, 34 and 35). different species and with other known glucosyl- According to one model (Fig. 2a; Ref. 36), glyco- transferases, suggesting that it probably contains the intermediates are retained in the ER catalytic domain. by a cycle of glucose removal and addition coupled The glucosyltransferase only recognizes non-native to chaperone binding and release.Glucosidase II per- glycoproteins as substratesz2J3. Among the proteins forms a dual role: it is needed to uncover the mono- capable of distinguishing native from non-native poly- glucosylated glycans for initial binding and afterwards peptides, it is the only one known to modify its sub- to de-glucosylate them completely, thus releasing strates covalently. As long as glycoproteins are incom- the glycoproteins from the chaperones.The glucosyl- pletely folded or misfolded, they are re-glucosylated transferase allows reassociation by re-glucosylating by the transferase and continue to be bound to cal- unbound non-native proteins. The glycoprotein nexin and calreticulin. This is illustrated by the ts045 exits the cycle when it has reached its native form vesicular stomatitis (VSV) G protein, which has and becomes ‘invisible’ to the glucosyltransferase. a thermoreversible folding defect. At the nonpermiss- Consistent with the need for glucose removal for ive temperature, the G protein undergoes continu- substrate release,inhibition of glucosidaseII activ- ous de- and re-glucosylation in the ER of infected ity inhibits release of substrates from calnexin and cells2* and it remains calnexin associatedz4. The ma- calreticulin9,13,28,37*38.It also prevents complete fold- jority of glycoproteins undergo re-glucosylation at ing and oxidation of bound influenza HA molecules, least once25. While it is not clear how the transferase suggestingthat cycling of the substrate on and off distinguishes between folded and unfolded substrate the chaperones is important for proper folding9. glycoproteins, the enzyme can detect very subtle dif- ferences between protein conformers (E. S. Trombetta Do calnexin and calreticulin interact with the and A. Helenius, unpublished). polypeptide moiety of substrates? Glucosidases I and II are sensitive to l-deoxynojiri- Most studieswith glycosylation and glucosidasein- mycin, castanospermine and related a-glucosidase hibitors, mutant cell lines and site-specific mutagen- inhibitors. By preventing glucose trimming, these esisof consensusglycosylation siteshave confirmed agents inhibit binding of newly synthesized glyco- that calnexin and calreticulin binding requires the proteins to the chaperones7,8,11,12,2~28. Therefore, presenceof one or more monoglucosylated N-linked they have been used to study the maturation of glycans6,7,1@14v37,39,40.In the model outlined above glycoproteins in the absence of calnexin and cal- (Fig. 2a), calnexin and calreticulin function as lectins, reticulin binding9f24,27,29. whereasthe glucosyltransferasemonitors the folding

194 trends in CELL BIOLOGY (Vol. 7) May 1997 TABLE I - PROTEINS INVOLVED IN THE CALNEXINKALRETICULIN CYCLE

Protein Cells defective In the respective gene

Mammals 5. cenwisiae Other

Clucosidase I (a1 LEC23 lb1 glsl-1 lC’ No growth defect N-r1800a.a. 1-c

Glucosidase II Id’ Phor 2.7 le) Agis2 ‘*’ No growth defect a 900 aa. a subunit

[i500 a.a.1 B subunit

Glucosyltransferase ‘f,g) ND# Akre5 l Ihl 5. pombe Agpt I @) Severe growth defect No growth defect 1500 a.a.

Calnexin (1’ CEM-NKR IJ) Acne1 l * lkli S. pombe cnxl (men) No growth defect Essential N-WI-C

Calreticulin ‘0’ ND l **

#None described. *KRES is the only gene found in the Saccharomyces cerevisiae genome showing moderate homology with other UDP-Clc: glycoprotein glucosyltransferases. No evidence has been obtained indicating that KreSp is a functional glycoprotein re-glucosylating enzyme [Ref. (p)]. **CNEI is the only gene found in the 5. cerevisiae genome showing some similarity to those encoding calnexin or calreticulin; Cnel p is a only marginally related to the calnexin family. ***No protein similar to calreticulin is found in the 5. cerevisioe genome. Abbreviations: a.a., amino acids; 5. pombe, Schizosoccharomyces pombe. References (ai Kalz-Fuller, B. et a/. (1995) Eur. 1. Biochem. 231, 344-351; w Ray, M. K. et ol. (1991) /. Biol. Chem. 266, 22818-22825; w Esmon, B. et a/. (1984) /. B/o/. Chem. 259, 10322-l 0327; Id) Trombetta, E. 5. et a/. (1996) 1. Biol. Chem. 271, 27509-27516; te) Reitman, M. L. et a/. (1982) /. Biol. Chem. 257, 10357-l 0363; (0 Parker, K. et a/. (1995) EMBO 1, 14, 1294-l 303; (g) Femandez, F. et a/. (1996) EM60 1. 15, 705-713; ihJ Meaden, P. et o/. (1990) Mol. Cell. t?io/. 10, 3013-3019; ‘I) Bergeron, J. J. et ol. (1994) Trends Biochem. Sci. 19, 124-l 28; iJi Scott, J. and Dawson, J. (1995) 1. Immunol. 155, 143-l 48; (H de Virgilio, C. et o/. (1993) Yeast 9, 185-l 88; (I) Parlati, F. et a/. (1995) /. Biol. Chem. 270, 244-253; cm’ Parlati, F. et a/. (1995) EMBO /. 14, 3064-3072; (“) Jannatipour, M. and Rokeach, L. A. (1995) /. Biol. Chem. 270, 48454853; ‘o) Michalak, M., ed. (1996) Co/reticu/in, R. C. Landes; ‘P) Fernandez, F. 5. et CJ/. (1994) 1. Biol. Chem. 269, 30701-30706.

status. However, there is an ongoing debate whether with ER chaperonesg5f4’j.The E subunit of the T-cell binding to the chaperones also involves protein- receptor also aggregates when expressed without protein interactions. other subunitsof the receptor (J. Hoppa and H. Ploegh, One of the reasonsfor the uncertainty is that some pers. commun.). If aggregation is the reason why glycoproteins artificially deprived of addition nonglycosylated proteins can coprecipitate with cal- by using tunicamycin, or by mutation of consensus nexin, it remains unclear whether there is a direct glycosylation sequences, have been shown to co- interaction between the nonglycosylated protein immunoprecipitate with calnexin4143.The E subunit and calnexin. of the T-cell receptor is also invoked as a naturally Another seriesof observations has suggestedthat a occurring nonglycosylated protein that binds to glycoprotein-calnexin interaction may be mediated calnexin because,when overexpressedtogether with both by -protein and protein-protein mutant calnexin, its distribution in cells is alteredg4. interactions. When isolated anti-calnexin immune Calreticulin has not been reported to bind to non- complexes were treated with endoglycosidaseH, the glycosylated proteins. oligosaccharides were removed but the substrate For VSV G-protein mutants, the coprecipitation polypeptides did not elute from the immuno- phenomenon was recently reported to depend on ag- beads1s,39,42.Clearly, the substratesremained bound gregation of the misfolded, nonglycosylated protein by interactions other than those occurring between in the ER40.It is commonly observed that, when ex- the oligosaccharides and the chaperone. Most of pressed without N-linked oligosaccharides, many these experiments were performed with membrane glycoproteins misfold, aggregate and coprecipitate proteins that may remain artificially trapped in a trends in CELL BIOLOGY (Vol. 7) May 1997 195 (4

Transferase / /x \ Glucosidase II

- q - q G,ucosidase,, (-Trg Glucosidases I and II 0

CHO-protein interaction

Glucosidases

CHO-protein Protein-protein interaction interaction (weak) (strong)

1 @ :“r’plete’y 0 Folded 1

FIGURE 2 Models for calnexin and calreticulin binding to their substrate glycoproteins. (a) Lectin-only modeP6. Glucose residues (G) in the core oligosaccharides are trimmed by glucosidases I and II. When trimmed to the monoglucosylated form, the oligosaccharides mediate the binding of the glycoprotein to calnexin (CNX) and calreticulin (CRT). These function as lectins. Release of the substrate glycoprotein depends on hydrolysis of the remaining glucose residues by glucosidase II. If the glycoproteins are not completely folded, the high- mannose glycans are selectively re-glucosylated by the glucosyltransferase, allowing the oligosaccharides to rebind to the chaperones. Once glycoproteins reach their mature conformation, they are no longer recognized by the glucosyltransferase. (b) Dual-mode model. According to this model, the monoglucosylated oligosaccharides are merely needed to bring glycoproteins into contact with calnexin (CHO-protein interaction). After the initial carbohydrate-mediated association, stronger protein-protein interactions occur between the chaperone and peptide elements exposed on the surface of the incompletely folded substrate protein. These stabilize the complex until the peptides are hidden in the folded protein. Thus, release of the substrate depends on conformational changes in the substrate glycoprotein that eliminate the protein-protein interaction. It may also involve ATP-mediated conformational changes in calnexin. The model is based in part on reports by Ware et a/.15 and Williams3.

common detergent micelle with calnexin after the the substrate polypeptide. As the peptide segments glycan is removed. Nonetheless, these results may responsible for binding are buried through folding suggest that protein-protein interactions are pres- of the protein, the complex becomes destabilized ent. Together with the observation that class-I heavy and dissociates. Release has also been proposed to be chains can be chemically crosslinked to calnexin at modulated by ATP binding to calnexin3Js,48. ATP has a site close to the transmembrane region4’, these been reported to bind to calnexin, but there is no findings have inspired a model in which the oligo- evidence for ATPase activiQ@. saccharides merely act to bring substrate and cal- In recent studies using bovine pancreatic ribonu- nexin into close proximity, whereafter a protein- clease (RNase) as a ligand, evidence for the lectin- protein interaction, similar to that seen for classical only model has been found37,49. RNase-calnexin and chaperones, takes over3Js (Fig. 2b). Once the com- RNase-calreticulin complexes were treated with plex has been established, the oligosaccharide com- N-glycanase F, which removed the N-linked oligosac- ponent is thought to be dispensable for binding. charides, or with glucosidase II, which just removed According to this model, release of the substrate the glucose residues 37. In either case, full dissociation does not occur through the action of glucosidase II, of the complexes occurred. It was concluded that it but rather as a result of a conformational change in was the interaction between the oligosaccharides and

196 trends in CELL BIOLOGY (Vol. 7) May 1997 FIGURE 3 the chaperones that held these complexes together. Mapping of calnexin- and This was further supported by observations that the calreticulin-binding domains on fully folded RNase binds to calnexin and calreticu- influenza haemagglutinin (HA). The lin3?, and the ectodomain of calnexin49, as efficiently location of N-linked glycans plays a as does unfolded RNase. Glucosidase-II-dependent role in differential calnexin and release from calnexln and calreticulin also occurred calreticulin binding to HA, suggesting irrespective of the folding status of the RNase3’. spatial constraints in determining chaperone specificity (D. N. Hebert The evidence for a lectin-like association both and A. Helenius, unpublished). The in vivo and in vitro is strong. It is based not only on N-linked glycosylation sites are the direct biochemical binding studies with isolated indicated by the red circles. oligosaccharides but also on extensive studies in cells Glycosylation sites in the stem domain and microsomes. As the need for glucosidase II to dis- were found to be involved in calnexin sociate the complexes also has been demonstrated binding, whereas the glycosylation experimentally, this suggests that an oligosaccharide- sites in the globular/hinge region mediated interaction is needed for establishing the were required for calreticulin binding. complexes and for maintaining them9J3f38. Further- The binding of the chaperones to more, experiments with microsomes have shown that distinct regions of the molecule re-glucosylation not only occurs but also results in permitted the formation of ternary substrate binding to calnexin13. More experiments will HA-calnexin-calreticulin complexes as be needed to evaluate the possible role of protein- depicted. The structure of HA was protein interactions - for which the evidence so far adapted from Ref. 59. is indirect. Only a few proteins have been analysed in detail. To obtain a more complete picture, detailed biochemical and structural information is needed for several soluble and membrane-bound substrate proteins. Both calnexin and calreticulin need to be included in these studies. It will also be important to design binding assays better than those based on co-immunoprecipitation.

Are calnexln and calreticulin functionally Such differences between the chaperones may re- distinct? flect differential accessibility of the glycans from the Since the lectin specificities of calnexin and cal- lumenal versus membrane-proximal side of a nascent reticulin are identical15,16, it is not surprising that or newly synthesized protein. The shift of the MHC there is extensive overlap between their substrate class-I heavy chain from calnexin to calreticulin glycoproteins. Not only do they bind to similar sets when it binds to B,-microglobulin may, for example, of proteins but they can also associate simultaneously reflect a conformational change that increases the with the same glycoproteinz8 (D. N. Hebert and lumenal exposure of the glycans. Alternatively, the A. Helenius, unpublished). specificity differences could be explained by exposure Closer analysis of the substrate specificity reveals of polypeptide determinants that interact differently interesting differences. VSV G protein, which has two with calnexin and calreticulin (see Fig. 2b). N-linked glycans, binds to calnexin but not calreticu- lin24. The protein associates first with BiP, an Hsp70 How important is the calnexin/calreticulin cycle? analogue, whereafter it is transferred to calnexin. In Calnexin and calreticulin associate with a wide va- the case of human class-I MHC molecules, calnexin riety of proteins, including soluble secretory proteins, binds to the free heavy chain but is soon replaced by complex membrane receptors and ion channels, calreticulin when the heavy chain oligomerizes with lysosomal hydrolases and com- the B,-microglobulin subunitso. Influenza HA, by ponents (Table 2). They also play a role in the biosyn- contrast, associates initially with both calnexin and thesis of viral glycoproteins, including those of calreticulin, but calreticulin dissociates from the HIV-l, virus and influenza virus. Their complex earlierg. Mutation of glycosylation consen- substrate-binding function has been linked to a num- sus sites in different combinations has shown that ber of diseases with an ER-storage phenotype, such calreticulin binding to HA depends on three glycans as cystic fibrosis, familial hypercholesterolaemia and in the top and hinge domain of the protein, whereas a,-antitrypsin deficiency34,53. In these disease states, calnexin binding depends on the four sites in the they serve as part of the machinery that retains the stem domain (D. N. Hebert and A. Helenius, unpub- misfolded glycoproteins in the ER. lished; Fig. 3). The top domain reaches its oxidized Calnexin/calreticulin binding increases the effi- form earlier than the stem domain, which explains ciency of glycoprotein folding both in live cells and why calreticulin binding ceases earlier than calnexin in microsomes. It is not difficult to find evidence of binding. Differences are also observed for the T-cell deleterious effects on the maturation of specific glyco- receptor (TCR) subunits: TCR a, TCR 0, CD36 and proteins when binding to the chaperones is inhib- CD3& bind to calnexin12,44J1,52, whereas only the o! ited. Addition of glucosidase inhibitors results in a and B subunits bind to calreticulin, and this for lowered rate and efficiency of for many shorter times than they bind to calnexin38. glycoproteins54. Also, the folding and subsequent

trends in CELL BIOLOGY (Vol. 7) May 1997 197 TABLE 2 - GLYCOPROTEINS THAT BIND TO CALNEXIN AND CALBEllCULlN

Ligands Calnexin Calretlculln RefS

Endogenous glycoproteins al -antitrypsin + ND 0 al -chymotrypsin + ND a Apo B-l 00 + ND a CFTR + ND C Complement 3 + ND a Fibrinogen + ND d GABA, receptor + ND e GLUT-l glucose transporter + + f gpB0 (MDCK glycoprotein) + ND 9 lntegrins + f h-j MHC class I + + k-u MHC class II + ND v-x Myeloperoxidase ND + Y Nicotinic acetylcholine receptor + ND z P-glycoprotein + ND aa T-cell receptor + + bb-dd Thyroglobulin + ND ee + + Of

Viral glycoproteins Cytomegalovirus glycoprotein B + ND 99 HIV gpl60 + + hh HA + + ii-II VW-c + - jj, mm,00 Hepatitis C virus El /E2 + ND PP

aO~, W. J. et al. (1993) Nature 364, 771-776; bLe, A. et al. (1994) /. Biol. C/rem. 269, 7514-7519; CPind, 5. et 01. (1994) /. Biol. Cbem. 269, 1278412788; dRoy, 5. et ol. (1996) /. Biol. Gem. 271, 24544-24550; %onnolly, C. N. et ol. (1996) /. Biol. Gem. 271,89-96; ‘Oliver, J. D. et ol. (1996) /. Biol. Chem. 271, 13691-l 3696; sWada, I. et ol. (1994) 1. Biol. Gem. 269, 7464-7472; hLenter, M. and Vestweber, D. (1994) /. Biol. Chem. 269,12263-l 2268; ‘Hotchin, N. A. et al. (1995)). Cell Biol. 128,1209-l 219; iCoppolino, M. et 01. (1995) /. Biol. Gem. 270, 23132-23138; kDegen, E. and Williams, D. B. (1991)/. Ce//Bio/. 112, 1099-l 115; ‘Capps, C. G. and Zuniga, M. C. (1994) /. Biol. Chem. 269, 11634-l 1639; mJackson, M. R. et ol. (1994) Science 263, 384-387; “Ortmann, B. et 01. (1994) Nature 368, 864-867; “Rajagopalan, S. and Brenner, M. B. (1994) /. Exp. Med. 180, 407412; PBalow, j. P. et 01. (1995) 1. Biol. Gem. 270, 29025-29029; ‘Xarreno, B. M. et al. (1995) /. Immunol. 155, 4726-I733; ‘Nossner, E. and Parham, P. (1995) 1. Exp. Med. 181, 327-337; Tector, M. and Salter, R. D. (1995) /. Biol. Chem. 270, 19638-l 9642; Yassilakos, A. et al. (1996) EMBO /. 15, 1495-l 506; “Sadasivan, B. et al. (1996) immunity 5, l-20; “Anderson, K. 5. and Cresswell, P. (1994) EMSO]. 13, 675-682; “Schreiber, K. L. et al. (1994) Int. Immunol. 6, 101-l 11; “Marks, M. S. et 01. (1995) J. Biol. Gem. 270, 10475-l 0481; YNauseef, W. M. et al. (1995) /. Biol. Chem. 270, 474111747; zGelman, M. S. et al. (1995) /. Biol. Chem. 270, 15085-l 5092; aaLo~, T. W. and Clarke, D. M. (1994) /. Biol. Chem. 269, 28683-28689; bbKearse, K. P. et al. (1994) EMSO]. 13, 3678-3686; ccRajagopalan, 5. et al. (1994) Science 263, 387-390; ddVan Leeuwen, J. and Kearse, K. (1996) /. Biol. Chem. 271, 25345-25349; =Kim, P. 5. and Arvan, P. (1995) /. Cell Biol. 128, 29-38; rrWada, I. et al. (1995) /. Biol. Cbem. 270, 2029820304; ssyamashita, Y. et ol. (1996) /. Viral. 70,2237-2246; hhOtteken, A. and Moss, B. (1996) /. Biol. Chem. 271, 97-l 03; “Hammond, C. et 01. (1994) hoc. Nat/. Acad. Sci. U. 5. A. 91, 913-917; IrPeterson, J. R. et al. (1995) Mol. Biol. Cell 6, 1173-l 184; “Then, W. et al. (1995) hoc. Not/. Acad. Sci. U. 5. A. 92,6229-6233; “Hebert, D. N. et al. (1996) FMBO]. 152961-2968; ““Hammond, C. and Helenius, A. (1994) Science266,456+58; ““Hammond, C. and Helenius, A. (1994)). Cell Biol. 126,41-52; %annon, K. 5. et of. (1996) /. Biol. Chem. 271, 14280-14284; PPDubuisson, I. and Rice, C. M. (1996) /. Viral. 70, 778-786. (+), association demonstrated; (-), does not associate; ND, not determined.

expression at the cell surface of HIV-1 glycoprotein, of KreSp, an ER protein with partial homology to the VSV G protein and both human and murine MHC glucosyltransferase, severely affects viability. How- class I molecules are severely inhibited24~26~27~29~ss, ever, there is no evidence that Kre5p or Cnelp are However, mammalian cells lacking glucosidases I functional homologues of the glucosyltransferase or II, or calnexin, are viable (Table 1). Apparently, and calnexin. As KreSp and glucosidases I and II af- most of the substrates can fold sufficiently well to sat- fect the synthesis of cell wall glucan structures, it is isfy the needs of these cells. possible that their primary function in S. cerevisiae is It also appears that most of the components of the not related to folding (Ref. 56, and J. F. Simons and cycle are dispensable in Saccharomyces cerevisiae as the A. Helenius, unpublished). genes for glucosidases I and II, as well as for the cal- In the fission yeast Schizosaccharomyces pombe, cal- nexin homologue Cnelp, can be disrupted without any nexin and glucosyltransferase are closely related to effect on growth (Table 1). By contrast, elimination their homologues in higher species. The calnexin

198 trends in CELL BIOLOGY (Vol. 7) May 1997 homologue, cnxl, is an essential protein, whereas Glycoproteins have no doubt evolved to make use no phenotype has been associated with disruption of of the chaperonesin the ERin many ways. They have the gene encoding the glucosyltransferase. The fact adjusted their glycosylation patterns to best exploit that mammalian cells devoid of calnexin are viable, theselectins. Being so different in structure and prop- whereas S. pombe are not, might be explained by the erties, each glycoprotein is likely to interact differ- presence of calreticulin in the former. Both S. pombe ently with the folding machinery in the ER. As more and S. cerevisiae seem to lack a calreticulin homologue. is learned about calnexin and calreticulin and their That proteins manage to fold, albeit less efficiently, accessory enzymes, and as different substrate pro- in certain cells that are devoid of a functional teins are analysed, great versatility in the system is calnexin/calreticulin cycle is probably due to redun- likely to emerge. Both of the two current models - dancy between folding factors in the ER. The ER is the lectin-only and the dual-mode model - may unique among the compartments of the cell in that apply, depending on the properties and complexity it contains a large number of different chaperones and of the specific substrateglycoproteins. folding enzymes at high concentiations. The notion of redundant functions is supported by the observation References that BiP is upregulated in castanospermine-treated or 1 HELENIUS, A. (1994) Mol. Biol. Cell 5, 253-265 glucosidase-II-deficient tissue culture cellW7. 2 BERCERON, J. M., BRENNER, M. B., THOMAS, D. Y. and WILLIAMS, D. B. (1994) Trends Biochem. Sci. 19, 124-l 28 Chaperones of a new kind 3 WILLIAMS, D. B. (1995) B&hem. Cell Biol. 73,123-l 32 Calnexin and calreticulin fulfil the most stringent 4 MICHALAK, M., ed. (1996) Colreticulin, R. C. Landes definitions of molecular chaperoness8.They associ- 5 SGNNICHSEN, B., FOLLEKRUG, J., VAN NGUYEN, P., ate transiently with folding intermediates of a large DIEKMANN, W., ROBINSON, D. C. and MIESKES, G. (1994) number of different proteins and promote proper j. Cell SC;. 107, 2705-2717 folding and assemblythrough a cycle of binding and 6 NAUSEEF, W. M., MCCORMICK, 5.1. and CLARK, R. A. (1995) releasev,2v.They help prevent aggregation of proteins, 1. Biol. Chem. 270, 4741-4747 suppressformation of non-native disulphide bonds, 7 PETERSON, J. R., ORA, A., VAN NGUYEN, P. and HELENIUS, A. inhibit premature degradation and provide retention (1995) Mol. Biol. Cell 6, 1173-l 184 for the quality-control function in the ER.Thus, their B WADA, I., IMAI, S-l., KAI, M., SAKANE, F. and KANOH, H. impact on protein biogenesis is similar to that de- (1995) 1. Biol. Gem. 270,20298-20304 scribedfor the classicalchaperones in the Hsp70 and 9 HEBERT, D. N., FOELLMER, B. and HELENIUS, A. (1996) EMBO /. chaperonin familiesss.However, their functional prin- l&2961 -2968 ciple appears to be different. Classical chaperones 10 OU, W-j., CAMERON, P. H., THOMAS, D. Y. and bind to hydrophobic sequencesexposed on the sur- BERGERON, ].I. M. (1993) Nature 364, 771-776 face of incompletely folded proteins. Substratebind- 11 HAMMOND, C., BRAAKMAN, I. and HELENIUS, A. (1994) ing and releaseis accompanied by a cycle of confor- Proc. Nat/. Acad. Sci. U. 5. A. 91, 913-917 mational changes regulated by ATP binding and 12 KEARSE, K. P., WILLIAMS, D. 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Dunford, R.P.and Dyer,T.A. (1337) A method for preparing Ouwerkerk, P.B.F.and Memelii, J. (19977)A simple method for PCR products for restriction enzymedireaed cloning directlord mdthnerization of DNA sequences Technical T@s without eqmic digestion Technical Ttps Online Online (http:l/w+vw.elsevier. com/locate/tto) TO1051 (http://www.elsevier. comilocateltto) T40075 Pusch, CM. (1997) High qual&y blotting of po@crylamide Gitelman, I. and Davis. C.A. (197) A novel, economical, gels Technical Tips Online (http://mw.elsevier. com/locate/tto) DNA molecular weight ladder Technical T@ Online TO1052 (http:l/www.elsevier. corn/locate/no) TO1053 Usman, N.J.,Tarabykin, VS., Matz, M.V., Bogdanova, E.A. and Humbert, J-F. and Elard, L. W97) A simple FCR method for Lukyanov,S.A. (1997) Whole-mount in si#u hybridization on r@dly detect@ defined point mutations Technical Tips freshwater planarian Technical Tips Online Online (http:/lwww.elsevier. com/locate/tto) T40076 (http://www.elsevier. com/locate/tto) TO1106

&litor Adrian Bkd, Institutefor Celland Molecular Biology at the Universityof Edinburgh

200 trends in CELL BIOLOGY (Vol. 7) May 1997