Protein Disulfide Isomerase and Assisted Protein Folding (1997)

The folding protein (and protein folder) is beset with a number of problems in translating the simple instructions encoded by DNA into the complex, three-dimensional structure of a correctly folded protein. Nature has solved these problems in an adequate, if not elegant, fashion, but we are just beginning to understand the variety of strategies that cells use to ensure efficient protein folding. This minireview will focus on several of the more general principles of assisted folding using the folding catalyst, protein disulfide isomerase (PDI), 1The abbreviations used are: PDI, protein disulfide isomerase; ER, endoplasmic reticulum; hCG, human chorionic gonadotropin. 1The abbreviations used are: PDI, protein disulfide isomerase; ER, endoplasmic reticulum; hCG, human chorionic gonadotropin. as an example. This remarkable resident of the endoplasmic reticulum (ER) inserts disulfides into folding proteins and provides a mechanism to correct errors in disulfide pairing when they occur. At high concentrations, it functions as an ATP-independent chaperone that inhibits aggregation; yet, at lower concentrations, it can also participate in an unusual interaction with substrate that leads to PDI-facilitated aggregation (anti-chaperone behavior 2At a recent Keystone Symposium on Protein Folding and Secretion, there was discussion of the use and misuse of the terms “chaperone” and “anti-chaperone.” A variety of terms for replacement of “anti-chaperone” were suggested. However, given the lack of a suitable replacement term, we would suggest that the term “anti-chaperone” be reserved to describe facilitated aggregation. 2At a recent Keystone Symposium on Protein Folding and Secretion, there was discussion of the use and misuse of the terms “chaperone” and “anti-chaperone.” A variety of terms for replacement of “anti-chaperone” were suggested. However, given the lack of a suitable replacement term, we would suggest that the term “anti-chaperone” be reserved to describe facilitated aggregation.).Proteins Fold in Different EnvironmentsAssisted protein folding occurs in at least three intracellular compartments, the cytosol, mitochondria, and the lumen of the ER. PDI is a resident of the endoplasmic reticulum, a specialized folding environment, replete with a variety of folding catalysts and chaperones (1Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3575) Google Scholar, 2Hebert D.N. Simons J.F. Peterson J.R. Helenius A. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 405-415Crossref PubMed Scopus (56) Google Scholar, 3Wittrup K.D. Curr. Opin. Biotechnol. 1995; 6: 203-208Crossref PubMed Scopus (34) Google Scholar). Folding in the ER is specialized to accommodate the structural features of membrane and secreted proteins, including glycosylation and disulfide formation (4Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (486) Google Scholar, 5Hammond C. Helenius A. J. Cell Biol. 1995; 126: 41-52Crossref Scopus (392) Google Scholar, 6Ruddon R.W. Sherman S.A. Bedows E. Protein Sci. 1996; 5: 1443-1452Crossref PubMed Scopus (69) Google Scholar). As proteins are synthesized and inserted into the ER, the sequence is presented vectorially to the ER lumen so that folding may begin as the protein emerges from the ribosome (7Hansen W.J. Lingappa V.R. Welch W.J. J. Biol. Chem. 1994; 269: 26610-26613Abstract Full Text PDF PubMed Google Scholar, 8Kudlicki W. Chirgwin J. Kramer G. Biochemistry. 1995; 34: 14284-14287Crossref PubMed Scopus (54) Google Scholar, 9Frydman J. Hartl F.U. Science. 1996; 272: 1497-1502Crossref PubMed Scopus (215) Google Scholar, 10Frydman J. Nimmersgern E. Ohtsuka K. Hartl F.U. Nature. 1994; 370: 111-117Crossref PubMed Scopus (562) Google Scholar). A glutathione redox buffer (a mixture of GSH and GSSG at a ratio of approximately 1:1–3:1) holds the redox state of the ER more oxidizing than that of the cytosol to allow disulfides to form and rearrange (11Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1574) Google Scholar).Folding catalysts such as PDI (12Goldberger R.F. Epstein C.J. Anfinsen C.B. J. Biol. Chem. 1963; 238: 628-635Abstract Full Text PDF PubMed Google Scholar, 13Noiva R. Protein Expression Purif. 1994; 5: 1-13Crossref PubMed Scopus (58) Google Scholar, 14Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (655) Google Scholar) and peptidylprolyl isomerase (15Kern G. Kern D. Schmid F.X. Fischer G. J. Biol. Chem. 1995; 270: 740-745Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Bose S. Freedman R.B. Biochem. J. 1994; 300: 865-870Crossref PubMed Scopus (24) Google Scholar) accelerate slow chemical steps that accompany folding. Disulfide formation can occur quite rapidly, even before the completion of synthesis (17Bergman L.W. Kuehl W.M. J. Biol. Chem. 1979; 254: 8869-8876Abstract Full Text PDF PubMed Google Scholar, 18Chen W. Helenius J. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6229-6233Crossref PubMed Scopus (220) Google Scholar), but for some proteins such as hCG-β and influenza virus hemagglutinin (19Segal M.S. Bye J.M. Sambrook J.F. Gething M.J. J. Cell Biol. 1992; 118: 227-244Crossref PubMed Scopus (70) Google Scholar), disulfide formation is delayed and occurs posttranslationally. Because of the high reactivity of the active site disulfide of PDI (20Walker K.W. Gilbert H.F. Biochemistry. 1995; 34: 13642-13650Crossref PubMed Scopus (35) Google Scholar), the delay in disulfide formation may be due to protection of certain cysteines against oxidation by folding of the nascent chain or by protection with other ER chaperones (19Segal M.S. Bye J.M. Sambrook J.F. Gething M.J. J. Cell Biol. 1992; 118: 227-244Crossref PubMed Scopus (70) Google Scholar). However, Ruddon and co-workers (21Huth J.R. Perini F. Lockridge O. Bedows E. Ruddon R.W. J. Biol. Chem. 1993; 268: 16472-16482Abstract Full Text PDF PubMed Google Scholar) have mapped the order of disulfide formation for human hCG-β during folding in the ER and for the same reaction in vitro, catalyzed by PDI. No differences were observed in the identity and order of disulfide formation suggesting, at least for hCG-β, that ER chaperones are not necessary for the inhibition of disulfide formation during folding (21Huth J.R. Perini F. Lockridge O. Bedows E. Ruddon R.W. J. Biol. Chem. 1993; 268: 16472-16482Abstract Full Text PDF PubMed Google Scholar) and that catalyzed and uncatalyzed folding proceeds by similar mechanisms.PDI Has Unique Structural and Functional FeaturesDuring protein folding in the ER, PDI catalyzes disulfide formation and rearrangement by thiol/disulfide exchange (22Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar). A member of the thioredoxin superfamily (14Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (655) Google Scholar), PDI has two independent (23Vuori K. Myllylä R. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1992; 267: 7211-7214Abstract Full Text PDF PubMed Google Scholar) but non-equivalent (24Lyles M.M. Gilbert H.F. J. Biol. Chem. 1994; 269: 30946-30952Abstract Full Text PDF PubMed Google Scholar) active sites, each with two cysteines (CGHC) that cycle between the dithiol and disulfide oxidation states. One thioredoxin domain is positioned near the N terminus, and another is poised near the C terminus (25Edman J.C. Ellis L. Blacher R.W. Roth R.A. Rutter W.J. Nature. 1985; 317: 267-270Crossref PubMed Scopus (473) Google Scholar). An NMR structure of the N-terminal thioredoxin domain shows that the backbone fold is comparable with that of thioredoxin (26Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Biochemistry. 1996; 35: 7684-7691Crossref PubMed Scopus (182) Google Scholar). The active site disulfide is a good oxidant (27Gilbert H.F. Biochemistry. 1989; 28: 7298-7305Crossref PubMed Scopus (50) Google Scholar,28Lundström J. Holmgren A. Biochemistry. 1993; 32: 6649-6655Crossref PubMed Scopus (157) Google Scholar) that directly introduces disulfides into protein substrates (29Walker K.W. Lyles M.M. Gilbert H.F. Biochemistry. 1996; 35: 1972-1980Crossref PubMed Scopus (142) Google Scholar,33Laboissière M.C.A. Sturley S.L. Raines R.T. J. Biol. Chem. 1995; 270: 28006-28009Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The dithiol redox state is essential for catalyzing disulfide rearrangements. The necessity of having oxidized and reduced active sites for catalysis of different steps results in a redox optimum (24Lyles M.M. Gilbert H.F. J. Biol. Chem. 1994; 269: 30946-30952Abstract Full Text PDF PubMed Google Scholar). The individual thioredoxin domains of PDI have been expressed alone, and they are catalytically active oxidants. However, neither isolated domain exhibits significant isomerase activity despite the fact that the chemical properties (reactivity and redox potential) are near those found for wild-type PDI (30Darby N.J. Creighton T.E. Biochemistry. 1995; 34: 3576-3587Crossref PubMed Scopus (90) Google Scholar).PDI is an essential gene in yeast (31Farquhar R. Honey N. Murant S.J. Bossier P. Schultz L. Montgomery D. Ellis R.W. Freedman R.B. Tuite M.F. Gene (Amst.). 1991; 108: 81-89Crossref PubMed Scopus (107) Google Scholar, 32LaMantia M.L. Lennarz W.J. Cell. 1993; 74: 899-908Abstract Full Text PDF PubMed Scopus (183) Google Scholar), and the isomerase activity is the critical feature (33Laboissière M.C.A. Sturley S.L. Raines R.T. J. Biol. Chem. 1995; 270: 28006-28009Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Overexpression of thioredoxin itself will not rescue PDI-null strains, but a mutant of thioredoxin in which the active site sequence (CGPC) has been changed to the PDI sequence (CGHC) can support yeast growth (34Chivers P.T. Laboissière M.C. Raines R.T. EMBO J. 1996; 15: 2659-2667Crossref PubMed Scopus (159) Google Scholar) even though this mutant thioredoxin (P34H) has only 10% of the isomerase activity of PDI in vitro (35Lundström J. Krause G. Holmgren A. J. Biol. Chem. 1992; 267: 9047-9052Abstract Full Text PDF PubMed Google Scholar).Mistakes Occur during FoldingProtein folding is error-prone. For some proteins like bovine pancreatic trypsin inhibitor, folding intermediates are dominated by native pairings of cysteines; however, the disulfides are formed in the “wrong” temporal order and must be rearranged before folding can proceed (36Creighton T.E. Goldenberg D.P. J. Mol. Biol. 1984; 179: 497-526Crossref PubMed Scopus (259) Google Scholar). In other cases such as RNase (37Creighton T.E. J. Mol. Biol. 1979; 129: 411-420Crossref PubMed Scopus (147) Google Scholar, 38Konishi Y. Ooi T. Scheraga H.A. Biochemistry. 1982; 21: 4734-4741Crossref PubMed Scopus (83) Google Scholar) or hirudin (39Chatrenet B. Chang J.Y. J. Biol. Chem. 1992; 267: 3038-3044Abstract Full Text PDF PubMed Google Scholar), disulfides are formed randomly in early folding intermediates. Thus, protein folding is a process of trial and error (40Todd M.J. Lorimer G.H. Thirumalai D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4030-4035Crossref PubMed Scopus (223) Google Scholar). PDI corrects these mistakes by catalyzing rearrangements to replace wrong disulfides with correct ones, a process that eventually leads to a “native” structure that is resistant to further rearrangement (41Gilbert H.F. Pain R. Protein Folding. IRL Press, Oxford1994: 104-136Google Scholar, 42Walker K.W. Gilbert H.F. J. Biol. Chem. 1997; 272: 8845-8848Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar).So far, no evidence has surfaced that PDI or other folding assistants directly guide the folding protein into a specific structure (21Huth J.R. Perini F. Lockridge O. Bedows E. Ruddon R.W. J. Biol. Chem. 1993; 268: 16472-16482Abstract Full Text PDF PubMed Google Scholar, 43Weissman J.S. Kim P.S. Nature. 1993; 365: 185-188Crossref PubMed Scopus (187) Google Scholar). PDI simply accelerates the formation and breaking of disulfides, providing cycles of PDI-catalyzed rearrangements to correct the frequent mistakes that occur. Turnover numbers for PDI-catalyzed disulfide isomerization are slow by normal enzyme standards (∼1 min−1), but given the high local concentration of PDI in the ER (about 0.2 mm) (44Lyles M.M. Gilbert H.F. Biochemistry. 1991; 30: 619-625Crossref PubMed Scopus (117) Google Scholar), this low turnover number is sufficient to make PDI-catalyzed folding kinetically competent in the cell. The value of K m for disulfide isomerization of protein substrates is near 10 μm (43Weissman J.S. Kim P.S. Nature. 1993; 365: 185-188Crossref PubMed Scopus (187) Google Scholar, 44Lyles M.M. Gilbert H.F. Biochemistry. 1991; 30: 619-625Crossref PubMed Scopus (117) Google Scholar).Disulfide rearrangements are initiated by the attack of the active site thiol of reduced PDI on a substrate disulfide (45Lambert N. Freedman R.B. Biochem. J. 1983; 213: 235-243Crossref PubMed Scopus (79) Google Scholar). The choice of which substrate disulfide to attack will depend on the kinetic reactivity and accessibility of the various substrate disulfides that are available. The formation of a PDI-substrate covalent intermediate disrupts one of the substrate disulfides, creating a free sulfhydryl in the substrate. This intermediate can be resolved by three alternative reactions (Fig.1). If PDI is displaced by reforming the same disulfide that was attacked initially, no disulfide rearrangement results. Rearrangement can occur by an intramolecular reaction of the newly created substrate thiol on another substrate disulfide or by reduction of the original disulfide followed by oxidation to form different disulfides (42Walker K.W. Gilbert H.F. J. Biol. Chem. 1997; 272: 8845-8848Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The second cysteine in the active site can serve as a clock to invoke reduction of the disulfide and escape from the complex if a substrate-PDI complex fails to rearrange or reverse in a timely fashion (Fig. 1). The offending substrate disulfide is reduced and released for further attempts at rearrangement or reduction. If reductive escape is inhibited by mutating the second active site cysteine to serine, PDI accumulates in mixed disulfide complexes with substrate, and the isomerization activity drops precipitously (<1% of wild type) (29Walker K.W. Lyles M.M. Gilbert H.F. Biochemistry. 1996; 35: 1972-1980Crossref PubMed Scopus (142) Google Scholar), suggesting that reduction/reoxidation is the dominant mechanism of disulfide rearrangement for RNase. The initial scanning of the substrate disulfides by PDI and the subsequent reactions of the PDI-substrate intermediate should favor the trial and error replacement of reactive disulfides of the substrate by less reactive ones (42Walker K.W. Gilbert H.F. J. Biol. Chem. 1997; 272: 8845-8848Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), a process similar to that suggested for repeated cycles of folding and unfolding catalyzed by ATP-dependent chaperones such as GroEL/ES (40Todd M.J. Lorimer G.H. Thirumalai D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4030-4035Crossref PubMed Scopus (223) Google Scholar).Folding Catalysts Recognize Many SubstratesIn keeping with the lack of obvious sequence cues that specify disulfide connectivity, PDI can catalyze thiol/disulfide exchange reactions involving a wide range of substrates, including proteins, peptides and low molecular weight thiols and disulfides (46Hillson D.A. Lambert N. Freedman R.B. Methods Enzymol. 1984; 107: 281-294Crossref PubMed Scopus (187) Google Scholar). PDI interacts relatively weakly with peptides (K d = 50–1000 μm) with no obvious correlation between binding and peptide charge, sequence, or hydrophobicity (47Morjana N.A. Gilbert H.F. Biochemistry. 1991; 30: 4985-4990Crossref PubMed Scopus (79) Google Scholar, 48Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar). A peptide-based photoaffinity probe labels one site near the C terminus and inhibits catalytic activity (49Noiva R. Freedman R.B. Lennarz W.J. J. Biol. Chem. 1993; 268: 19210-19217Abstract Full Text PDF PubMed Google Scholar). In contrast to glutaredoxin (50Lundström J. Holmgren A. J. Biol. Chem. 1995; 270: 7822-7828Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), PDI does not exhibit any special affinity for glutathione as a substrate (27Gilbert H.F. Biochemistry. 1989; 28: 7298-7305Crossref PubMed Scopus (50) Google Scholar). However, the dominant mechanism of PDI-dependent oxidation of small peptide substrates involves the formation of peptide-glutathione mixed disulfides (51Darby N.J. Freedman R.B. Creighton T.E. Biochemistry. 1994; 33: 7937-7947Crossref PubMed Scopus (80) Google Scholar), and protein-glutathione mixed disulfides are good PDI substrates (52Ruoppolo M. Freedman R.B. Biochemistry. 1995; 34: 9380-9388Crossref PubMed Scopus (28) Google Scholar,53Ruoppolo M. Freedman R.B. Pucci P. Marino G. Biochemistry. 1996; 35: 13636-13646Crossref PubMed Scopus (35) Google Scholar). Although mutants of PDI with a single, more N-terminal cysteine might be expected to catalyze oxidation by glutathione-mixed disulfide formation, they do not (29Walker K.W. Lyles M.M. Gilbert H.F. Biochemistry. 1996; 35: 1972-1980Crossref PubMed Scopus (142) Google Scholar). In addition, substrate oxidation occurs rapidly in the absence of a redox buffer, where stoichiometric amounts of PDI supply all of the oxidizing equivalents (44Lyles M.M. Gilbert H.F. Biochemistry. 1991; 30: 619-625Crossref PubMed Scopus (117) Google Scholar). Although glutathione mixed disulfide formation does occur, it is not an obligate oxidation pathway.Aggregation Is a Major Complication to Productive FoldingFolding intermediates with a less organized and more exposed hydrophobic core have an increased tendency to aggregate, sometimes imposing stingy limits on solubility (54Jaenicke R. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1995; 348: 97-105Crossref PubMed Scopus (90) Google Scholar). At least for some proteins, aggregation can occur from specific folding intermediates through specific associations (55DeFelippis M.R. Alter L.A. Pekar A.H. Havel H.A. Brems D.N. Biochemistry. 1993; 32: 1555-1562Crossref PubMed Scopus (69) Google Scholar, 56Oberg K. Chrunyk B.A. Wetzel R.B. Fink A.L. Biochemistry. 1994; 33: 2628-2634Crossref PubMed Scopus (197) Google Scholar). Yet hydrophobic aggregation can also be nonspecific, involving the interaction of multiple proteins (57Sawyer J.T. Lukaczyk T. Yilla M. J. Biol. Chem. 1994; 269: 22440-22445Abstract Full Text PDF PubMed Google Scholar). PDI has two somewhat paradoxical roles in substrate aggregation. It can behave as a chaperone and inhibit aggregation, or under certain conditions, it can behave as an anti-chaperone and facilitate the formation of aggregates.As a chaperone, PDI inhibits substrate aggregation by two mechanisms, rapid disulfide formation and a chaperone-buffer effect. At concentrations near 10 μm, reduced, denatured hen egg white lysozyme tends to aggregate (58Goldberg M.E. Rudolph R. Jaenicke R. Biochemistry. 1991; 30: 2790-2796Crossref PubMed Scopus (410) Google Scholar), compromising the yield during refolding. If present when lysozyme is diluted into solution, high concentrations (near 100 μm) of catalytically active PDI are required to inhibit aggregation, suggesting that very rapid disulfide formation can divert substrate molecules onto productive folding pathways and decrease the amount of aggregation (59Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 25889-25896Abstract Full Text PDF PubMed Google Scholar, 60Puig A. Lyles M.M. Noiva R. Gilbert H.F. J. Biol. Chem. 1995; 269: 19128-19135Abstract Full Text PDF Google Scholar). However, PDI also inhibits the aggregation of proteins that do not form disulfides (61Cai H. Wang C.C. Tsou C.L. J. Biol. Chem. 1994; 269: 24550-24552Abstract Full Text PDF PubMed Google Scholar, 62Song J.L. Wang C.C. Eur. J. Biochem. 1995; 2: 312-316Crossref Google Scholar, 63Primm T.P. Walker K.W. Gilbert H.F. J. Biol. Chem. 1996; 271: 33664-33669Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). inhibit the chaperone activity suggesting that the binding of PDI may with the substrate protein and aggregation by complexes that sites H. G. Wang C.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google of the more features of folding is that PDI under certain conditions, facilitate aggregation, a behavior that has been anti-chaperone activity (59Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 25889-25896Abstract Full Text PDF PubMed Google Scholar, 60Puig A. Lyles M.M. Noiva R. Gilbert H.F. J. Biol. Chem. 1995; 269: 19128-19135Abstract Full Text PDF Google Scholar, 63Primm T.P. Walker K.W. Gilbert H.F. J. Biol. Chem. 1996; 271: 33664-33669Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). conditions, low concentrations of PDI to a of reduced lysozyme to form that lysozyme and PDI (59Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 25889-25896Abstract Full Text PDF PubMed Google Scholar). PDI at low concentrations accelerates the aggregation of during but concentrations of PDI aggregation T.P. Walker K.W. Gilbert H.F. J. Biol. Chem. 1996; 271: 33664-33669Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). This paradoxical of a folding from anti-chaperone to chaperone has an in the of a by a of (Fig. (59Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 25889-25896Abstract Full Text PDF PubMed Google Scholar). For a protein that has some tendency to into with PDI can an mechanism for to in At some comparable with the in an the number of is formed to and PDI as an At PDI concentrations, to of it more that an protein will to a PDI that is not to another the of formation, and chaperone The that aggregation are relatively PDI is with the protein in a approximately proteins PDI (59Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 25889-25896Abstract Full Text PDF PubMed Google Scholar, 63Primm T.P. Walker K.W. Gilbert H.F. J. Biol. Chem. 1996; 271: 33664-33669Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). ER chaperone, also exhibits and it is into at a ratio and in with PDI A. Gilbert H.F. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google behavior of PDI may be to a At low concentrations concentrations of PDI can to solubility by providing to the substrate protein into the ratio of PDI to substrate is the number of will the complex will and PDI will behave as an anti-chaperone At PDI concentrations formation by will be inhibited by PDI, and PDI will behave as a is as a folding and facilitated aggregation is a to using the folding assistants in However, there are that between behavior be to the as a mechanism to proteins in the ER when the stoichiometric chaperone is or to proteins from In contrast to the aggregation so observed in vitro, aggregation can be in the I. Helenius J. Helenius A. Nature. 1992; PubMed Scopus (223) Google Scholar). The inhibition of disulfide formation in cells by including in the results in the of that are in the ER. the is these are correctly and released from the ER as proteins U. Braakman I. Helenius A. EMBO J. 1993; PubMed Scopus Google Scholar, I. Helenius J. Helenius A. EMBO J. 1992; PubMed Scopus Google Scholar). The may multiple substrate proteins and multiple M. D. J.T. J. Cell Biol. 1992; 118: PubMed Scopus Google Scholar) it is not these are complexes or of The normal folding of in cells by and the of S. M. L. Myllylä R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (34) Google Scholar) proceeds through P.S. P. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, P.S. P. J. Cell Biol. 1995; PubMed Scopus Google Scholar), and mutants of human chorionic can also be in the ER as that on to fold at a W. Bedows E. Ruddon R.W. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus (24) Google and other folding catalysts and molecular chaperones the with an to the protein folding The of folding can be by the of slow chemical steps that by the formation of and by the folding protein through the formation of The in the near will be to understand assisted folding at the molecular and structural and to assisted folding are in the cell. The folding protein (and protein folder) is beset with a number of problems in translating the simple instructions encoded by DNA into the complex, three-dimensional structure of a correctly folded protein. Nature has solved these problems in an adequate, if not elegant, fashion, but we are just beginning to understand the variety of strategies that cells use to ensure efficient protein folding. This minireview will focus on several of the more general principles of assisted folding using the folding catalyst, protein disulfide isomerase (PDI), 1The abbreviations used are: PDI, protein disulfide isomerase; ER, endoplasmic reticulum; hCG, human chorionic gonadotropin. 1The abbreviations used are: PDI, protein disulfide isomerase; ER, endoplasmic reticulum; hCG, human chorionic gonadotropin. as an example. This remarkable resident of the endoplasmic reticulum (ER) inserts disulfides into folding proteins and provides a mechanism to correct errors in disulfide pairing when they occur. At high concentrations, it functions as an ATP-independent chaperone that inhibits aggregation; yet, at lower concentrations, it can also participate in an unusual interaction with substrate that leads to PDI-facilitated aggregation (anti-chaperone behavior 2At a recent Keystone Symposium on Protein Folding and Secretion, there was discussion of the use and misuse of the terms “chaperone” and “anti-chaperone.” A variety of terms for replacement of “anti-chaperone” were suggested. However, given the lack of a suitable replacement term, we would suggest that the term “anti-chaperone” be reserved to describe facilitated aggregation. 2At a recent Keystone Symposium on Protein Folding and Secretion, there was discussion of the use and misuse of the terms “chaperone” and “anti-chaperone.” A variety of terms for replacement of “anti-chaperone” were suggested. However, given the lack of a suitable replacement term, we would suggest that the term “anti-chaperone” be reserved to describe facilitated Fold in Different EnvironmentsAssisted protein folding occurs in at least three intracellular compartments, the cytosol, mitochondria, and the lumen of the ER. PDI is a resident of the endoplasmic reticulum, a specialized folding environment, replete with a variety of folding catalysts and chaperones (1Gething M.J. Sambrook J. Nature. 1992; 355: