ANTI-CHAPERONE BEHAVIOR OF BIP DURING THE PROTEIN DISULFIDE ISOMERASE-CATALYZED REFOLDING OF REDUCED DENATURED LYSOZYME

Folding catalysts of the endoplasmic reticulum (ER), such as protein d isulfide isomerase (PDI), accelerate the slow chemical steps, such as disulfide bond formation, that accompany protein folding. Molecular ch aperones of the ER, notably the heavy chain-binding protein, BiP (grp7 8), bind and release unfolded proteins in an ATP-dependent fashion. In vitro, the fate of reduced, denatured lysozyme is dependent on whethe r the substrate interacts first with BiP or PDI. Depending on the rati o of PDI to substrate and order in which the components of the reactio n are mixed, PDI can exhibit a foldase/ chaperone activity, which incr eases the rate and extent of lysozyme refolding, or it can function as an anti-chaperone that promotes the formation of inactive, disulfide linked lysozyme aggregates (Puig, A., and Gilbert, H. F. (1994) J. Bio l. Chem. 269, 7764-7771). Reduced, denatured lysozyme, but not the nat ive protein, interacts with BiP and efficiently stimulates its peptide -dependent ATPase activity. When present at substoichiometric amounts, BiP, like PDI, facilitates the formation of large, inactive lysozyme aggregates that are non-covalently associated with BiP. BiP and PDI co mpete for a limited number of sites in these insoluble aggregates. If Bip is present at a high molar excess, the chaperone binds unfolded ly sozyme and inhibits its aggregation by maintaining it in a soluble, ye t inactive, conformation, both in the presence or absence of ATP. Incr easing concentrations of BiP decrease the extent, but not the initial rate, of refolding, suggesting that Bip and PDI compete for unfolded l ysozyme and that the BiP-lysozyme complex is not a very good substrate for PDI either in the presence or absence of ATP. Depending on the Bi P and PDI concentrations, unfolded lysozyme may either be efficiently refolded into the native conformation in a PDI-catalyzed reaction, or it may form both soluble and insoluble BiP-lysozyme complexes. In vitr o, PDI- and BiP-facilitated aggregation, as well as the competition of the two proteins for substrate, reproduces many of the features of th e quality control system of the ER.