The In Vivo Association of BiP with Newly Synthesized Proteins Is Dependent on the Rate and Stability of Folding and Not Simply on the Presence of Sequences That Can Bind to BiP

Abstract

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine λI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as “kinetic traps.” Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.

Mutation of a Single Conserved Residue in VH Complementarity-Determining Region 2 Results in a Severe Ig Secretion Defect

“…Thus, these results provide no evidence for the creation of a better BiP site, but rather predict reduced or no change in BiP binding to this putative site. Although the predicted BiP score does not suggest increased BiP binding, this result should be interpreted with caution given that Hellman et al (42) demonstrated that there was no correlation between the presence of predicted BiP binding sequences and actual BiP binding to murine I variable and constant domains. Further analysis is required to determine whether Ile 51 is part of a BiP-binding site used in vivo and whether the single mutations affect this interaction.…”

Age-Associated Changes in Histology and Gene-Expression Profile in the Rat Ventral Prostate

“…Lau et al hsp70 family of chaperones, is one of the most abundant proteins in the ER lumen. BiP has been implicated in cotranslational folding of nascent polypeptides and in the recognition and disposal of aberrant polypeptides (Hatano et al, 1997;Hellman et al, 1999). Interestingly, calnexin and calreticulin, two closely related molecular chaperones in rough ER, when associated with BiP, suppress aggregation and promote refolding of protein and glycoprotein substrates (Stronge et al, 2001).…”

Modulation of the ATPase Cycle of BiP by Peptides and Proteins

“…15 In vivo, the accessibility of a binding site may be determined by the folding kinetics of the respective domain as it had been shown that stronger BiP interactions are observed if the folding of a protein is decelerated in vivo. 16,17 Like other Hsp70 chaperones, BiP assists folding of the substrate by repeated cycles of binding and release, driven by ATP-dependent conformational changes of the chaperone. 18 The regulation of this ATPase cycle is best understood for the E. coli Hsp70 homologue DnaK.…”