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

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 f amily 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 ente r the ER and more stably with protein subunits produced in stoichiometric e xcess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To be gin to understand what controls the likelihood that a nascent protein enter ing the ER will associate with BiP, we have examined the in vivo folding of a murine XI immunoglobulin (Ig) light chain (LC). This LC is composed of t wo 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 d uring folding, we used BiP ATPase mutants, which bind irreversibly to prote ins, as "kinetic traps." Although both the wild-type and mutant BiP dearly associated with the unoxidized variable region domain, we were unable to de tect binding of either BiP protein to the constant region domain. A combina tion of in vivo and in vitro folding studies revealed that the constant dom ain folds rapidly and stably even in the absence of an intradomain disulfid e 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 wheth er or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.