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© The Rockefeller University Press, 0021-9525/1999//21 $5.00
The Journal of Cell Biology, Volume 144, Number 1, , 1999 21-30

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

Rachel Hellman^*, Marc Vanhove^*, Annabelle Lejeune, Fred J. Stevens, and Linda M. Hendershot^*,¶

^* Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105; Laboratoire d'Enzymologie and Centre d'Ingénierie des Protéines, Institut de Chimie B6, Université de Liège, Sart-Tilman, B-4000 Liège, Belgium; Argonne National Laboratories, Argonne, Illinois 60439; and ^¶ Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163

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 I 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.

Key Words: BiP • endoplasmic reticulum • chaperone • protein folding

Abbreviations used in this paper: 2-ME, 2-mercaptoethanol; BiP, immunoglobulin heavy chain-binding protein; C, constant; C_H1, first constant domain of the heavy chain; C_L, single constant domain of the light chain; cWT, pSV eukaryotic expression vector; ER-C, a 1 constant domain that could be translocated to the ER lumen; gWT, pJDEI genomic clone; LC, light chain; V, variable; WT, wild-type.

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