Proc. Natl. Acad. Sci. USA Vol. 93, pp. 5269-5274, May 1996 Cell Bio ogy Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants LINDA HENDERSHOT*tt, JUEYANG WEI*t, JAMES GAUT§, JEFFREY MELNICKI, SIGAL AVIELI, AND YAIR ARGONSII *Department of Tumor Cell Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105; tDepartment of Biochemistry, University of Tennessee, 800 Madison Avenue, Memphis, TN 38163; §Institute of Gerontology and Department of Biological Chemistry, University of Michigan, 300 North Ingalls, Ann Arbor, MI 48109; and $Department of Immunology, Duke University Medical Center, Jones Building, Durham, NC 27710; and IlDepartment of Pathology, University of Chicago, 5841 South Maryland Ave, Chicago, IL 60637 Communicated by D. Bernard Amos, Duke University Medical Center, February 5, 1996 (received for review September 20, 1995) ABSTRACT A group of resident ER proteins have been activity (13). Affinity screening of two peptide display libraries identified that are proposed to function as molecular chap- revealed that BiP preferentially binds peptides containing erones. The best characterized of these is BiP/GRP78, an hydrophobic amino acids in every other position, which would hsp7O homologue that binds peptides containing hydrophobic place them on a single side of an extended polypeptide chain residues in vitro and unfolded or unassembled proteins in vivo. (14). Together, these studies are consistent with the possibility However, evidence that mammalian BiP plays a direct role in that BiP binds to hydrophobic stretches on nascent or unas- protein folding remains circumstantial. In this study, we sembled polypeptides that would become inaccessible after examine how BiP interacts with a particular substrate, im- folding and assembly are complete. munoglobulin light chain (A LC), during its folding. Wild-type In vitro folding studies with dnaK (15), the bacterial hsp70 hamster BiP and several well-characterized BiP ATPase mu- homologue, and human hsp7O (16), a cytosolic family member, tants were used in transient expression experiments. We demonstrate that these chaperones bind to unfolded or dena- demonstrate that wild-type A LCs showed prolonged associ- tured proteins and, in concert with other chaperones, promote ation with mutant BiP which inhibited their secretion. Both their correct folding. Most recently, the folding and oxidation wild-type and mutant BiP bound only to unfolded and par- of the yeast vacuolar protein plCPY was inhibited when tially folded LCs. The wild-type BiP was released from the dominant negative Kar2 (BiP) mutants were expressed (17). incompletely folded LCs, allowing them to fold and be se- Although the Kar2 mutants have not been characterized creted, whereas the mutant BiP was not released. As a result, biochemically, the mutations map to the ATP binding domain the LCs that were bound to BiP mutants were unable to and are assumed to inhibit nucleotide binding or hydrolysis. It undergo complete disulfide bond formation and were retained remains to be shown that the in vivo binding ofmammalian BiP in the ER. Our experiments suggest that LCs undergo both to unfolded proteins in any way affects their folding. Although BiP-dependent and BiP-independent folding steps, demon- yeast and mammalian BiP share a high degree of sequence strating that both ATP binding and hydrolysis activities ofBiP homology and are thought to perform some of the same are essential for the completion ofLC folding in vivo and reveal functions, it is increasingly clear that mammalian BiP does not that BiP must release before disulfide bond formation can do everything that yeast BiP does. Therefore, determining occur in that domain. which functions they share is important to understanding which of the functions attributed to yeast are actually general Nascent proteins that enter the endoplasmic reticulum (ER) characteristics of hsp70 family members. must fold and assemble into multimeric complexes in an All hsp7O proteins bind and hydrolyze ATP, and ATP is oxidizing milieu that contains millimolar concentrations of required for the in vitro folding of proteins by dnaK (15) and protein and calcium (1). In the past decade, proteins that hsp70 (16). This folding is thought to occur through ATP- interact transiently with incompletely folded and assembled driven cycles of binding and release from substrate proteins. proteins have been identified and characterized. Some of these BiP, like other family members, can be released from target associated proteins are enzymes responsible for glycosylation, proteins in vitro with ATP (3, 18). Recent experiments have disulfide bond formation, and isomerization of peptide bonds established that ATP binding, but not hydrolysis, is required (1). Others, termed "molecular chaperones," appear to stabi- for the in vitro release of dnaK and hsp70 from proteins (19, lize protein folding intermediates and are thought to prevent 20). It has been hypothesized that protein folding occurs incorrect interactions between protein domains or promote during the release phase of hsp7O binding. If folding does not correct ones (1, 2). The hsp70 family of proteins are the most occur, hsp7O would rebind and prevent aggregation of the abundant and best characterized of the molecular chaperones. protein. Another cycle of ATP-mediated release would pro- They are present in all cellular organelles and in all organisms. vide another opportunity for folding. However, direct data for BiP is identical to a 78-kDa glucose-regulated protein, grp78 cycles of BiP binding and release or for BiP release to occur (3-5) and is the ER member of the hsp70 family. The evidence before protein can fold have not been obtained. for the role of BiP in protein folding is largely circumstantial. To investigate the role of BiP in the in vivo folding of It was first identified stably and noncovalently associated with proteins, we have examined its interactions with murine im- the unassembled, nontransported Ig heavy chains in Abelson munoglobulin lambda light chains (A LCs). In vivo, newly virus transformed pre-B cell lines (6). BiP also associates synthesized LCs interact transiently with several molecular transiently with nascent proteins that are often unassembled chaperones, including BiP and GRP94 (6, 21, 22). A LCs were and may be incompletely folded (7-10). BiP also binds more transiently coexpressed with a series of hamster BiP mutants stably to mutants proteins that are in many cases thought to be with well-characterized point mutations in the ATP binding incorrectly folded (11, 12). Peptides containing hydrophobic domain. These mutations affect the ability of the BiP mutants amino acids are the most effective stimulators of BiP's ATPase Abbreviations: ER, endoplasmic reticulum; LC, light chain; NEM, The publication costs of this article were defrayed in part by page charge N-ethylmaleimide; DTT, dithiothreitol; hsp7o, 70-kDa heat shock pro- payment. This article must therefore be hereby marked "advertisement" in tein. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5269 Downloaded by guest on September 28, 2021 5270 Cell Biology: Hendershot et al. Proc. Natl. Acad. Sci. USA 93 (1996) to bind ATP, to undergo an ATP-mediated conformational RESULTS change, or to hydrolyze ATP (20). All of these mutants are able to bind substrate proteins in vivo (23, 24) and in vitro (20, 23), Effects ofBiP Mutant Expression on the Secretion ofA LCs. but the ATP binding and conformational change mutants are To assess the role of BiP in protein folding in vivo, we unable to release bound proteins in vitro when ATP is added characterized its interactions with murine A LCs. Wild-type or to isolated BiP-protein complexes (20, 23). The folding status mutant BiP was expressed in COS monkey fibroblasts along of LCs bound to both wild-type and mutant BiP was deter- with either wild-type LCs or a nonsecreted LC point mutant, mined, and the effects of the BiP mutants on LC release from DK82. The wild-type LCs were not readily detected in asso- BiP in vivo, disulfide bond formation of the individual LC ciation with wild-type BiP in coimmunoprecipitation experi- domains, and secretion of the LCs were assessed. ments and were secreted into the culture medium (Fig. 1A). However, when we coexpressed wild-type LCs with two dif- ferent BiP ATPase mutants (T229G or T37G), a significant MATERIALS AND METHODS proportion of the LCs were coprecipitated with the BiP LC and BiP DNA Clones. DNAs encoding either wild-type mutants and fewer LCs were secreted relative to the control AI LC (25) or a nonsecreted A LC point mutant, DK82, in cultures (Fig. 1A). Because endogenous wild-type BiP is also which aspartic acid at position 82 in the VL domain is mutated present in the cells transfected with BiP mutants, we propose to lysine (S.A., J.M., J.D., and Y.A., unpublished data) were that the inhibition of LC secretion must be due to BiP mutants used. A hamster BiP cDNA clone was obtained from Amy S. competing with endogenous BiP for the LC and acting as Lee (University of Southern California, Los Angeles) and was "chaperone traps" that capture and retain their targets. placed under the control of an adenovirus promoter in the When the LC mutant DK82 was similarly analyzed, readily pMT vector which contains an simain virus 40 origin of detectable amounts of wild-type BiP coprecipitated with the replication. Single point mutations in the BiP cDNA clone LCs (Fig. 1B) due to the mutation in the VL domain. Expres- have been made and characterized previously (20, 23). The sion of DK82 with mutant BiP resulted in a further increase in T229G mutant is defective in ATP hydrolysis, the T37G the amount of BiP coprecipitating with the LCs (Fig. 1B), mutant is unable to undergo an ATP-induced conformational showing that wild-type BiP binding to these mutant LCs is not change, and the G227D mutant does not bind ATP (20).