The modification and assembly of proteins in the endoplasmic reticulum.

Proteins fold and assemble in the endoplasmic reticulum in an environment that is very different from the cytosol. The presence of relatively high concentrations of calcium, an oxidizing state, ATP and lumenal proteins are all important in mediating these events.

Giving protein traffic the green light.

Most proteins that are secreted or expressed on a cell surface are synthesized on membrane polysomes and enter the endoplasmic reticulum (ER) as unfolded polypeptide chains. A complex series of interactions with resident enzymes and molecular chaperones ensure that these proteins are folded and assembled to achieve their correct tertiary structures before being transported to the Golgi and along the secretory pathway. However, the mechanism by which properly folded molecules are sorted from incompletely or improperly folded proteins and from the resident proteins that guide this process remains unclear.

Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response.

PERK and IRE1 are type-I transmembrane protein kinases that reside in the endoplasmic reticulum (ER) and transmit stress signals in response to perturbation of protein folding. Here we show that the lumenal domains of these two proteins are functionally interchangeable in mediating an ER stress response and that, in unstressed cells, both lumenal domains form a stable complex with the ER chaperone BiP. Perturbation of protein folding promotes reversible dissociation of BiP from the lumenal domains of PERK and IRE1. Loss of BiP correlates with the formation of high-molecular-mass complexes of activated PERK or IRE1, and overexpression of BiP attenuates their activation. These findings are consistent with a model in which BiP represses signalling through PERK and IRE1 and protein misfolding relieves this repression by effecting the release of BiP from the PERK and IRE1 lumenal domains.

Immunoglobulin heavy chain and binding protein complexes are dissociated in vivo by light chain addition.

Immunoglobulin heavy chain binding protein (BiP, GRP78) associates stably with the free, nonsecreted Ig heavy chains synthesized by Abelson virus transformed pre-B cell lines. In cells synthesizing both Ig heavy and light chains, the Ig subunits assemble rapidly and are secreted. Only incompletely assembled Ig molecules can be found bound to BiP in these cells. In addition to Ig heavy chains, a number of mutant and incompletely glycosylated transport-defective proteins are stably complexed with BiP. When normal proteins are examined for combination with BiP, only a small fraction of the intracellular pool of nascent, unfolded, or unassembled proteins can be found associated. It has been difficult to determine whether these BiP-associated molecules represent assembly intermediates which will be displaced from BiP and transported from the cell, or whether these are aberrant proteins that are ultimately degraded. In order for BiP to monitor and aid in normal protein transport, its association with these proteins must be reversible and the released proteins should be transport competent. In the studies described here, transient heterokaryons were formed between a myeloma line producing BiP-associated heavy chains and a myeloma line synthesizing the complementary light chain. Introduction of light chain synthesis resulted in assembly of prelabeled heavy chains with light chains, displacement of BiP from heavy chains, and secretion of Ig into the culture supernatant. These data demonstrate that BiP association can be reversible, with concordant release of transportable proteins. Thus, BiP can be considered a component of the exocytic secretory pathway, regulating the transport of both normal and abnormal proteins.

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas.

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.

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 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 lambdaI 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.

Protein-specific chaperones: the role of hsp47 begins to gel.

Collagen biosynthesis involves a complex series of post-translational modifications, controlled by a number of general and specific molecular chaperones. A recent study has shed new light on the role played in this process by the procollagen-specific chaperone Hsp47.

Identity of the immunoglobulin heavy-chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function.

The 78,000-dalton glucose-regulated protein (GRP78) and the immunoglobulin heavy-chain-binding protein (BiP) were shown to be the same protein by NH2-terminal sequence comparison. Immunoprecipitation of GRP78-BiP induced by glucose starvation and a temperature-sensitive mutation in a hamster fibroblast cell line demonstrated the association of GRP78-BiP with other cellular proteins. In both fibroblasts and lymphoid cells, GRP78-BiP was found to label with 32Pi and [3H]adenosine. Phosphoamino acid analysis demonstrated that GRP78-BiP is phosphorylated on serine and threonine residues. Conditions which induce increased production of GRP78-BiP resulted in decreased incorporation of 32Pi and [3H]adenosine into GRP78-BiP. Furthermore, we report here that the phosphorylated form of BiP resides in the endoplasmic reticulum and that BiP which is associated with heavy chains is not phosphorylated or labeled with [3H]adenosine, whereas free BiP is. This suggests that posttranslational modifications may be important in regulating the synthesis and binding of BiP.

Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153).

The gene encoding C/EBP-homologous protein (CHOP), also known as growth arrest and DNA-damage-inducible gene 153 (GADD153), is activated by agents that adversely affect the function of the endoplasmic reticulum (ER). Because of the pleiotropic effects of such agents on other cellular processes, the role of ER stress in inducing CHOP gene expression has remained unclear. We find that cells with conditional (temperature-sensitive) defects in protein glycosylation (CHO K12 and BHK tsBN7) induce CHOP when cultured at the nonpermissive temperature. In addition, cells that are defective in initiating the ER stress response, because of overexpression of an exogenous ER chaperone, BiP/GRP78, exhibit attenuated inducibility of CHOP. Surprisingly, attenuated induction of CHOP was also noted in BiP-overexpressing cells treated with methyl methanesulfonate, an agent thought to activate CHOP by causing DNA damage. The roles of DNA damage and growth arrest in the induction of CHOP were therefore reexamined. Induction of growth arrest by culture to confluence or treatment with the enzymatic inhibitor N-(phosphonacetyl)-L-aspartate did not induce CHOP. Furthermore, both a DNA-damage-causing nucleoside analog (5-hydroxymethyl-2'-deoxyuridine) and UV light alone did not induce CHOP. These results suggest that CHOP is more responsive to ER stress than to growth arrest or DNA damage and indicate a potential role for CHOP in linking stress in the ER to alterations in gene expression.

BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly.

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP-heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo.

A role for human heavy chain binding protein in the developmental regulation of immunoglobin transport.

Human EBV transformed lymphoblastoid cell lines and lymphomas representing various stages of B cell development were examined for heavy chain binding protein (BiP) expression and its association with immunoglobin (Ig) heavy chains. Human BiP was shown to migrate with an apparent mol. wt of 79,000 and to have a pI of approximately 5.5 in all the human cell lines examined. Both the mum and the mus heavy chains synthesized in a pre-B cell line (mu+, LC-) remained associated with BiP and were all found to be endo H sensitive, suggesting that this association occurred in the endoplasmic reticulum (ER). Surface Ig+ B cell lines produce membrane type heavy chains which are expressed on the cell surface and secretory type heavy chains which remain intracellular. The membrane type mu heavy chains produced by a surface Ig+ B cell line were not associated with BiP after assembling with light chains and processing in the Golgi. However, the secretory type mu heavy chains synthesized by these same cells did not combine efficiently with LC and a significant quantity remained associated with BiP and were not secreted suggesting that BiP is involved in the divergent transport of membrane and secretory mu heavy chains in surface Ig+ B cell lines. In Ig secreting plasmacytoid lines the heavy chains were only associated with BiP prior to assembling with LC. When LC assembly was inhibited, the association of heavy chains with BiP was prolonged and Ig secretion was blocked. Therefore, BiP was found to participate in the post-translational processing of mu heavy chains synthesized by human lymphoid cell lines representing all stages of B cell development. Further, heavy chains that remained associated with BiP were not transported to the cell surface or secreted while heavy chains that were only transiently associated with BiP chains were expressed on the cell surface or secreted.

Association of transport-defective light chains with immunoglobulin heavy chain binding protein.

Immunoglobulin light chains are usually secreted from cells when they are synthesized alone or in molar excess of heavy chains, but, there have been reports of nonsecreted light chains. We wished to determine whether immunoglobulin heavy chain binding protein (BiP), which blocks the transport of free heavy chains, might be responsible for the lack of secretion of some light chains. In two murine lymphoid cell lines that synthesize but do not secrete immunoglobulin light chains, the free light chain polymers were found bound to BiP. Examination of 20 other cell lines and hybridomas failed to disclose any cells synthesizing free or excess light chains that associated with BiP, in all cases the free light chains were secreted as dimers. Despite their association with BiP and their blocked secretion, the aberrant light chains could combine with heavy chains and could be secreted as intact Ig molecules. Thus, while light chains do not usually express signals which allow them to bind to BiP, it appears that such signals can be expressed on certain light chains, resulting in their combination with BiP and blocked secretion. When single chain mutant cell lines are isolated from parental lines producing both heavy and light chains, they are almost always light chain producers suggesting that free heavy chains are much more toxic than free light chains. In both PC700 and P3X63Ag cells, however, clones that have lost either heavy chains or transport-defective light chains are present at the same frequency. Our findings that the light chains in both of these lines are associated with BiP raise the possibility that BiP actually contributes to heavy chain toxicity instead of preventing it.

The rabbit B cell antigen receptor is non-covalently associated with unique heteromeric protein complexes: possible insights into the membrane IgM/IgD coexpression paradox.

We describe several proteins that are components of the rabbit B cell receptor complex. Two proteins (37 kDa and 42 kDa) were found in non-covalent association with IgM expressed on B cells from peripheral blood. These proteins were also immunoprecipitated by anti-B29 (Ig-beta) and anti-mb1 (Ig-alpha) monoclonal antibodies. As in the mouse and human, the IgM associated molecules were found as heteromeric structures with non-reduced apparent molecular weights of approximately 70-75 kDa. On rabbit B cells we also found these proteins in a 100-135 kDa complex which may represent trimeric or tetrameric structures. By Western blot, the 37 kDa protein was identified as rabbit Ig-beta (B29), suggesting that the 42 kDa protein is rabbit Ig-alpha. These data suggest that rabbit IgM is associated with both Ig-alpha/beta and Ig-(alpha beta)2 or alpha beta gamma complexes. When similar immunoprecipitation studies were performed on lysates made from B cells isolated from appendix follicles, we found two additional IgM associated protein complexes containing 34 kDa and 36 kDa proteins.

Protein folding and assembly in the endoplasmic reticulum.

The newly synthesized protein emerging through the ER membrane enters a unique environment for folding and assembly. Unlike the cytosol, the ER provides an oxidizing environment, has high levels of calcium, and contains enzymes for N-linked glycosylation. The growing nascent polypeptide chain is in many cases modified co-translationally with N-linked sugars and begins to fold while still attached to the ribosome. Disulfide bond formation stabilizes the tertiary structure of the protein. The in vivo folding and assembly of nascent proteins requires a delicate balance between allowing folding to occur and preventing incorrect interactions that would ultimately lead to improper folding and/or aggregation. In the past several years, two groups of proteins that interact transiently with incompletely folded and assembled proteins in the ER have been identified and characterized. The first group consists of enzymes that promote or stabilize protein folding. The second is composed of proteins termed "molecular chaperones" that bind transiently to nascent polypeptides and apparently prevent misfolding by masking those regions that could lead to incorrect interactions between protein domains or aggregation.

Mutations within the nucleotide binding site of immunoglobulin-binding protein inhibit ATPase activity and interfere with release of immunoglobulin heavy chain.

Immunoglobulin-binding protein (BiP), a 70-kDa heat shock protein in the endoplasmic reticulum, binds transiently to nascent proteins, releasing them upon folding and assembly. The in vitro release of bound proteins from BiP requires ATP hydrolysis. Recently, the three-dimensional structure was solved for an ATP-hydrolyzing proteolytic 44-kDa fragment of a 71-kDa heat shock cognate protein, HSC71. Because of the high degree of homology in this region, BiP presumably forms a similar ATP binding structure. Amino-terminal deletions in BiP eliminated ATP-agarose binding. Alteration of a second potential ATP binding site had no effect, suggesting that only the HSC71-like site was capable of ATP binding. Crystallographic data from HSC71 implicated certain amino acids in interactions with the beta-phosphate, gamma-phosphate, and divalent cation of ATP. Mutation of each corresponding residue in BiP (Thr-37, Thr-229, and Glu-201) severely inhibited its ATPase activity. These BiP mutants were still capable of binding ATP and immunoglobulin heavy chains, suggesting that these mutations did not drastically alter the structure of BiP. They did however block the ATP-mediated release of heavy chains from BiP. Our results demonstrate that the structure of BiP in this region must be extremely similar to that elucidated for HSC71 and that mutations of residues proposed to interact with ATP block the ATP-mediated release of bound protein by inhibiting ATP hydrolysis.

The immunoglobulin-binding protein in vitro autophosphorylation site maps to a threonine within the ATP binding cleft but is not a detectable site of in vivo phosphorylation.

In vitro incubation of immunoprecipitated immunoglobulin-binding protein (BiP) complexes with calcium and [gamma-32P]ATP resulted in the phosphorylation of BiP on a threonine residue. This autophosphorylation activity did not occur in the presence of magnesium but had the same pH optimum as reported for its magnesium-dependent ATPase activity. This suggested the possibility that both activities could occur through ATP hydrolysis at the same site. In support of this, mutation of either Thr-37 or Thr-229 to a glycine eliminated both autophosphorylation and ATPase activities, and mutation of either residue to a serine significantly reduced both activities. Glutamic acid 175 in HSC71 has been hypothesized to flank the divalent cation complexed with ATP. Mutation of the analogous glutamic acid, Glu-201, in BiP abolished ATPase activity but still supported some autophosphorylation. The in vitro phosphorylation site was mapped to Thr-229 by mutational analysis. This threonine has been hypothesized to interact with the gamma-phosphate of ATP through a polarized water molecule and would be in a position to act as a phosphate acceptor in the ATP hydrolysis reaction. These data imply that both ATPase and autophosphorylation result from ATP hydrolysis at the same site and that the cation associated with BiP determines which activity is observed. Comparison of partial protease digestion or cyanogen bromide cleavage products of in vitro and in vivo phosphorylated BiP demonstrated that Thr-229 is not a detectable site of phosphorylation in cells. Therefore, whatever functional role phosphorylation may have in vivo, it cannot be attributed to autophosphorylation of Thr-229.

Characterization of the nucleotide binding properties and ATPase activity of recombinant hamster BiP purified from bacteria.

HSP70 family proteins bind ATP and hydrolyze it, but the precise role of these activities in their in vivo chaperoning function has not been determined. In this report, we characterized wild-type hamster BiP isolated from bacteria in terms of its ATP binding and ATPase activities. Recombinant BiP behaved essentially the same as endogenous BiP in terms of oligomeric status, protease digestion patterns, and ATPase properties. By engineering a Factor Xa cleavable site following the His tag which was used for affinity purification, we demonstrated that the six histidines had no effect on either the structural or ATPase properties of recombinant BiP. We also found that bacteria-synthesized BiP had a tightly bound ADP that was resistant to dialysis. Removal of the bound nucleotide allowed us to directly measure the binding affinity of ATP and ADP to BiP (Kd of 0.2 microM for ATP and 0.29 microM for ADP) by equilibrium dialysis. Careful characterization of wild-type BiP will allow us to use this system to characterize BiP ATP binding site mutants that can be used to probe the role of ATP binding and ATPase activity in BiP functions.