Unassembled Ig Heavy Chains Do Not Cycle from BiP In Vivo but Require Light Chains to Trigger Their Release

Vanhove, Marc; Usherwood, Young-Kwang; Hendershot, Linda M.

doi:10.1016/s1074-7613(01)00163-7

Cited by 109 publications

(119 citation statements)

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“…It is known to interact with the immunoglobulins HCs and prevent their aggregation before LCs are attached (Vanhove et al 2001). It was observed previously, that with increasing specific mAb productivity in recombinant cells, number of molecular chaperones known to directly interact with nascent immunoglobulins exhibit a significant increase in abundance as well.…”

Section: Discussionmentioning

confidence: 99%

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

et al. 2014

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Despite the fact, that monoclonal antibodies are the fastest growing group of biopharmaceuticals in development, this is not true for the IgM class, which remains as enigmatic as ever. While more examples of usefulness of IgMs for medical applications are emerging, their recombinant production is still not common. In our study, stable monoclonal IgM producing CHO DG44 and HEK 293 cell lines, expressing two model IgM molecules (IgM-617 and IgM-012) were established. Recombinant cell lines were compared in regard of specific productivity, specific growth rate, maximal achieved antibody titer, gene copy numbers and transcription levels of transgene. IgM-617 cell lines were identified as high while IgM-012 clones were low producers. Although differences in gene copy numbers as well as in transcription levels were observed, they did not seem to be a limitation. Levels of relevant endoplasmic reticulumstress related proteins were analyzed and no indications of unfolded protein response were detected. This could indicate that the difference in the intrinsic protein stability of our model proteins (as was previously observed on purified samples) might cause lower yields of IgM-012. Transcriptomics and/or proteomics follow up studies might be necessary for identification of potential bottlenecks in IgM producing cell lines.

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Section: Discussionmentioning

confidence: 99%

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

et al. 2014

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“…The trigger for BiP dissociation may be proteins unfolded in response to reducing agent or a specific ϪSH reactive sensor protein, either of which may expose BiP to ATP binding and release from ATF6. By analogy, antibody light chain binding to heavy chain causes ATP release of BiP from heavy chain (20).…”

Section: Discussionmentioning

confidence: 99%

In vitro reconstitution of ER-stress induced ATF6 transport in COPII vesicles

Schindler

Schekman

2009

Proc. Natl. Acad. Sci. U.S.A.

188

148

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The transcription factor ATF6 is held as a membrane precursor in the endoplasmic reticulum (ER), and is transported and proteolytically processed in the Golgi apparatus under conditions of unfolded protein response stress. We show that during stress, ATF6 forms an interaction with COPII, the protein complex required for vesicular traffic of cargo proteins from the ER. Using an in vitro budding reaction that recapitulates the ER-stress induced transport of ATF6, we show that no cytoplasmic proteins other than COPII are necessary for transport. ATF6 is retained in the ER by association with the chaperone BiP (GRP78). In the in vitro reaction, the ATF6-BiP complex disassembles when membranes are treated with reducing agent and ATP. A hybrid protein with the ATF6 cytoplasmic domain replaced by a constitutive sorting signal (Sec22b SNARE) retains stress-responsive transport in vivo and in vitro. These results suggest that unfolded proteins or an ER luminal ؊SH reactive bond controls BiP-ATF6 stability and access of ATF6 to the COPII budding machinery.regulated transport ͉ unfolded protein response ͉ BiP ͉ prebudding complex E ndoplasmic reticulum (ER) homeostasis can be perturbed by up-regulation of secretory proteins or by disruption of protein processing, a condition termed ER stress (1). ER stress causes increased protein misfolding and leads to activation of the unfolded protein response (UPR), an evolutionarily conserved pathway to alleviate ER stress (2). The UPR leads to a slowdown in protein synthesis, an upregulation of ER chaperones, and an upregulation of ER-associated protein degradation.The three effector proteins for the UPR are IRE1, PERK, and ATF6. During the UPR, IRE and PERK remain in the ER and act via cytoplasmic effectors, whereas ATF6 is transported to the Golgi complex. In the Golgi, ATF6 is cleaved by Site-1 and Site-2 proteases (3). These cleavages release the N-terminal cytoplasmic domain, which contains a bZIP motif that binds DNA at ER stress response elements that control expression of the ER chaperones BiP (GRP78) and GRP94 (4).Transport of ATF6 and other cargo proteins likely involves the COPII coat, a complex of five cytoplasmic proteins that selects cargo at the ER membrane and pinches off membranes to form vesicles. The five proteins are the GTPase Sar1, which is recruited to the ER membrane and initiates coat formation by GDP to GTP exchange; the heterodimeric complex Sec23/Sec24, which binds to Sar1⅐GTP; and a second dimeric complex, Sec13/Sec31, which binds to Sec23/24 and provides the curvature necessary for vesicle fission (5). Cargo is selected by interactions between domains on the Sec24 subunit and cytoplasmic motifs on cargo proteins (6). Inhibition of COPII by overexpression of dominant negative Sar1 has been shown to block transport of ATF6 (7).A central question in ATF6 function is how luminal stress is converted into recognition by the cytoplasmic COPII complex. The luminal chaperone BiP binds ATF6 stably in unstressed cells and dissociates specifically during stress (8)...

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“…to aggregation of the unfolded substrate (38,39). Thus, it is reasonable to assume that the timing or conditions of release might be important and that overexpression of a nucleotide exchange factor could have either positive or negative effects on protein folding.…”

Section: Discussionmentioning

confidence: 99%

BAP, a Mammalian BiP-associated Protein, Is a Nucleotide Exchange Factor That Regulates the ATPase Activity of BiP

Chung

Shen

Hendershot

2002

Journal of Biological Chemistry

170

123

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We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an ϳ54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.

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Unassembled Ig Heavy Chains Do Not Cycle from BiP In Vivo but Require Light Chains to Trigger Their Release

Cited by 109 publications

References 50 publications

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

In vitro reconstitution of ER-stress induced ATF6 transport in COPII vesicles

BAP, a Mammalian BiP-associated Protein, Is a Nucleotide Exchange Factor That Regulates the ATPase Activity of BiP

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