BiP, a major chaperone protein of the endoplasmic reticulum lumen, plays a direct and important role in the storage of the rapidly exchanging pool of Ca2+

The activity of BiP, the major chaperone of the endoplasmic reticulum (ER) lumen, is known to be Ca2+-regulated; however, the participation of this protein in the ER storage of the cation has not yet been investigated. Here such a role is demonstrated in human epithelial (HeLa) cells transiently transfected with the hamster BiP cDNA and incubated in Ca2+-free medium, as revealed by two different techniques. In the first, co-transfected aequorin was employed as a probe for assaying either the cytosolic of the mitochondrial free Ca2+ concentration. By this approach higher Ca2+ release responses were revealed in BiP-transfected cells by experiments in which extensive store depletion was induced either by repetitive stimulation with inositol 1,4,5-trisphosphate-generating agonists or by treatment with the Ca2+ ionophore, A23187. In the second technique the cells were loaded at the equilibrium with 45Ca, and the release of the tracer observed upon treatment with thapsigargin, a blocker of the ER Ca2+ ATPases, was larger in BiP-transfected than in control cells. The latter results were obtained also when BiP was overexpressed not via transfection but as a response to ER stress by tunicamycin. These results are sustained by increases of the ER Ca2+ storage capacity rather than by artifacts or indirect readjustments induced in the cells by the overexpression of the chaperone since (a) the exogenous and endogenous BiP were both confined to the ER, (b) the expression levels of other proteins active in the ER Ca2+ storage were not changed, and (c) effects similar to those of wild type BiP were obtained with a deletion mutant devoid of chaperone activity. The specificity of the results was confirmed by parallel 45Ca experiments carried out in HeLa cells transfected with two other Ca2+-binding proteins, calreticulin and CaBP2(ERp72), only the first of which induced increases of Ca2+ capacity. We conclude that BiP has a dual function, in addition to its chaperone role it is a bona fide ER lumenal Ca2+ storage protein contributing, under resting cell conditions, to around 25% of the store, with a stoichiometry of 1-2 moles of calcium/mole of BiP

The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum

The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain–binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding–induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain–binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates

First virtual international congress on cellular and organismal stress responses, november 5–6, 2020

Members of the Cell Stress Society International (CSSI), Patricija van Oosten-Hawle (University of Leeds, UK), Mehdi Mollapour (SUNY Upstate Medical University, USA), Andrew Truman (University of North Carolina at Charlotte, USA) organized a new virtual meeting format which took place on November 5–6, 2020. The goal of this congress was to provide an international platform for scientists to exchange data and ideas among the Cell Stress and Chaperones community during the Covid-19 pandemic. Here we will highlight the summary of the meeting and acknowledge those who were honored by the CSSI

Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for Monoclonal Antibody Expression Level and Quality in CHO Cells

10.1371/journal.pone.0063247PLoS ONE85