Lièvremont J P, Rizzuto R, Hendershot L, Meldolesi J
Department of Pharmacology and B. Ceccarelli Center of Neurobiology, University of Milano, CNR Center of Molecular and Cellular Pharmacology, and DIBIT, Scientific Institute San Raffaele, 20132 Milano, Italy.
J Biol Chem. 1997 Dec 5;272(49):30873-9. doi: 10.1074/jbc.272.49.30873.
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.
内质网（ER）腔的主要伴侣蛋白BiP的活性已知受Ca2+调节；然而，该蛋白在阳离子的内质网储存中的参与情况尚未得到研究。在此，通过两种不同技术表明，在转染了仓鼠BiP cDNA并在无Ca2+培养基中孵育的人上皮（HeLa）细胞中存在这样一种作用。首先，共转染的水母发光蛋白用作检测胞质或线粒体游离Ca2+浓度的探针。通过这种方法，在用产生肌醇1,4,5-三磷酸的激动剂重复刺激或用Ca2+离子载体A23187处理诱导大量储存耗尽的实验中，在转染BiP的细胞中发现了更高的Ca2+释放反应。在第二种技术中，细胞在平衡状态下加载45Ca，在用内质网Ca2+ ATP酶阻滞剂毒胡萝卜素处理后观察到的示踪剂释放，在转染BiP的细胞中比对照细胞更大。当BiP不是通过转染而是作为对衣霉素内质网应激的反应而过表达时，也得到了后者的结果。这些结果是由内质网Ca2+储存能力的增加所支持的，而不是由伴侣蛋白过表达在细胞中诱导的假象或间接重新调整所支持的，因为（a）外源性和内源性BiP都局限于内质网，（b）在内质网Ca2+储存中起作用的其他蛋白的表达水平没有改变，并且（c）用缺乏伴侣蛋白活性的缺失突变体获得了与野生型BiP相似的效果。在用另外两种Ca2+结合蛋白钙网蛋白和CaBP2（ERp72）转染的HeLa细胞中进行的平行45Ca实验证实了结果的特异性，其中只有第一种蛋白诱导了Ca2+容量的增加。我们得出结论，BiP具有双重功能，除了其伴侣蛋白作用外，它还是一种真正的内质网腔Ca2+储存蛋白，在静息细胞条件下，对储存的贡献约为25%，化学计量比为1-2摩尔钙/摩尔BiP。
