Morris J A, Dorner A J, Edwards C A, Hendershot L M, Kaufman R J
Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1997 Feb 14;272(7):4327-34. doi: 10.1074/jbc.272.7.4327.
BiP/GRP78 is a lumenal stress protein of the endoplasmic reticulum (ER) that interacts with polypeptide folding intermediates transiting the secretory compartment. We have studied the secretion and the stress response in Chinese hamster ovary (CHO) cells that overexpress either wild-type immunoglobulin binding protein (BiP) or a BiP deletion molecule (residues 175-201) that can bind peptides and ATP but is defective in ATP hydrolysis and concomitant peptide release. Overexpressed wild-type BiP was localized to the ER and unique vesicles within the nucleus, whereas overexpressed ATPase-defective BiP was localized to the ER and cytoplasmic vesicles but was absent from the nucleus. Compared with wild-type CHO cells, overexpression of ATPase-defective BiP prevented secretion of factor VIII, a coagulation factor that extensively binds BiP in the lumen of the ER. Under these conditions factor VIII was stably associated with the ATPase-defective BiP. In contrast, the secretion of monocyte/macrophage colony stimulating factor, a protein that is not detected in association with BiP, was not affected by overexpression of ATPase-defective BiP. These results show that BiP function is not required for secretion of some proteins and suggest that some proteins do not interact with BiP upon transport through the ER. The presence of unfolded protein in the ER induces transcription of BiP and also elicits a general inhibition of protein synthesis. Overexpression of wild-type BiP prevented the stress-mediated transcriptional induction of BiP in response to either calcium ionophore A23187 treatment or tunicamycin treatment. In contrast, overexpression of ATPase-defective BiP did not prevent the stress induction of BiP, showing that the ATPase activity is required to inhibit transcriptional induction. Overexpression of wild-type BiP, but not ATPase-defective BiP, increased survival of cells treated with A23187. The increased survival mediated by overexpressed wild-type BiP correlated with reduced translation inhibition in response to the stress condition. These results indicate that overexpressed BiP alleviated the stress in the ER to prevent BiP transcriptional induction and permit continued translation of cellular mRNAs.
BiP/GRP78是内质网（ER）的一种腔内应激蛋白，它与通过分泌区室转运的多肽折叠中间体相互作用。我们研究了过表达野生型免疫球蛋白结合蛋白（BiP）或BiP缺失分子（第175 - 201位氨基酸残基）的中国仓鼠卵巢（CHO）细胞中的分泌和应激反应，该缺失分子能够结合肽和ATP，但在ATP水解及伴随的肽释放方面存在缺陷。过表达的野生型BiP定位于内质网和细胞核内的独特囊泡，而过表达的ATP酶缺陷型BiP定位于内质网和细胞质囊泡，但细胞核中没有。与野生型CHO细胞相比，ATP酶缺陷型BiP的过表达阻止了凝血因子VIII的分泌，凝血因子VIII在内质网腔中与BiP广泛结合。在这些条件下，凝血因子VIII与ATP酶缺陷型BiP稳定结合。相反，单核细胞/巨噬细胞集落刺激因子（一种未检测到与BiP结合的蛋白质）的分泌不受ATP酶缺陷型BiP过表达的影响。这些结果表明，某些蛋白质的分泌不需要BiP发挥功能，并提示某些蛋白质在通过内质网转运时不与BiP相互作用。内质网中未折叠蛋白的存在会诱导BiP的转录，同时也会引发蛋白质合成的普遍抑制。野生型BiP的过表达可防止因钙离子载体A23187处理或衣霉素处理而导致的应激介导的BiP转录诱导。相反，ATP酶缺陷型BiP的过表达并不能阻止BiP的应激诱导，表明ATP酶活性是抑制转录诱导所必需的。野生型BiP的过表达而非ATP酶缺陷型BiP的过表达可提高经A23187处理的细胞的存活率。过表达的野生型BiP介导的存活率提高与应激条件下翻译抑制的降低相关。这些结果表明，过表达的BiP减轻了内质网中的应激，以防止BiP转录诱导并允许细胞mRNA的持续翻译。
