Nigam S K, Goldberg A L, Ho S, Rohde M F, Bush K T
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1994 Jan 21;269(3):1744-9.
The major proteins in the lumen of the endoplasmic reticulum (ER) are thought to function in Ca2+ sequestration or as "molecular chaperones" in the folding and assembly of membrane or secreted proteins. Based on the ability of many chaperones to bind selectively to unfolded proteins and to dissociate from them upon ATP hydrolysis, we developed an affinity chromatography method to isolate proteins with these characteristics from pancreatic or liver ER. Seven ER proteins bound selectively to denatured protein columns and were specifically eluted by ATP (10(-6) M) but not by a nonhydrolyzable ATP analog. These proteins were identified with antibodies and microsequencing as the ER chaperone BiP (grp78), grp94, calreticulin, a novel 46-kDa protein that binds azido-ATP, as well as three members of the thioredoxin superfamily: protein-disulfide isomerase, ERp72, and a previously reported 50-kDa protein (p50). This set of seven proteins bound to and was eluted with ATP from a variety of denatured proteins, including histone, gelatin, alpha fetoprotein, thyroglobulin, lysozyme, casein, and IgG. The release of grp94, protein-disulfide isomerase, ERp72, calreticulin, and p50 was stimulated by Ca2+ in the presence of ATP. These proteins thus appear to function as Ca(2+)-dependent chaperones, which may account for the Ca2+ and ATP requirement for protein folding in the ER.
内质网（ER）腔中的主要蛋白质被认为在Ca2+螯合中发挥作用，或在膜蛋白或分泌蛋白的折叠和组装中作为“分子伴侣”。基于许多伴侣蛋白能够选择性地结合未折叠蛋白并在ATP水解时与之解离的能力，我们开发了一种亲和色谱方法，从胰腺或肝脏内质网中分离具有这些特性的蛋白质。七种内质网蛋白选择性地结合到变性蛋白柱上，并被ATP（10^(-6) M）特异性洗脱，但不被不可水解的ATP类似物洗脱。这些蛋白质通过抗体和微测序鉴定为内质网伴侣蛋白BiP（grp78）、grp94、钙网蛋白、一种结合叠氮ATP的新型46 kDa蛋白，以及硫氧还蛋白超家族的三个成员：蛋白二硫键异构酶、ERp72和一种先前报道的50 kDa蛋白（p50）。这七种蛋白质结合到包括组蛋白、明胶、甲胎蛋白、甲状腺球蛋白、溶菌酶、酪蛋白和IgG在内的多种变性蛋白上，并被ATP洗脱。在ATP存在的情况下，Ca2+刺激了grp94、蛋白二硫键异构酶、ERp72、钙网蛋白和p50的释放。因此，这些蛋白质似乎作为Ca(2+)依赖性伴侣蛋白发挥作用，这可能解释了内质网中蛋白质折叠对Ca2+和ATP的需求。
