Kaji E H, Lodish H F
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
J Biol Chem. 1993 Oct 15;268(29):22188-94.
In cultured hepatoma HepG2 cells, serum retinol-binding protein (RBP) is secreted more rapidly in the presence of retinol than in its absence (Tosetti, F., Ferrari, N., Pfeffer, U., Brigati, C., and Vidali, G. (1992) Exp. Cell Res. 200, 467-472). In the presence of millimolar concentration of DTT, HepG2 cells synthesize fully reduced RBP within the endoplasmic reticulum (ER) which, upon removal of DTT, forms disulfide bonds post-translationally. Secretion of this post-translationally folded RBP is also dependent on the presence of retinol. Using nonreducing gel electrophoresis, we resolved disulfide-bonded RBP folding intermediates. In addition, two other intracellular folding intermediates, compact I and II, which co-migrate with mature RBP were resolved by their different sensitivity to DTT-induced unfolding. Retinol, as well as retinoic acid, stabilized both compact I and II RBP intermediates to DTT-induced unfolding, suggesting that RBP assumes different conformations in the ER in the presence and absence of a ligand. However, only RBP synthesized in the presence of retinol is rapidly secreted, indicating that the ER export quality control system recognizes RBP containing retinol, but not retinoic acid, as fully folded and competent for export. Folding of RBP so that it is stabilized to DTT reduction is not a sufficient condition for ER exit.
在培养的肝癌HepG2细胞中，与无视黄醇时相比，视黄醇存在时血清视黄醇结合蛋白（RBP）的分泌更快（托塞蒂，F.，法拉利，N.，普费弗，U.，布里加蒂，C.，和维达利，G.（1992年）《实验细胞研究》200，467 - 472）。在毫摩尔浓度的二硫苏糖醇（DTT）存在下，HepG2细胞在内质网（ER）中合成完全还原的RBP，去除DTT后，其在翻译后形成二硫键。这种翻译后折叠的RBP的分泌也依赖于视黄醇的存在。使用非还原凝胶电泳，我们解析了二硫键结合的RBP折叠中间体。此外，另外两种细胞内折叠中间体，紧密型I和II，它们与成熟RBP共迁移，通过它们对DTT诱导的解折叠的不同敏感性得以解析。视黄醇以及视黄酸使紧密型I和II RBP中间体对DTT诱导的解折叠稳定，这表明在有或没有配体存在的情况下，RBP在内质网中呈现不同的构象。然而，只有在视黄醇存在下合成的RBP才会迅速分泌，这表明内质网输出质量控制系统将含有视黄醇而非视黄酸的RBP识别为完全折叠且有能力输出。RBP的折叠使其对DTT还原稳定并不是内质网输出的充分条件。
