Le Bivic A, Sambuy Y, Mostov K, Rodriguez-Boulan E
Department of Cell Biology and Anatomy, Cornell University Medical College, New York 10021.
J Cell Biol. 1990 May;110(5):1533-9. doi: 10.1083/jcb.110.5.1533.
We studied the cell-surface delivery pathways of newly synthesized membrane glycoproteins in MDCK cells and for this purpose we characterized an endogenous apical integral membrane glycoprotein. By combining a pulse-chase protocol with domain-selective cell-surface biotinylation, immune precipitation, and streptavidin-agarose precipitation (Le Bivic et al. 1989. Proc. Natl. Acad. Sci USA. 86:9313-9317), we followed the appearance at the cell surface of a major apical sialoglycoprotein, gp114, a basolateral protein, uvomorulin, and a transcytosing protein, the polyimmunoglobulin receptor (pIg-R). We determined that both gp114 and uvomorulin appeared to be delivered directly to their respective surface, with mistargeting levels of 8 and 2%, respectively. Using the same technique, the pIg-R was first detected on the basolateral domain and then on the apical domain, to be finally released into the apical medium, as described (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). To directly determine whether the gp114 pool present on the basolateral surface was a precursor of the apical gp114, we compared it with the equivalent pIg-R pool, by labeling with sulfo-NHS-SS-biotin, a cleavable, tight junction-impermeable probe, and by following the fraction of this probe that became resistant to basal glutathione and accessible to apical glutathione during incubation at 37 degrees C. We found that, contrary to pIg-R, basolateral gp114 was poorly endocytosed and was not transcytosed to the apical side. These results demonstrate that an endogenous apical integral membrane glycoprotein of Madin-Darby canine kidney cells is sorted intracellularly and is vectorially targeted to the apical surface.
我们研究了新合成的膜糖蛋白在MDCK细胞中的细胞表面递送途径，为此我们对一种内源性顶端整合膜糖蛋白进行了表征。通过将脉冲追踪方案与结构域选择性细胞表面生物素化、免疫沉淀和链霉亲和素-琼脂糖沉淀相结合（勒·比维克等人，1989年。美国国家科学院院刊。86:9313 - 9317），我们追踪了一种主要的顶端唾液酸糖蛋白gp114、一种基底外侧蛋白桥粒芯蛋白以及一种转胞吞蛋白多聚免疫球蛋白受体（pIg - R）在细胞表面的出现情况。我们确定gp114和桥粒芯蛋白似乎都直接递送至它们各自的表面，错误靶向水平分别为8%和2%。使用相同技术，如前所述（莫斯托夫，K. E.，和D. L. 迪彻尔。1986年。细胞。46:613 - 621），pIg - R首先在基底外侧结构域被检测到，然后在顶端结构域被检测到，最终释放到顶端培养基中。为了直接确定存在于基底外侧表面的gp114库是否是顶端gp114的前体，我们通过用可裂解的、紧密连接不可渗透的探针磺基 - NHS - SS - 生物素标记，并追踪该探针在37℃孵育期间对基底谷胱甘肽产生抗性且可被顶端谷胱甘肽作用的部分，将其与等效的pIg - R库进行比较。我们发现，与pIg - R相反，基底外侧gp114很少被内吞，也不会转胞吞至顶端侧。这些结果表明，Madin - Darby犬肾细胞的一种内源性顶端整合膜糖蛋白在细胞内被分选，并以向量方式靶向至顶端表面。
