Yu J, Manabe M, Wu X R, Xu C, Surya B, Sun T T
Department of Dermatology, Kaplan Cancer Center, New York University School of Medicine, New York 10016.
J Cell Biol. 1990 Sep;111(3):1207-16. doi: 10.1083/jcb.111.3.1207.
The luminal surface of mammalian urothelium is covered with numerous plaques (also known as the asymmetric unit membrane or AUM) composed of semi-crystalline, hexagonal arrays of 12-nm protein particles. Despite the presumed importance of these plaques in stabilizing the urothelial surface during bladder distention, relatively little is known about their protein composition. Using a mouse mAb, AE31, we have identified a 27-kD protein that is urothelium-specific and is differentially expressed in superficial umbrella cells. This protein (pI approximately 5.8) partitions into the detergent phase during Triton X-114 phase separation. Pulse-chase experiments using cultured bovine urothelial cells showed that this protein is synthesized as a 32-kD precursor that is processed through a 30-kD intermediate, to the mature 27-kD form. In cytoplasmic vesicles containing immature AUM, the AE31 epitope is detected in patches on the cytoplasmic side, but in mature, apical AUM it is detected exclusively on the luminal side. This suggests an unusual translocation of the AE31 epitope during AUM maturation; more data are required, however, to substantiate this interpretation. Immunoaffinity purification of the 27-kD protein results in the copurification in approximately molar ratio of a 15-kD protein, as well as a small and variable amount of a 47-kD protein. Immunoblotting data indicate that these three proteins are immunologically distinguishable. This copurified 15-kD protein is relative basic (pI approximately 8.0). Like the 27-kD protein, it is urothelium-specific and is present mainly in the umbrella cells. Together, our data indicate that a 27-kD protein is urothelial plaque-associated (uroplakin I). Based on complex formation data, we provisionally name the 15-kD protein uroplakin II; additional data will be required to determine whether this and the 47-kD protein are integral parts of AUM. The identification of these AUM-associated and -related proteins, plus the availability of a culture system capable of synthesizing and processing some of these molecules, offer new opportunities for studying the detailed structure, assembly, and function of asymmetrical unit membrane.
哺乳动物尿路上皮的管腔表面覆盖着许多斑块（也称为不对称单位膜或AUM），这些斑块由12纳米蛋白质颗粒的半结晶六边形阵列组成。尽管这些斑块在膀胱扩张过程中对稳定尿路上皮表面的重要性已得到推测，但对其蛋白质组成的了解相对较少。利用小鼠单克隆抗体AE31，我们鉴定出一种27-kD的蛋白质，它是尿路上皮特异性的，在表层伞细胞中差异表达。这种蛋白质（pI约为5.8）在Triton X-114相分离过程中分配到去污剂相中。使用培养的牛尿路上皮细胞进行的脉冲追踪实验表明，这种蛋白质以32-kD的前体形式合成，经过30-kD的中间体加工成成熟的27-kD形式。在含有未成熟AUM的细胞质小泡中，AE31表位在细胞质侧的斑块中被检测到，但在成熟的顶端AUM中，它仅在管腔侧被检测到。这表明在AUM成熟过程中AE31表位发生了异常转运；然而，需要更多数据来证实这一解释。对27-kD蛋白质进行免疫亲和纯化后，会以大约摩尔比共纯化出一种15-kD的蛋白质，以及少量且数量可变的47-kD蛋白质。免疫印迹数据表明这三种蛋白质在免疫学上是可区分的。这种共纯化的15-kD蛋白质相对呈碱性（pI约为8.0）。与27-kD蛋白质一样，它是尿路上皮特异性的，主要存在于伞细胞中。我们的数据共同表明，一种27-kD的蛋白质与尿路上皮斑块相关（uroplakin I）。根据复合物形成数据，我们暂时将15-kD的蛋白质命名为uroplakin II；需要更多数据来确定这种蛋白质以及47-kD蛋白质是否是AUM的组成部分。这些与AUM相关及相关蛋白质的鉴定，加上能够合成和加工其中一些分子的培养系统的可用性，为研究不对称单位膜的详细结构、组装和功能提供了新的机会。
