Walz T, Häner M, Wu X R, Henn C, Engel A, Sun T T, Aebi U
M. E. Müller Institute for Structural Biology Biozentrum, University of Basel, Switzerland.
J Mol Biol. 1995 May 19;248(5):887-900. doi: 10.1006/jmbi.1995.0269.
The asymmetric unit membrane (AUM) forms numerous plaques covering the apical surface of mammalian urinary bladder epithelium. These plaques contain four major integral membrane proteins called uroplakins Ia, Ib, II and III, which form particles arranged in a well-ordered hexagonal lattice with p6 symmetry and a lattice constant of 16.5 nm. Bovine AUM plaques negatively stained with anionic sodium silicotungstate revealed structural detail to 3.1 nm resolution. Correlation averaging resolved each particle into 12 stain-excluding domains arranged in two concentric rings (inner ring radius (rm) = 3.7 nm, outer ring radius (rout) = 6.6 nm), each with six domains which were rotated by roughly 30 degrees relative to each other. Negative staining with cationic uranyl formate increased the resolution to 2.2 nm and unveiled distinct connections between adjacent AUM particles. These connections may provide a molecular basis for the observed insolubility of the plaques in many detergents. Examination of the luminal face of freeze-dried/unidirectionally metal-shadowed AUM plaques established a left-handed vorticity of the 16 nm protein particles, whereas the cytoplasmic face exhibited no significant surface corrugations. Three-dimensional reconstruction from sodium silicotungstate-stained specimens revealed the AUM particles to be built of six "V-shaped" subunits anchored upright in the membrane. The mass density distribution within uranyl formate-stained AUM particles was similar except that the inner tip of each V was bridged to the outer tip of an adjacent V, so that the 16 nm AUM particle appeared as a continuous, "twisted ribbon" embracing a central cavity. Finally, mass measurements of unstained/freeze-dried plaques by scanning transmission electron microscopy yielded a total mass of 1,120 kDa per membrane-bound AUM particle. By imposing constraints on the possible uroplakin stoichiometries within AUM plaques, these data provide a first glimpse of the molecular architecture of the 16 nm particles constituting the plaques.
不对称单位膜（AUM）形成许多斑块，覆盖哺乳动物膀胱上皮的顶端表面。这些斑块包含四种主要的整合膜蛋白，称为uroplakins Ia、Ib、II和III，它们形成排列成具有p6对称性和16.5 nm晶格常数的有序六边形晶格的颗粒。用阴离子硅钨酸钠负染的牛AUM斑块显示出3.1 nm分辨率的结构细节。相关平均将每个颗粒解析为12个不染色区域，排列成两个同心环（内环半径（rm）= 3.7 nm，外环半径（rout）= 6.6 nm），每个环有六个区域，彼此相对旋转约30度。用阳离子甲酸铀酰负染将分辨率提高到2.2 nm，并揭示了相邻AUM颗粒之间的明显连接。这些连接可能为观察到的斑块在许多去污剂中的不溶性提供分子基础。对冻干/单向金属阴影的AUM斑块腔面的检查确定了16 nm蛋白质颗粒的左旋涡度，而细胞质面没有明显的表面波纹。从硅钨酸钠染色标本进行的三维重建显示，AUM颗粒由六个“V形”亚基组成，这些亚基垂直锚定在膜中。甲酸铀酰染色的AUM颗粒内的质量密度分布相似，只是每个V的内尖端与相邻V的外尖端相连，因此16 nm的AUM颗粒呈现为围绕中心腔的连续“扭曲带”。最后，通过扫描透射电子显微镜对未染色/冻干斑块进行质量测量，得出每个膜结合的AUM颗粒的总质量为1120 kDa。通过对AUM斑块内可能的uroplakin化学计量施加限制，这些数据首次展示了构成斑块的16 nm颗粒的分子结构。
