Ybe Joel A, Ruppel Nicholas, Mishra Sanjay, VanHaaften Eric
Department of Biology, Indiana University, Bloomington, IN 47405, USA.
Traffic. 2003 Dec;4(12):850-6. doi: 10.1046/j.1600-0854.2003.0139.x.
The three-legged or triskelion shape of clathrin is critical for the formation of polyhedral lattices around clathrin-coated vesicles. Filamentous legs radiate from a common vertex, with amino acids 1550-1615 contributed by each leg to define the trimerization domain (Liu S-H, Wong ML, Craik CS, Brodsky FM. Cell 1995; 83: 257-267). Within this amino acid stretch there are 3 cysteines at positions 1565, 1569 and 1573 which are completely conserved in higher mammals from humans to C. elegans. The cysteine-to-serine mutation at position 1573 was observed to have the largest impact on clathrin structure and self-assembly. We have also found that Cysteine 1528 located near the boundary between the proximal region and trimerization domain mediated the formation of nonproductive clathrin aggregates when bound light chain subunits were removed. However, when light chains were added back, the ability of this cysteine to form disulfide bridges between individual clathrin molecules was blocked, suggesting bound light chain interacted with Cysteine 1528 to prevent aggregation. This new information serves to map the orientation of the light chain subunit in the vicinity of the trimerization domain and supports previous models that indicate involvement of the trimerization domain in LC binding (Chen C-Y, Reese ML, Hwang PK, Ota N, Agard D, Brodsky FM. EMBO J 2002; 21: 6072-6082; Pishvaee B, Munn A, Payne GS. EMBO J 1997; 16: 2227-2239).
网格蛋白的三足或三歧形状对于在网格蛋白包被小泡周围形成多面体晶格至关重要。丝状的腿从一个共同的顶点辐射出来，每条腿贡献氨基酸1550 - 1615来定义三聚化结构域（Liu S-H, Wong ML, Craik CS, Brodsky FM. Cell 1995; 83: 257 - 267）。在这个氨基酸片段中，位置1565、1569和1573处有3个半胱氨酸，在从人类到秀丽隐杆线虫的高等哺乳动物中是完全保守的。观察到位置1573处的半胱氨酸到丝氨酸的突变对网格蛋白结构和自组装的影响最大。我们还发现，位于近端区域和三聚化结构域边界附近的半胱氨酸1528在去除结合的轻链亚基时介导了无活性网格蛋白聚集体的形成。然而，当重新添加轻链时，这个半胱氨酸在单个网格蛋白分子之间形成二硫键的能力被阻断，这表明结合的轻链与半胱氨酸1528相互作用以防止聚集。这一新信息有助于确定轻链亚基在三聚化结构域附近的方向，并支持先前表明三聚化结构域参与轻链结合的模型（Chen C-Y, Reese ML, Hwang PK, Ota N, Agard D, Brodsky FM. EMBO J 2002; 21: 6072 - 6082; Pishvaee B, Munn A, Payne GS. EMBO J 1997; 16: 2227 - 2239）。
