Clathrin self‐assembly is regulated by three light‐chain residues controlling the formation of critical salt bridges
Clathrin self‐assembly is regulated by three light‐chain residues controlling the formation of critical salt bridges
复制标题
DOI：
10.1093/emboj/17.5.1297
复制
发表时间：
1998-03
期刊：
影响因子：
--
通讯作者：
J. Ybe;B. Greene;Shu-Hui Liu;Ursula M. Pley;P. Parham;F. Brodsky
中科院分区：
文献类型：
--
作者：
J. Ybe;B. Greene;Shu-Hui Liu;Ursula M. Pley;P. Parham;F. Brodsky
网格蛋白自组装成多面体晶格介导内吞作用和细胞器生物发生期间的膜蛋白分选。晶格形成在体外低pH下自发发生，在细胞内由生理pH下的衔接子触发。为了开始了解网格蛋白聚合的细胞调控，我们分析了网格蛋白重链重组中心片段自发组装期间的分子相互作用，这些片段结合网格蛋白轻链亚基并模拟完整网格蛋白的自组装。使用轻链亚基的缺失和取代突变体重建枢纽表明，网格蛋白自组装的pH依赖性仅由网格蛋白轻链亚基中的三个酸性残基控制。枢纽组装的盐抑制鉴定了两类涉及的盐桥，缺失分析绘制了参与其形成的网格蛋白重链区。这些综合观察结果表明，在轻链亚基中鉴定的带负电荷的调节残基抑制高亲和力盐桥的形成，否则盐桥将诱导网格蛋白重链在生理pH下组装。在轻链存在下，网格蛋白自组装依赖于仅在低pH下形成的盐桥，但对调节非常敏感。我们提出，细胞网格蛋白组装是通过逆转轻链调控序列的抑制作用的简单生化机制来控制的，从而促进高亲和力盐桥形成。
Clathrin self‐assembly into a polyhedral lattice mediates membrane protein sorting during endocytosis and organelle biogenesis. Lattice formation occurs spontaneously in vitro at low pH and, intracellularly, is triggered by adaptors at physiological pH. To begin to understand the cellular regulation of clathrin polymerization, we analyzed molecular interactions during the spontaneous assembly of recombinant hub fragments of the clathrin heavy chain, which bind clathrin light‐chain subunits and mimic the self‐assembly of intact clathrin. Reconstitution of hubs using deletion and substitution mutants of the light‐chain subunits revealed that the pH dependence of clathrin self‐assembly is controlled by only three acidic residues in the clathrin light‐chain subunits. Salt inhibition of hub assembly identified two classes of salt bridges which are involved and deletion analysis mapped the clathrin heavy‐chain regions participating in their formation. These combined observations indicated that the negatively charged regulatory residues, identified in the light‐chain subunits, inhibit the formation of high‐affinity salt bridges which would otherwise induce clathrin heavy chains to assemble at physiological pH. In the presence of light chains, clathrin self‐assembly depends on salt bridges that form only at low pH, but is exquisitely sensitive to regulation. We propose that cellular clathrin assembly is controlled via the simple biochemical mechanism of reversing the inhibitory effect of the light‐chain regulatory sequence, thereby promoting high‐affinity salt bridge formation.