Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants
Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants
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DOI：
10.1073/pnas.93.11.5269
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发表时间：
1996-05-28
影响因子：
11.1
通讯作者：
Argon, Y
中科院分区：
文献类型：
--
作者：
Hendershot, L;Wei, JY;Argon, Y
一组常驻内质网蛋白已被确定为分子伴侣。其中最具特征的是BiP/GRP78，这是一种hsp70同源物，在体外结合含有疏水残基的肽，在体内结合未折叠或未组装的蛋白。然而，哺乳动物BiP在蛋白质折叠中起直接作用的证据仍然是间接的。在这项研究中，我们研究了BiP在折叠过程中如何与特定的底物免疫球蛋白轻链（lambda LC）相互作用。在瞬时表达实验中使用了野生型仓鼠BiP和几种具有良好特征的BiP atp酶突变体。我们证明野生型lambda LCs与突变体BiP的长期关联抑制了它们的分泌。野生型和突变型BiP都只与未折叠和部分折叠的LCs结合。野生型BiP从未完全折叠的LCs中释放出来，使它们能够折叠和分泌，而突变型BiP则不被释放。结果，与BiP突变体结合的LCs不能完全形成二硫键，并保留在内质网中。我们的实验表明，LC经历了依赖于BiP和不依赖于BiP的折叠步骤，表明ATP结合和BiP的水解活性对于在体内完成LC折叠是必不可少的，并且揭示在该结构域形成二硫键之前，BiP必须释放。
A group of resident ER proteins have been identified that are proposed to function as molecular chaperones. The best characterized of these is BiP/GRP78, an hsp70 homologue that binds peptides containing hydrophobic residues in vitro and unfolded or unassembled proteins in vivo. However, evidence that mammalian BiP plays a direct role in protein folding remains circumstantial. In this study, we examine how BiP interacts with a particular substrate, immunoglobulin light chain (lambda LC), during its folding. Wild-type hamster BiP and several well-characterized BiP ATPase mutants were used in transient expression experiments. We demonstrate that wild-type lambda LCs showed prolonged association with mutant BiP which inhibited their secretion. Both wild-type and mutant BiP bound only to unfolded and partially folded LCs. The wild-type BiP was released from the incompletely folded LCs, allowing them to fold and be secreted, whereas the mutant BiP was not released. As a result, the LCs that were bound to BiP mutants were unable to undergo complete disulfide bond formation and were retained in the ER. Our experiments suggest that LCs undergo both BiP-dependent and BiP-independent folding steps, demonstrating that both ATP binding and hydrolysis activities of BiP are essential for the completion of LC folding in vivo and reveal that BiP must release before disulfide bond formation can occur in that domain.