Liu Chuan Yin, Xu Zhaohui, Kaufman Randal J
Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0650, USA.
J Biol Chem. 2003 May 16;278(20):17680-7. doi: 10.1074/jbc.M300418200. Epub 2003 Mar 13.
Accumulation of unfolded proteins in the lumen of the endoplasmic reticulum activates a signal transduction cascade that culminates in the transcriptional induction of genes encoding adaptive functions. One proximal sensor for this unfolded protein response is the protein kinase/endoribonuclease IRE1alpha. IRE1alpha is a type-I transmembrane glycoprotein for which the N-terminal luminal domain (NLD) senses the accumulation of unfolded proteins. Previously we demonstrated that the NLD forms a stable ligand-independent dimer linked by disulfide bridges. In this report we have identified the cysteine residues responsible for intermolecular disulfide bonding. However, this covalent interaction was not required for dimerization and/or signaling, suggesting that a cryptic dimer interface exists in the NLD that is independent of covalent disulfide interactions. Limited proteolysis of the NLD revealed characteristic fragments, all retaining the same N-terminal sequences as full-length NLD. Biochemical and functional studies using NLD truncation mutants indicated that the dimerization domain of the NLD is confined to the conserved motifs at the N-terminal regions where putative hydrophobic interactions exist. In addition, the peptide binding domain of the endoplasmic reticulum protein chaperone BiP interacted with the N-terminal region within the NLD. Our findings suggest that the NLD has at least two distinct types of interactions mediating dimerization and function in signaling, i.e. covalent interactions involving disulfide bond formation and hydrophobic interactions, with the hydrophobic interaction being the driving force for dimerization.
内质网腔中未折叠蛋白的积累激活了一个信号转导级联反应，最终导致编码适应性功能的基因的转录诱导。这种未折叠蛋白反应的一个近端传感器是蛋白激酶/核糖核酸内切酶IRE1α。IRE1α是一种I型跨膜糖蛋白，其N端腔内结构域（NLD）感知未折叠蛋白的积累。此前我们证明，NLD形成了一个由二硫键连接的稳定的非配体依赖性二聚体。在本报告中，我们确定了负责分子间二硫键形成的半胱氨酸残基。然而，这种共价相互作用对于二聚化和/或信号传导不是必需的，这表明在NLD中存在一个独立于共价二硫键相互作用的隐蔽二聚体界面。对NLD的有限蛋白酶解揭示了特征性片段，所有片段都保留了与全长NLD相同的N端序列。使用NLD截短突变体的生化和功能研究表明，NLD的二聚化结构域局限于N端区域的保守基序，这些区域存在假定的疏水相互作用。此外，内质网蛋白伴侣BiP的肽结合结构域与NLD内的N端区域相互作用。我们的研究结果表明，NLD至少有两种不同类型的相互作用介导二聚化和信号传导功能，即涉及二硫键形成的共价相互作用和疏水相互作用，其中疏水相互作用是二聚化的驱动力。
