Kono Nozomu, Amin-Wetzel Niko, Ron David
Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom
Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom.
Mol Biol Cell. 2017 Aug 15;28(17):2318-2332. doi: 10.1091/mbc.E17-03-0144. Epub 2017 Jun 14.
Altered cellular lipid composition activates the endoplasmic reticulum unfolded protein response (UPR), and UPR signaling effects important changes in lipid metabolism. Secondary effects on protein folding homeostasis likely contribute to UPR activation, but deletion of the unfolded protein stress-sensing luminal domain of the UPR transducers PERK and IRE1α does not abolish their responsiveness to lipid perturbation. This finding suggests that PERK and IRE1α also directly recognize the membrane aberrancy wrought by lipid perturbation. However, beyond the need for a transmembrane domain (TMD), little is known about the features involved. Regulation of the UPR transducers entails changes in their oligomeric state and is easily corrupted by overexpression. We used CRISPR/Cas9-mediated gene editing of the locus to study the role of the TMD in the ability of the endogenous IRE1α protein to recognize membrane aberrancy in mammalian cells. Conducted in the background of a point mutation that isolated the response to membrane aberrancy induced by palmitate from unfolded protein stress, our analysis shows that generic membrane-spanning features of the TMD are sufficient for IRE1α's responsiveness to membrane aberrancy. Our data suggest that IRE1α's conserved TMD may have been selected for features imparting a relatively muted response to acyl-chain saturation.
细胞脂质组成的改变会激活内质网未折叠蛋白反应（UPR），并且UPR信号传导会影响脂质代谢的重要变化。对蛋白质折叠稳态的次级影响可能有助于UPR的激活，但删除UPR转导因子PERK和IRE1α的未折叠蛋白应激感应腔内结构域并不能消除它们对脂质扰动的反应性。这一发现表明，PERK和IRE1α也能直接识别由脂质扰动引起的膜异常。然而，除了需要一个跨膜结构域（TMD）外，人们对其中涉及的特征知之甚少。对UPR转导因子的调控需要改变它们的寡聚状态，并且很容易因过表达而被破坏。我们使用CRISPR/Cas9介导的基因编辑来研究TMD在哺乳动物细胞内源性IRE1α蛋白识别膜异常能力中的作用。在一个点突变的背景下进行研究，该突变将对棕榈酸诱导的膜异常的反应与未折叠蛋白应激分开，我们的分析表明，TMD的一般跨膜特征足以使IRE1α对膜异常产生反应。我们的数据表明，IRE1α保守的TMD可能因其赋予对酰基链饱和度相对较弱反应的特征而被选择。
