Université de Nice Sophia-Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, Equipe labellisée Fondation pour la Recherche Médicale et Laboratoire d'Excellence Distalz, Sophia-Antipolis, Valbonne, France.
J Alzheimers Dis. 2014;40(1):161-76. doi: 10.3233/JAD-131268.
γ-Secretase is involved in the regulated intramembrane proteolysis of amyloid-β protein precursor (AβPP) and of many other important physiological substrates. γ-secretase is a multiproteic complex made of four main core components, namely presenilin 1 or 2, APH-1, PEN-2, and Nicastrin. Since APH-1 exists as different variants, combinations of these proteins can theoretically yield distinct γ-secretase complexes. Whether γ-secretase complexes trafficking and targeting to either similar or distinct subcellular compartments depend upon their molecular composition remains unknown. A differential complex-specific distribution may drive a narrow specificity for a subset of substrates that would traffic within the same cellular compartments. Here, we generated bigenic expression vectors to co-express untagged nicastrin or presenilin 1 together with either PEN-2 or distinct variants of APH-1 (aL, aS and b) tagged with complementary fragments of the fluorescent protein Venus. We show that these constructs allow the formation of functional γ-secretase complexes and their visualization with bimolecular fluorescence complementation (BiFC). BiFC can be detected at the plasma membrane as well as in endosomes/lysosomes in addition to the endoplasmic reticulum (ER) of COS-7 cells transfected with the different variants of APH-1. However, the majority of cells co-transfected with APH-1b presented BiFC signal only in the ER, suggesting enhanced retention/retrieval of APH-1b-containing γ-secretase complexes. Therefore, the new tools described here should be helpful to decipher the precise subcellular trafficking of γ-secretase complexes and to delineate the distinct variant-linked pathways in various cellular systems.
γ-分泌酶参与淀粉样β蛋白前体（AβPP）和许多其他重要生理底物的调节性膜内蛋白水解。γ-分泌酶是一种由四个主要核心成分组成的多蛋白复合物，即早老素 1 或 2、APH-1、PEN-2 和 Nicastrin。由于 APH-1 存在不同的变体，这些蛋白质的组合理论上可以产生不同的 γ-分泌酶复合物。γ-分泌酶复合物是否通过其分子组成而在相似或不同的亚细胞隔室中运输和靶向尚不清楚。不同的复合物特异性分布可能导致一组亚基的狭窄特异性，这些亚基将在相同的细胞隔室中运输。在这里，我们生成了双基因表达载体，共同表达未标记的 Nicastrin 或早老素 1 与标记有荧光蛋白 Venus 的互补片段的 PEN-2 或不同变体 APH-1（aL、aS 和 b）。我们表明，这些构建体允许功能性 γ-分泌酶复合物的形成，并通过双分子荧光互补（BiFC）对其进行可视化。BiFC 可在转染了不同 APH-1 变体的 COS-7 细胞的质膜以及内体/溶酶体中检测到，除了内质网（ER）之外。然而，与 APH-1b 共转染的大多数细胞仅在 ER 中呈现 BiFC 信号，这表明 APH-1b 包含的 γ-分泌酶复合物的保留/回收增强。因此，这里描述的新工具应该有助于破译 γ-分泌酶复合物的精确亚细胞运输，并描绘各种细胞系统中不同变体相关的途径。
