BACKGROUND The γ-secretase complex, composed of transmembrane proteins termed presenilin (PS), anterior pharynx defective (APH), nicastrin (NCT), and presenilin enhancer-2 (Pen-2) catalyzes intramembranous hydrolysis of a variety of Type I membrane protein substrates. In order to understand aspects of subunit assembly, interactions, dynamics and catalysis, it is essential to clarify the membrane topology of each polypeptide. Hydophathicity plots predict that the 101 amino acid Pen-2 molecule has two hydrophobic domains (HP1 and HP2) that may serve as transmembrane spanning domains. Earlier reports indicated that transiently overexpressed Pen-2 uses these two hydrophobic domains as transmembrane helices that generates a "U-shaped" hairpin topology with both amino- (N-) and carboxyl-(C-) termini facing the lumen. In this report, we have reexamined the topology of endogenous Pen-2 and Pen-2 chimeras that are stably expressed in mammalian cells, and have assessed the function of these molecules in rescuing γ-secretase activity in Pen-2-deficient fibroblasts. RESULTS We confirm that the Pen-2 C-terminus is lumenal, but the N-terminus of Pen-2 is exposed to the cytoplasm, thus indicating that HP1 does not traverse the lipid bilayer as a transmembrane domain. Domain swapping studies reveal the importance of specific regions within the first hydrophobic domain of Pen-2 that are critical for generating the topology that is a prerequisite for mediating PS1 endoproteolysis and γ-secretase activity. Finally, we report that the first fourteen amino acids of the Pen-2 HP1 are required for γ-secretase activity. CONCLUSIONS We propose that the first hydrophobic domain of Pen-2 forms a structure similar to a reentrant loop while the second hydrophobic domain spans the lipid bilayer.

中文翻译：

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重新探讨了pen-2（一种γ-分泌酶亚基）的拓扑结构：一个折返环和单程跨膜结构域的证据。

背景γ-分泌酶复合物，由称为早老素（PS），咽部前缺陷（APH），尼卡斯特林（NCT）和早老素增强剂2（Pen-2）的跨膜蛋白组成，可催化多种I型膜蛋白的膜内水解。基材。为了理解亚基组装，相互作用，动力学和催化的方面，阐明每种多肽的膜拓扑结构是必不可少的。疏水性图预测101个氨基酸的Pen-2分子具有两个疏水结构域（HP1和HP2），可以用作跨膜结构域。较早的报道表明，瞬时过表达的Pen-2使用这两个疏水域作为跨膜螺旋，产生一个“ U形”发夹形拓扑结构，氨基（N-）和羧基（C-）末端都面向内腔。在这份报告中，我们已经重新审查了在哺乳动物细胞中稳定表达的内源Pen-2和Pen-2嵌合体的拓扑结构，并评估了这些分子在挽救Pen-2缺陷成纤维细胞中γ分泌酶活性中的功能。结果我们确认Pen-2的C末端是管腔，而Pen-2的N末端暴露于细胞质，因此表明HP1没有作为跨膜结构域穿过脂质双层。域交换研究揭示了Pen-2的第一个疏水域中特定区域的重要性，这些区域对于生成拓扑至关重要，而拓扑是介导PS1内蛋白水解和γ分泌酶活性的前提。最后，我们报告说Pen-2 HP1的前14个氨基酸是γ-分泌酶活性所必需的。