Lehmann S, Chiesa R, Harris D A
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1997 May 2;272(18):12047-51. doi: 10.1074/jbc.272.18.12047.
Mutations in genes encoding presenilin 1 and presenilin 2 account for the majority of cases of early-onset familial Alzheimer's disease. The presenilins have been localized to the endoplasmic reticulum and Golgi, but which of the multiple hydrophobic segments of the polypeptide chain span the lipid bilayer is unclear. To address this question, we have constructed a series of chimeric molecules in which a topologically neutral reporter protein (a C-terminal fragment of prolactin) containing three artificial glycosylation sites is fused to presenilin 1 following each of the 10 potential transmembrane domains identified in hydrophobicity plots. We have expressed these chimeras by translation in reticulocyte lysate containing canine pancreatic microsomes and by synthesis in transfected COS cells. Based on utilization of the glycosylation sites and sensitivity of the reporter to protease digestion, we provide evidence that presenilin 1 has six transmembrane segments with the N and C termini in the cytoplasm. This model provides important clues to the potential functions of different parts of the presenilin molecule and how these might relate to the pathogenesis of Alzheimer's disease.
编码早老素1和早老素2的基因突变是早发性家族性阿尔茨海默病的主要病因。早老素定位于内质网和高尔基体，但多肽链的多个疏水片段中哪些跨越脂质双层尚不清楚。为了解决这个问题，我们构建了一系列嵌合分子，其中包含三个人工糖基化位点的拓扑中性报告蛋白（催乳素的C末端片段）在疏水性图谱中确定的10个潜在跨膜结构域中的每一个之后与早老素1融合。我们通过在含有犬胰腺微粒体的网织红细胞裂解物中翻译以及在转染的COS细胞中合成来表达这些嵌合体。基于糖基化位点的利用情况和报告蛋白对蛋白酶消化的敏感性，我们提供证据表明早老素1有六个跨膜片段，N端和C端位于细胞质中。该模型为早老素分子不同部分的潜在功能以及这些功能如何与阿尔茨海默病的发病机制相关提供了重要线索。
