Phosphorylation, subcellular localization, and membrane orientation of the Alzheimer's disease-associated presenilins
Phosphorylation, subcellular localization, and membrane orientation of the Alzheimer's disease-associated presenilins
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DOI：
10.1074/jbc.272.6.3590
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发表时间：
1997-02-07
影响因子：
4.8
通讯作者：
VanLeuven, F
中科院分区：
文献类型：
--
作者：
DeStrooper, B;Beullens, M;VanLeuven, F
早老素1和早老素2是在转染COS-1和中国仓鼠卵巢细胞中的非糖基化蛋白，表观分子质量分别为45和50 kDa。在转染和未转染的细胞中，它们与来自内质网和凝胶装置的蛋白质共定位。在COS-1细胞中，检测到少量以45 kDa迁移的完整内源性早老素1片段，以及相对大量迁移在18 - 30 kDa之间的早老素1片段。早老素在SDS-聚丙烯酰胺凝胶电泳中有很强的形成聚集体的倾向（质量为100-250 kDa），在37℃而不是95℃的SDS变性下可以部分分解，但未观察到早老素的磺化，糖胺聚糖修饰或酰化，但两种蛋白质在翻译后都在丝氨酸残基上磷酸化。导致阿尔茨海默病的突变Ala-246 -> Glu或Cys-410 -> Tyr不会干扰早老素1的生物合成或磷酸化。最后，使用低浓度的洋地黄苷选择性地渗透细胞膜而不是内质网膜，证明了早老素1的两个主要亲水结构域面向细胞质。目前的研究记录了早老素的翻译后修饰和亚细胞定位，并表明与淀粉样蛋白前体蛋白代谢的假设相互作用应该发生在生物合成途径的早期区室中。
Presenilins 1 and 2 are unglycosylated proteins with apparent molecular mass of 45 and 50 kDa, respectively, in transfected COS-1 and Chinese hamster ovary cells. They colocalize with proteins from the endoplasmic reticulum and the Gels apparatus in transfected and untransfected cells. In COS-1 cells low amounts of intact endogeneous presenilin 1 migrating at 45 kDa are detected together with relative larger amounts of presenilin 1 fragments migrating between 18 and 30 kDa. The presenilins have a strong tendency to form aggregates (mass of 100-250 kDa) in SDS-polyacrylamide gel electrophoresis, which can be partially resolved when denatured by SDS at 37 degrees C instead of 95 degrees C. Sulfation, glycosaminoglycan modification, or acylation of the presenilins was not observed, but both proteins are posttranslationally phosphorylated on serine residues. The mutations Ala-246 --> Glu or Cys-410 --> Tyr that cause Alzheimer's disease do not interfere with the biosynthesis or phosphorylation of presenilin 1. Finally, using low concentrations of digitonin to selectively permeabilize the cell membrane but not the endoplasmic reticulum membrane, it is demonstrated that the two major hydrophilic domains of presenilin 1 are oriented to the cytoplasm. The current investigation documents the posttranslational modifications and subcellular localization of the presenilins and indicates that postulated interactions with amyloid precursor protein metabolism should occur in the early compartments of the biosynthetic pathway.