Early lipid intermediates in glycosyl-phosphatidylinositol anchor assembly are synthesized in the ER and located in the cytoplasmic leaflet of the ER membrane bilayer.
Early lipid intermediates in glycosyl-phosphatidylinositol anchor assembly are synthesized in the ER and located in the cytoplasmic leaflet of the ER membrane bilayer.
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DOI：
10.1083/jcb.121.5.987
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发表时间：
1993-06
影响因子：
7.8
通讯作者：
Menon, A K
中科院分区：
文献类型：
--
作者：
Vidugiriene, J;Menon, A K
糖基化磷酸肌苷作为许多真核细胞表面糖蛋白的膜锚点。最近的生物化学和遗传学研究表明，糖脂是通过连续添加组分（单糖和磷酸乙醇胺）到磷脂酰肌醇来组装的。生物合成步骤被认为发生在内源性，但缺乏正式的证据。我们描述了旨在建立糖基磷脂酰肌醇（GPI）锚定生物合成初始步骤亚细胞定位的实验，并定义了早期生物合成脂质中间体的跨膜分布。实验采用胸腺瘤细胞系BW5147.3进行。亚细胞分离方案显示GPI组装的早期生物合成步骤，即n -乙酰氨基葡萄糖酰磷脂酰肌醇的合成和去乙酰化发生在内质网中。用UDP-[3H]GlcNAc孵育微粒体合成GPI生物合成中间体，用磷脂酰肌醇特异性磷脂酶C （PI-PLC）检测标记脂质的跨膜分布。用PI-PLC对放射性标记的微粒体进行处理，发现约70%的n -乙酰氨基葡萄糖酰磷脂酰肌醇和葡萄糖氨基磷脂酰肌醇可被水解，表明这两种脂质主要分布在微粒体的胞质（外）小叶中。用链溶素o渗透性胸腺瘤细胞获得了类似的裂解结果。当通透化细胞与UDP-[3H]GlcNAc孵育并与PI-PLC处理时，大约85%的放射性标记的n -乙酰氨基葡萄糖酰磷脂酰肌醇和氨基葡萄糖酰磷脂酰肌醇可以被裂解，表明酶可以接近它们。累积数据表明，早期GPI中间体主要位于内质网的胞质小叶中，可能是由胞质小叶中的PI和胞质溶胶中合成的UDP-GlcNAc合成的。
Glycosylated phosphoinositides serve as membrane anchors for numerous eukaryotic cell surface glycoproteins. Recent biochemical and genetic studies indicate that the glycolipids are assembled by sequential addition of components (monosaccharides and phosphoethanolamine) to phosphatidylinositol. The biosynthetic steps are presumed to occur in the ER, but formal proof of this is lacking. We describe experiments designed to establish the subcellular location of the initial steps in glycosyl-phosphatidylinositol (GPI) anchor biosynthesis and to define the transmembrane distribution of early biosynthetic lipid intermediates. The experiments were performed with the thymoma cell line BW5147.3. A subcellular fractionation protocol was used to show that early biosynthetic steps in GPI assembly, i.e., synthesis and deacetylation of N-acetylglucosaminyl phosphatidylinositol, occur in the ER. GPI biosynthetic intermediates were synthesized by incubating the microsomes with UDP-[3H]GlcNAc, and the transmembrane distribution of the labeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). Treatment of the radiolabeled microsomes with PI-PLC showed that > 70% of the N-acetylglucosaminyl phosphatidylinositol and glucosaminyl phosphatidylinositol could be hydrolyzed, indicating that the two lipids were primarily distributed in the cytoplasmic (outer) leaflet of the microsomes. Similar cleavage results were obtained using Streptolysin O-permeabilized thymoma cells. When permeabilized cells were incubated with UDP-[3H]GlcNAc and treated with PI-PLC, approximately 85% of the radiolabeled N-acetylglucosaminyl phosphatidylinositol and glucosaminyl phosphatidylinositol could be cleaved, indicating that they were accessible to the enzyme. The cumulative data indicate that early GPI intermediates are primarily located in the cytoplasmic leaflet of the ER, and are probably synthesized from PI located in the cytoplasmic leaflet and UDP-GlcNAc synthesized in the cytosol.