Cholesterol-dependent retention of GPI-anchored proteins in endosomes
Cholesterol-dependent retention of GPI-anchored proteins in endosomes
复制标题
DOI：
10.1093/emboj/17.16.4626
复制
发表时间：
1998-08-17
期刊：
影响因子：
11.4
通讯作者：
Maxfield, FR
中科院分区：
文献类型：
--
作者：
Mayor, S;Sabharanjak, S;Maxfield, FR
一些细胞表面的真核蛋白在C末端有一个糖基磷脂酰肌醇(GPI)修饰，这是它们唯一的膜锚定手段。利用荧光标记的配体和数字荧光显微镜，我们发现与细胞凋亡模型相反，GPI锚定的蛋白内化到含有受体介导的摄取(例如转铁蛋白)和液相内吞(例如右旋糖苷)标志物的内体中。免疫金电子显微镜证实了这一点，并观察到与GPI锚定的叶酸受体结合的荧光叶酸衍生物与共内化的辣根过氧化物酶-转铁蛋白内化到同一隔室；当细胞随后与二氨基联胺和过氧化氢孵育时，叶酸荧光被猝灭。大多数GPI锚定的蛋白质被循环回到质膜，但其速度至少比C-6-NBD-鞘磷脂或回收受体慢3倍，这种内吞保留受细胞膜中胆固醇水平的调节；在胆固醇耗竭的细胞中，GPI锚定的蛋白质回收回到细胞表面的速度与回收转铁蛋白受体和C6-NBD-鞘磷脂的速度相同。依赖胆固醇的GPI锚定蛋白的内吞分选与特定的脂类结构域或“筏”参与内吞分选是一致的。这些结果为一些GPI锚定蛋白需要GPI的功能提供了另一种解释。
Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the C-terminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI-anchored proteins are internalized into endosomes that contain markers for both receptor-mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI-anchored folate receptor is internalized into the same compartment as co-internalized horseradish peroxidase-transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI-anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3-fold slower than C-6-NBD-sphingomyelin or recycling receptors, This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI-anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6-NBD-sphingomyelin in cholesterol-depleted cells. Cholesterol-dependent endocytic sorting of GPI-anchored proteins is consistent with the involvement of specialized lipid domains or 'rafts' in endocytic sorting. These results provide an alternative explanation for GPI-requiring functions of some GPI-anchored proteins.