Wall D A, Hubbard A L
J Cell Biol. 1985 Dec;101(6):2104-12. doi: 10.1083/jcb.101.6.2104.
The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin.
利用分离灌注肝脏的形态学和生物化学相结合的方法，对大鼠肝细胞中去唾液酸糖蛋白（ASGP）途径的内吞区室进行了研究。使用电子显微镜示踪剂和温度转换方案来同步配体进入，证实了我们之前体内研究中观察到的ASGP内化途径（1），并建立了相关条件，在此条件下我们可以标记该途径中连续区室的内容物，用于亚细胞分级分离研究。研究证实了三个内体区室，ASGPs通过被膜小窝和小泡进入细胞后，但在到达溶酶体中的降解位点之前，会出现在这些区室中。这三个区室可以通过它们在肝细胞内的位置、原位的形态外观以及在蔗糖梯度中的密度来区分。在亚细胞分级分离过程中，还检测了ASGP受体（可及的和潜在的，通过去污剂通透化显示）的分布，并与配体的分布进行了比较。大多数可及的ASGP受体与传统的质膜标记物共分布。然而，肝细胞含有大量潜在的ASGP结合位点的细胞内池，其数量超过细胞表面受体的数量，并且其存在不依赖于ASGP的暴露。这些潜在的ASGP受体在蔗糖梯度上的分布（通过免疫检测或结合测定检测）与早期内体区室中隔离的配体的分布一致。此外，早期内体和含有潜在ASGP受体的膜泡都含有高胆固醇含量，因为两者在暴露于洋地黄皂苷后密度都有明显变化。
