Isolation and preliminary characterization of the major membrane boundaries of the endocytic pathway in lymphocytes.
Isolation and preliminary characterization of the major membrane boundaries of the endocytic pathway in lymphocytes.
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DOI：
10.1083/jcb.111.5.1811
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发表时间：
1990-11
期刊：
影响因子：
--
通讯作者：
Hopkins CR
中科院分区：
文献类型：
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作者：
Beaumelle BD;Gibson A;Hopkins CR
使用密度变动方案从小鼠T淋巴瘤细胞系中分离质膜、包被小凹、内体和溶酶体，其中这些隔室选择性地装载有金缀合物。质膜制备后，选择性标记为1小时，在2摄氏度与金蓖麻毒素，并给出了40%的产率根据酶和抗原标记。通过在22 ℃下用与抗小鼠转铁蛋白受体mAb络合的金加载细胞2小时来获得内体。使用类似的程序分离包被的小凹，但是在10摄氏度下孵育之后，这允许小凹的深内陷但防止内化。产率（使用[125I]转铁蛋白的回收率计算）为32%的内涵体和10%的包被坑。最后，通过在37 ℃下用金低密度脂蛋白（LDL）加载细胞18小时，然后在37 ℃下用单独的LDL追踪3小时来制备溶酶体。最终溶酶体产率（基于溶酶体酶的回收率）为16%。二维凝胶上这些细胞区室的蛋白质组成的研究表明，虽然一些主要蛋白质存在于整个途径中，但可以在每个分离的组分中鉴定出特定的蛋白质。蛋白质成分的模式中看到的最大变化沿着途径之间的内体和溶酶体的准备工作。
Plasma membrane, coated pits, endosomes, and lysosomes were isolated from a mouse T lymphoma cell line using a density shift protocol in which these compartments were selectively loaded with gold conjugates. The plasma membrane was prepared after selective labeling for 1 h at 2 degrees C with gold-ricin and gave a yield of 40% according to enzymatic and antigenic markers. Endosomes were obtained by loading the cells for 2 h at 22 degrees C with gold complexed to an antimouse transferrin receptor mAb. Coated pits were isolated using a similar procedure, but after an incubation at 10 degrees C, which allowed deep invagination of the pits but prevented internalization. The yield (calculated using the recovery of [125I]transferrin) was 32% for endosomes and 10% for coated pits. Finally lysosomes were prepared by loading the cells for 18 h at 37 degrees C with gold low density lipoproteins (LDLs) followed by a 3-h chase at 37 degrees C with LDL alone. The final lysosome yield (based on the recovery of lysosomal enzymes) was 16%. Studies of the protein composition of these cellular compartments on two-dimensional gels showed that while some major proteins are present throughout the pathway, specific proteins can be identified in each of the isolated fractions. The greatest change in the pattern of protein constituents seen along the pathway was between endosomal and lysosomal preparations.