Bernier-Valentin F, Kostrouch Z, Rabilloud R, Munari-Silem Y, Rousset B
Institut National de la Santé et de la Recherche Médicale Unit 197, Faculté de Médecine Alexis Carrel, Lyon, France.
J Biol Chem. 1990 Oct 5;265(28):17373-80.
We have tried to identify iodinated thyroglobulin molecules in purified thyroid-coated vesicles to determined whether the internalization of the thyroid prohormone could proceed via a mechanism of receptor-mediated endocytosis. Coated vesicles isolated from pig thyroids by differential centrifugation and centrifugation on 2H2O-sucrose cushion were characterized by transmission electron microscopy and analyses of the polypeptide composition by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and Western blot using anti-clathrin heavy chain and anti-thyroglobulin antibodies. Clathrin and thyroglobulin (Tg) appeared as the two major components of the purified thyroid coated vesicles (TCV). Purified TCV fraction was homogeneous when analyzed by isopycnic centrifugation on 30% Percoll gradient. TCV had an apparent buoyant density of 1.035 g/ml. The presence of Tg molecules inside TCV was ascertained by (a) immunogold labeling on cryosections of TCV pellet and (b) identification by gel electrophoresis and radio-immunoassay of a definite fraction of Tg (3-5% of total protein) in TCV treated by Triton X-100. The detergent-treated TCV also contained protein-bound iodine: 0.5-0.7 micrograms of iodine/mg protein. Pulse-chase experiments on in vitro reconstituted thyroid follicles have been used to further document the presence of iodinated Tg molecules in coated vesicles. TCV were isolated from reconstituted thyroid follicles previously labeled with [125I]iodide to radioiodinate Tg of the follicular lumen (the pre-endocytotic compartment) and incubated with or without thyrotropin or dibutyryl cyclic AMP to activate intraluminal 125I-Tg endocytosis. Autoradiographic analyses revealed the presence of 125I-Tg in purified TCV and Triton X-100-treated TCV. 125I-Tg present in TCV represented 1-2% of the total intracellular protein-bound radioactivity. Thyrotropin and dibutyryl cyclic AMP increased 2-3-fold the 125I-Tg content of TCV. Our results clearly show that iodinated Tg, the molecular form of the thyroid prohormone known to be internalized, is present into TCV. The data suggest that coated vesicles are involved in the uptake and transport of Tg from the follicular lumen to the lysosomal compartment and therefore, that the internalization of Tg could proceed, at least for a part, via a mechanism of receptor-mediated endocytosis.
我们试图在纯化的甲状腺被膜小泡中鉴定碘化甲状腺球蛋白分子，以确定甲状腺前激素的内化是否可通过受体介导的内吞作用机制进行。通过差速离心和在2H2O-蔗糖垫层上离心从猪甲状腺中分离出的被膜小泡，通过透射电子显微镜进行表征，并在十二烷基硫酸钠存在下通过聚丙烯酰胺凝胶电泳分析多肽组成，以及使用抗网格蛋白重链和抗甲状腺球蛋白抗体进行蛋白质印迹分析。网格蛋白和甲状腺球蛋白（Tg）是纯化的甲状腺被膜小泡（TCV）的两个主要成分。当通过在30% Percoll梯度上进行等密度离心分析时，纯化的TCV组分是均匀的。TCV的表观浮力密度为1.035 g/ml。通过以下方法确定了TCV内部存在Tg分子：（a）对TCV沉淀的冷冻切片进行免疫金标记，以及（b）通过凝胶电泳和放射免疫测定法对经Triton X-100处理的TCV中一定比例的Tg（占总蛋白的3-5%）进行鉴定。经去污剂处理的TCV还含有与蛋白结合的碘：0.5-0.7微克碘/毫克蛋白。对体外重建的甲状腺滤泡进行脉冲追踪实验，以进一步证明被膜小泡中存在碘化Tg分子。从先前用[125I]碘化物标记以对滤泡腔（内吞前区室）的Tg进行放射性碘化的重建甲状腺滤泡中分离出TCV，并在有或没有促甲状腺激素或二丁酰环磷酸腺苷的情况下孵育，以激活腔内125I-Tg的内吞作用。放射自显影分析显示，在纯化的TCV和经Triton X-100处理的TCV中存在125I-Tg。TCV中存在的125I-Tg占细胞内与蛋白结合的总放射性的1-2%。促甲状腺激素和二丁酰环磷酸腺苷使TCV中125I-Tg的含量增加了2-3倍。我们的结果清楚地表明，已知会被内化的甲状腺前激素的分子形式碘化Tg存在于TCV中。数据表明，被膜小泡参与了Tg从滤泡腔到溶酶体区室的摄取和运输，因此，Tg的内化至少部分可以通过受体介导的内吞作用机制进行。
