Connolly C N, Futter C E, Gibson A, Hopkins C R, Cutler D F
Medical Research Council Laboratory for Molecular Cell Biology, University College London, United Kingdom.
J Cell Biol. 1994 Nov;127(3):641-52. doi: 10.1083/jcb.127.3.641.
We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.
我们开发了一种新技术，用于研究胞吐运输的形态学基础。我们将cDNA中的HRP在多种细胞中表达，并结合过氧化物酶细胞化学，在电子显微镜水平以非常高的灵敏度勾勒出进出高尔基体的运输过程。过氧化物酶的一种分泌形式（ssHRP）从分泌途径开始就具有活性，并且其活性能从细胞中有效清除。使用带有内质网滞留基序（ssHRPKDEL）标记的ssHRP对参与可溶性内质网蛋白行程的形态学元件进行研究表明，它在高尔基体堆栈中的前进不超过最顺面元件。由ssHRPKDEL勾勒出的粗面内质网和高尔基体堆栈之间的运输通过囊泡载体以及管状元件进行。ssHRP也被用于研究高尔基体复合体的反面高尔基体网络，在低温下孵育时，HRP反应产物勾勒出反面高尔基体网络。在与ssHRP的双标记实验中，用转铁蛋白受体追踪内体区室，未发现这两个区室之间有任何重叠。
