分泌蛋白的追踪翻译

1、 17.1跟随一个蛋白质进出细胞 电子显微镜的问世使研究人员能够看到细胞和其结构在细节上的前所未有的水平。乔治帕拉德利用这个工具不仅看到细胞的精细细节，而且还分析了分泌过程。通过结合电子显微镜与脉冲追踪实验，帕拉德发现蛋白质跟着离开了细胞的路径。 Background In addition to synthesizing proteins to carry out cellular functions, many cells must also produce and secrete additional proteins that perform their duties outside

2、of the cell. The mechanism of protein secretion fascinated many cell biologists, including Palade. In early research on secretion,cells were disrupted and the various organelles separated by centrifugation. In early research on secretion cells were disrupted and the various organelles separated by c

3、entrifugation. These cell fractionation studies had shown that secreted proteins are present in membranebound vesicles associated with the endoplasmic reticulum (ER), where they are synthesized, and with the plasma membrane, where they are eventually released from the cell. Unfortunately, results fr

4、om these studies were hard to interpret due to difficulties in obtaining clean separations between different organelles. To further clarify the pathway, Palade turned to a newly developed technique, highresolution autoradiography, that allowed him to follow radioactively labeled proteins as they mov

5、ed within the cell. His work led to the seminal finding that secreted proteins travel within vesicles from the ER to the Golgi complex, and then to the plasma membrane除了合成蛋白质来行使细胞功能，许多细胞还必须产生和分泌其他蛋白到细胞外履行职责。蛋白分泌的机制迷住了许多细胞生物学家，包括帕拉德。在分泌的研究的早期，细胞被破坏，各种细胞器被离心分离。这些细胞分级分离的研究表明，分泌的蛋白是存在于与内质网（ER）相关联的膜结合囊泡细

6、胞和除了合成蛋白质来行使细胞功能，许多细胞还必须产生和分泌其他蛋白到细胞外履行职责。蛋白分泌的机制迷住了许多细胞生物学家，包括帕拉德。在分泌的研究的早期，细胞被破坏，各种细胞器被离心分离。这些细胞分级分离的研究表明，分泌的蛋白是存在于与内质网（ER）相关联的膜结合的囊泡细胞和质膜，在那里它们被合成，并被最终从释放. 不幸的是，这些研究结果很难解释，由于在取得不同的细胞器之间的清洁分离很困难。为了进一步阐明的途径，帕拉德转向了新开发的技术，高分辨率放射自显影，放射性标记的蛋白质在细胞内移动时, 使他可以跟踪它们。他的工作产生深远意义的发现是该分泌蛋白在囊泡内的移动是从ER到高尔基复合体，然后到质

7、膜。The ExperimentPalade wanted to identify which cell structures and organelles participate in protein secretion. To study such a complex process, he carefully chose an appropriate model system for his studies, the pancreatic exocrine cell, which is responsible for producing and secreting large amoun

8、ts of digestive enzymes.Palade first examined the protein secretion pathway in vivo by injecting guinea pigs with 3H leucine, which was incorporated into newly made proteins, thereby radioactively labeling them. At time points from 4 minutes to 15 hours, the animals were sacrificed, and the pancreat

9、ic tissue was fixed. By subjecting the specimens to autoradiography and viewing them in an electron microscope, Palade could trace where the labeled proteins were in cells at various times. As expected, the radioactivity localized in vesicles at the ER at time points immediately following the 3Hleuc

10、ine injection, and at the plasma membrane at the later time points. The surprise came in the middle time points. Rather than traveling straight from the ER to the plasma membrane, the radioactively labeled proteins appeared to stop off at the Golgi complex in the middle of their journey. In addition

11、, there never was a time point where the radioactively labeled proteins were not confined to vesicles.帕拉德想要确定哪些细胞结构和细胞器参与蛋白质的分泌。要研究这样一个复杂的过程中，他精心选择了与所研究合适的模型系统，胰腺外分泌细胞，它负责制造和分泌大量的消化酶。帕拉德检测了在体内的蛋白分泌途径。首先通过给豚鼠注射3H亮氨酸，使它纳入新合成的蛋白质中，从而放射性标记它们。在从4分钟到15小时的时间点，将动物处死，并且胰腺组织是固定的。通过使所述试样用放射自显影和在电子显微镜观看，帕拉德可以跟踪

12、在细胞中在不同的时间，被标记蛋白质何处。正如所料，紧接3H亮氨酸注射后，放射性定位ER的囊泡在时间点，在稍后质膜的时间点。令人惊讶的排在中间的时间点。而不是直接从ER行进到质膜中，放射性标记的蛋白出现停留在其旅途中的高尔基复合体。此外，从来没有一个时间点的放射性标记的蛋白质不局限于囊泡。The observation that the Golgi complex was involved in protein secretion was both surprising and intriguing. To thoroughly address the role of this organell

13、e in protein secretion, Palade turned to in vitro pulse-chase experiments, which permitted more precise monitoring of thefate of labeled proteins. In this labeling technique, cells are exposed to a radiolabeled precursor, in this case 3Hleucine, for a short period of time known as the“pulse.” The ra

14、dioactive precursor is then replaced with itsnonlabeled form for a subsequent “chase” period.高尔基复合体参与蛋白质的分泌的观察是既令人惊讶和有趣。为了彻底解决这一细胞器的蛋白分泌的作用，帕拉德转向体外脉冲追踪实验，这允许更精确的监控标记的蛋白质的去留。在这个标签技术,细胞暴露于放射性标记的前体中,在这种情况下,H3亮氨酸,短时间内被称为“脉冲”。 随后的“追”的时期,放射性前体用未标记的形式取代.Proteins synthesized during the pulse period will be

15、labeled and detected by autoradiography, while those synthesized during the chase period, being nonlabeled, will not be detected. Palade began by cutting the guinea pig pancreas into thick slices, which were then incubated for 3 minutes in media containing 3Hleucine. At the end of the pulse, he adde

16、d excess unlabeled leucine. The tissue slices were then either fixed for autoradiography or used for cell fractionation. To assure that his results were an accurate reflection of protein secretion in vivo, Palade meticulously characterized the system. Once convinced that his in vitro system accurate

17、ly mimicked protein secretion in vivo, he proceeded to the critical experiment.蛋白质在脉冲期间合成将被标记并通过放射自显影检测，而那些在追逐期间合成的，被未标记，将不被检测到。帕拉德开始通过削减豚鼠胰腺切成厚片，然后将其培养含有3H亮氨酸的媒体中3分钟。在脉冲结束时，他加入过量未标记的亮氨酸。组织切片然后或者固定为放射自显影或用于细胞分离。为了确保他的结果是蛋白分泌在体内的准确反映，帕拉德精心设计系统。一旦确信，他在体外系统准确地模仿蛋白分泌的体内，他进行关键的实验。He pulse-labeled tissue

18、slices with 3H leucine for 3 minutes, then chased the label for 7, 17, 37, 57, and 117 minutes with unlabeled leucine. Radioactivity, again confined in vesicles, began at the ER, then traveled in vesicles to the Golgi complex, and remained in the vesicles as they passed through the Golgi and onto th

19、e plasma membrane (see Figure17.1). As the vesicles traveled farther along the pathway, they became more densely packed with radioactive protein. From his remarkable series of autoradiograms at different chase times, Palade concluded that secreted proteins travel in vesicles from the ER to the Golgi

20、 and onto the plasmamembrane, and that throughout this process they remain in vesicles and do not mix with the rest of the cell.他 用3H亮氨酸脉冲标记组织切片3分钟，然后用未标记的亮氨酸追逐的标签7，17，37，57，和117分钟。放射性，又局限在小泡，开始在ER，然后在囊泡内,运到高尔基复合体，当他们经过高尔基体和到质膜时留在囊泡（见Figure17.1）。随着囊泡沿通路走的更远，他们放射性蛋白质变得更加密集。在不同追时期,从他的显着一系列放射自显影，Palade

21、的结论是分泌蛋白在囊泡旅行从ER到高尔基体和到质膜，并且在这个过程中它们保持在囊泡，并且不与细胞的其余部分混合。DiscussionPalades experiments gave biologists the first clear look at proteins traveling through the secretory pathway. His studies on the pancreatic exocrine cells yielded two seminal observations. First, that secreted proteins pass through th

22、e Golgi complex on their way out of the cell. This was the first function assigned to the Golgi complex. Second, secreted proteins never mix with other cellular proteins; they are segregated into vesicles throughout the pathway. These findings were predicated on two important aspects of the experime

23、ntal design. 拉德的实验给了生物学家首先明确一下蛋白质通过分泌途径移动。他对胰腺外分泌细胞的研究结果产生了两个开创性的看法。首先，该分泌蛋白穿过他们的方式进出细胞的高尔基复合体。这是分配到高尔基复合体第一个功能。第二，分泌蛋白从来不会与其他细胞蛋白. 它们在整个通路分隔成囊泡。这些研究结果是实验设计的两个重要方面的前提.Palades careful use of electron microscopy and autoradiography allowed him to look at the fine details of the pathway. Of equal importance was the choice of a cell type devote