分泌蛋白的追蹤翻譯

1、 17.1跟隨一個(gè)蛋白質(zhì)進(jìn)出細(xì)胞 電子顯微鏡的問世使研究人員能夠看到細(xì)胞和其結(jié)構(gòu)在細(xì)節(jié)上的前所未有的水平。喬治帕拉德利用這個(gè)工具不僅看到細(xì)胞的精細(xì)細(xì)節(jié)，而且還分析了分泌過程。通過結(jié)合電子顯微鏡與脈沖追蹤實(shí)驗(yàn)，帕拉德發(fā)現(xiàn)蛋白質(zhì)跟著離開了細(xì)胞的路徑。 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除了合成蛋白質(zhì)來行使細(xì)胞功能，許多細(xì)胞還必須產(chǎn)生和分泌其他蛋白到細(xì)胞外履行職責(zé)。蛋白分泌的機(jī)制迷住了許多細(xì)胞生物學(xué)家，包括帕拉德。在分泌的研究的早期，細(xì)胞被破壞，各種細(xì)胞器被離心分離。這些細(xì)胞分級(jí)分離的研究表明，分泌的蛋白是存在于與內(nèi)質(zhì)網(wǎng)（ER）相關(guān)聯(lián)的膜結(jié)合囊泡細(xì)

6、胞和除了合成蛋白質(zhì)來行使細(xì)胞功能，許多細(xì)胞還必須產(chǎn)生和分泌其他蛋白到細(xì)胞外履行職責(zé)。蛋白分泌的機(jī)制迷住了許多細(xì)胞生物學(xué)家，包括帕拉德。在分泌的研究的早期，細(xì)胞被破壞，各種細(xì)胞器被離心分離。這些細(xì)胞分級(jí)分離的研究表明，分泌的蛋白是存在于與內(nèi)質(zhì)網(wǎng)（ER）相關(guān)聯(lián)的膜結(jié)合的囊泡細(xì)胞和質(zhì)膜，在那里它們被合成，并被最終從釋放. 不幸的是，這些研究結(jié)果很難解釋，由于在取得不同的細(xì)胞器之間的清潔分離很困難。為了進(jìn)一步闡明的途徑，帕拉德轉(zhuǎn)向了新開發(fā)的技術(shù)，高分辨率放射自顯影，放射性標(biāo)記的蛋白質(zhì)在細(xì)胞內(nèi)移動(dòng)時(shí), 使他可以跟蹤它們。他的工作產(chǎn)生深遠(yuǎn)意義的發(fā)現(xiàn)是該分泌蛋白在囊泡內(nèi)的移動(dòng)是從ER到高爾基復(fù)合體，然后到質(zhì)

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.帕拉德想要確定哪些細(xì)胞結(jié)構(gòu)和細(xì)胞器參與蛋白質(zhì)的分泌。要研究這樣一個(gè)復(fù)雜的過程中，他精心選擇了與所研究合適的模型系統(tǒng)，胰腺外分泌細(xì)胞，它負(fù)責(zé)制造和分泌大量的消化酶。帕拉德檢測了在體內(nèi)的蛋白分泌途徑。首先通過給豚鼠注射3H亮氨酸，使它納入新合成的蛋白質(zhì)中，從而放射性標(biāo)記它們。在從4分鐘到15小時(shí)的時(shí)間點(diǎn)，將動(dòng)物處死，并且胰腺組織是固定的。通過使所述試樣用放射自顯影和在電子顯微鏡觀看，帕拉德可以跟蹤

12、在細(xì)胞中在不同的時(shí)間，被標(biāo)記蛋白質(zhì)何處。正如所料，緊接3H亮氨酸注射后，放射性定位ER的囊泡在時(shí)間點(diǎn)，在稍后質(zhì)膜的時(shí)間點(diǎn)。令人驚訝的排在中間的時(shí)間點(diǎn)。而不是直接從ER行進(jìn)到質(zhì)膜中，放射性標(biāo)記的蛋白出現(xiàn)停留在其旅途中的高爾基復(fù)合體。此外，從來沒有一個(gè)時(shí)間點(diǎn)的放射性標(biāo)記的蛋白質(zhì)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.高爾基復(fù)合體參與蛋白質(zhì)的分泌的觀察是既令人驚訝和有趣。為了徹底解決這一細(xì)胞器的蛋白分泌的作用，帕拉德轉(zhuǎn)向體外脈沖追蹤實(shí)驗(yàn)，這允許更精確的監(jiān)控標(biāo)記的蛋白質(zhì)的去留。在這個(gè)標(biāo)簽技術(shù),細(xì)胞暴露于放射性標(biāo)記的前體中,在這種情況下,H3亮氨酸,短時(shí)間內(nèi)被稱為“脈沖”。 隨后的“追”的時(shí)期,放射性前體用未標(biāo)記的形式取代.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.蛋白質(zhì)在脈沖期間合成將被標(biāo)記并通過放射自顯影檢測，而那些在追逐期間合成的，被未標(biāo)記，將不被檢測到。帕拉德開始通過削減豚鼠胰腺切成厚片，然后將其培養(yǎng)含有3H亮氨酸的媒體中3分鐘。在脈沖結(jié)束時(shí)，他加入過量未標(biāo)記的亮氨酸。組織切片然后或者固定為放射自顯影或用于細(xì)胞分離。為了確保他的結(jié)果是蛋白分泌在體內(nèi)的準(zhǔn)確反映，帕拉德精心設(shè)計(jì)系統(tǒng)。一旦確信，他在體外系統(tǒng)準(zhǔn)確地模仿蛋白分泌的體內(nèi)，他進(jìn)行關(guān)鍵的實(shí)驗(yàn)。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亮氨酸脈沖標(biāo)記組織切片3分鐘，然后用未標(biāo)記的亮氨酸追逐的標(biāo)簽7，17，37，57，和117分鐘。放射性，又局限在小泡，開始在ER，然后在囊泡內(nèi),運(yùn)到高爾基復(fù)合體，當(dāng)他們經(jīng)過高爾基體和到質(zhì)膜時(shí)留在囊泡（見Figure17.1）。隨著囊泡沿通路走的更遠(yuǎn)，他們放射性蛋白質(zhì)變得更加密集。在不同追時(shí)期,從他的顯著一系列放射自顯影，Palade

21、的結(jié)論是分泌蛋白在囊泡旅行從ER到高爾基體和到質(zhì)膜，并且在這個(gè)過程中它們保持在囊泡，并且不與細(xì)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. 拉德的實(shí)驗(yàn)給了生物學(xué)家首先明確一下蛋白質(zhì)通過分泌途徑移動(dòng)。他對(duì)胰腺外分泌細(xì)胞的研究結(jié)果產(chǎn)生了兩個(gè)開創(chuàng)性的看法。首先，該分泌蛋白穿過他們的方式進(jìn)出細(xì)胞的高爾基復(fù)合體。這是分配到高爾基復(fù)合體第一個(gè)功能。第二，分泌蛋白從來不會(huì)與其他細(xì)胞蛋白. 它們在整個(gè)通路分隔成囊泡。這些研究結(jié)果是實(shí)驗(yàn)設(shè)計(jì)的兩個(gè)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