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研究生:陳映潔
研究生(外文):In-Jie Chen
論文名稱:真核生物核醣蛋白L35與內質網之交互作用
論文名稱(外文):The Interaction between the Ribosomal Protein and Endoplasmic Reticulum (ER): The role of large subunit ribosomal protein L35 in making rough ER
指導教授:林茂榮林茂榮引用關係
指導教授(外文):Alan Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:核醣體內質網
外文關鍵詞:ribosomendoplasmic reticulum
相關次數:
  • 被引用被引用:4
  • 點閱點閱:122
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  • 下載下載:4
  • 收藏至我的研究室書目清單書目收藏:0
由之前的冷凍電子顯微鏡結構指出,核醣體和內質網上的蛋白質引導管道 (translocon) 之間具有四個結合點,其中包括核醣蛋白L19、L25、L26、L35。本篇論文針對其中的L35核醣蛋白來測試其內質網結合能力。首先,我們先直接以L35重組蛋白質與內質網微粒作結合測試,發現核醣蛋白L35單獨存在時就具有和內質網結合的能力,但若其羧基端的11個胺基酸序列缺失時則會失去此能力,可見其羧基端對於這個結合作用是很重要的。另一方面,使重組蛋白質在細胞中表現,產生重組核醣體。這些重組蛋白質包括人類核醣蛋白L35,以及羧基端缺失的突變L35蛋白質,並且在其羧基末端都設計有可以被HMK磷酸激酶標定上32P的,以區別細胞中原生性的核醣體以及重組核醣體。利用觀察這些重組核醣體的的功能,來研究L35蛋白質在核醣體中所扮演的角色。本研究分別觀察重組核醣體在試管中以及細胞中與內質網的結合作用,結果顯示不論L35蛋白的羧基端是否缺失此重組核醣體仍具有內質網結合的能力,可見只破壞Ribosome -translocon四個接觸點中的一個,並不足以使核醣體完全失去和內質網結合的能力。而由HMK磷酸激酶標定實驗顯示,當核醣體和內質網結合時,L35核醣蛋白和內質網之間有相當緊密的作用,以致於HMK酵素無法對L35蛋白上的RRASV序列做標定。綜合以上結果,無論是從單一蛋白質來看或是經由整個重組核醣體來看,都顯示L35核醣蛋白應該在核醣體-內質網結合機制上具有協助的功能。最後,我利用交叉結合化學反應劑找到了一個約10kD大小的分子會和L35蛋白質有結合反應,由其分子量大小猜測可能是Sec61β蛋白質。
An early cryo-EM data has proposed that ribosome has four connecting sites to the translocon of the ER. These sites are ribosomal proteins L19, L25, L26 and L35. In this study, I have focused on the study of the interaction between L35 and the ER. Firstly, I use recombinant protein L35 as a ligand to perform microsome binding assay, and the result shows that ribosomal protein L35 alone is able to bind the ER, but L35 lack of the last 11 residues at carboxyl terminal end is not, suggesting that the C-terminal end is essential for the ER-binding. Secondary, I have made a recombinant ribosome by overexpressing a phosphorylation-tagged ribosomal protein in cell. A phosphorylation peptide, RRASV was used as the tag and being inserted at C-terminal end of recombinant proteins. With this insertion the recombinant ribosome is distinguishable from native ribosome in cell. By examine the function of these recombinant ribosomes, the role of L35 protein in ribosome is defined. Using this approach, L35 and it’s truncated L35 recombinant ribosomes carried the ER-binding property. I also found that defection on one of the four connecting sites did not affect the binding ability of ribosome to the ER. Third, in the kinase labeling in vivo and in vitro experiments, I also observed that the interaction between L35 in ribosome and the translocon is very tightly because the association has prevented kinase labeling on the RRASV site of L35 protein. Finally, using cross-linking reagent I was able to find that a 10kD molecule, a possible Sec61β, is associated with L35.
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