生命科學院: 生化科技學系指導教授: 常怡雍洪孟如Hung, Meng-JuMeng-JuHung2017-03-022018-07-062017-03-022018-07-062015http://ntur.lib.ntu.edu.tw//handle/246246/272227在原核細胞中 DnaK、DnaJ 與GrpE 蛋白質構成一個協助蛋白質正確摺疊的伴護蛋白(以下簡稱KJE 伴護蛋白)。DnaK 由一個ATP 水解酶區及一個蛋白質受質結合區所組成。透過cochaperone DnaJ 的幫助，DnaK 與受質結合，並水解ATP 成ADP 而改變受質結合區構型，增加與受質的親和力，促進蛋白質摺疊效率。接著藉由核苷酸交換因子GrpE 將ADP 置換成ATP，降低與受質親和力以釋放出摺疊完成的蛋白質。這套KJE 伴護蛋白在演化過程中也保留在葉綠體及粒線體中，參與將細胞質內合成的蛋白質運送入這兩種胞器內。阿拉伯芥的核基因組裡具有多個編碼DnaJ 的基因、兩個編碼粒線體DnaK 的同源基因mtHSC70-1 與mtHSC70-2 以及編碼粒線體GrpE 的MGE1 與MGE2。mtHSC70-2 及MGE2的轉錄皆受到高溫誘導，但mtHSC70-1 及MGE1 則否。mtHSC70-1、mtHSC70-2及MGE2 失能突變株幼苗在不同高溫逆境下呈現不同的耐熱缺陷，顯示在演化的過程中mtHSC70 或MGE 伴護蛋白在功能上的分化，然而其詳細的分工仍不清楚，進一步的研究有助於瞭解植物如何適應高溫逆境。首先，本研究分析在常溫及高溫狀態下的蛋白質運輸是否由不同的mtHSC70 與MGE 組合負責，亦即mtHSC70-1 及MGE1 主要負責常溫而mtHSC70-2 及MGE2 負責高溫下的運送。利用富集的粒線體蛋白質分劃(mitochondria enriched fraction) 作為材料，以西方墨點分析植物在高溫逆境下位於粒線體基質的特定蛋白質輸送情形。結果顯示mtHSC70-2 或MGE2 的失能並不會明顯地影響這些蛋白質的輸送，因此推測mtHSC70-1 及MGE1 在高溫逆境下亦可參與蛋白質的輸送。而mtHSC70-1 的失能亦不會明顯地影響蛋白質在高溫下的輸送，顯示mtHSC70-1 與mtHSC70-2 在這功能上有部分的重疊性。許多高等植物可透過pre-mRNA 選擇性剪接保留 MGE2 的第二內含子並編碼出富含絲胺酸、精胺酸及離胺酸(SR/K-rich) 的保守序列。為了進一步瞭解此段序列是否對於阿拉伯芥耐受高溫逆境有貢獻，本實驗構築了含阿拉伯芥 MGE2 啟動子及蛋白質編碼區域基因組核苷酸序列的載體pMGE2::MGE2 做為對照組與去除SR/K-rich 保守序列的MGE2D 載體pMGE2::MGE2D 做為實驗組，結果發現對照組與實驗組的阿拉伯芥互補轉殖株轉錄產物皆可被熱誘導表現，隨著熱處理時間增長其相對含量會下降，但其蛋白質產物卻可以持續累積，顯示SR/K-rich 保守序列存在與否不影響 MGE2 蛋白質的穩定性，且其對於阿拉伯芥耐受慢性長期高溫並非必需。In prokaryotes, DnaK, DnaJ, and GrpE form a set of molecular chaperone machine to facilitate protein folding. The DnaK protein consists of an ATPase domain and a substrate binding domain. With the help of the cochaperone DnaJ, DnaK binds to the protein substrate and change the conformation by hydrolyzing ATP to ADP. The ADP-bound DnaK has increased affinity for the substrate, which promotes the folding of its protein substrate. GrpE serves as a nucleotide exchange factor that replaces ADP with ATP on DnaK and decreases its affinity to the substrate, leading to release of the folded protein. The DnaK-DnaJ-GrpE chaperone (or KJE chaperone in short) also exists in the chloroplasts and mitochondria in eukaryotes, involving in protein import as well as protein folding. In Arabidopsis nuclear genome, there are two homologous genes encoding mitochondrial DnaK, named mtHSC70-1 and mtHSC70-2. Similarly, mitochondrial GrpE is also encoded by two different genes, named MGE1 and MGE2. Previous studies showed that mtHSC70-2 and MGE2 are heat-inducible and that the Arabidopsis mutants without mtHSC70-1, mtHSC70-2, or MGE2 could not tolerate certain heat stress conditions. It is possible that mtHSC70 or MGE have evolved to cope with different heat stress conditions. However, the molecular functions of these components are not clear. In this study, I investigated whether mtHSC70-1/MGE1 and mtHSC70-2/MGE2 are differentially responsible for mitochondrial protein import. The levels of certain mitochondria matrix proteins in the mitochondrial enriched fraction were analyzed by Western blot. The results show that the defect in mtHSC70-2 or MGE2 do not obviously affect the import of the tested proteins at high temperature, suggesting that mtHSC70-1 and MGE1 also function under high temperature. The defect in mtHSC70-1 also does not affect the protein import at high temperature, suggesting functional redundancy of the two mtHSC70 chaperones encoding genes. Another interesting aspect concerns the structure of MGE2. In many higher plants structural variant of MGE2 can be produced due to pre-mRNA alternative splicing and the retention of intron 2, which encodes a conserved serine and arginine/lysine (SR/K)-rich sequence. To understand whether the SR/K-rich sequence is important for heat tolerance in plants, I constructed pMGE2::MGE2 plasmid containing the full length MGE2 genomic DNA and pMGE2::MGE2D that encodes MGE2 without the SR/K-rich sequence for transforming the MGE2 knockout mutant. The results show that the MGE2 or MGE2D transcripts in the transgenic lines were heat inducible and down regulated when the heat treatment prolonged. The MGE2 or MGE2D proteins were similarly accumulated during the heat stress treatment. Moreover, both pMGE2::MGE2 and pMGE2::MGE2D rescued the heat sensitivity of the MGE2 knockout mutant from prolonged exposure to moderately high temperature. In conclusion, the results suggest that the SR/K-rich sequence is not required for the stability of MGE2 and is also not required for the thermotolerance to moderately high temperature.2589335 bytesapplication/pdf論文公開時間: 2016/7/23論文使用權限: 同意有償授權(權利金給回饋本人)粒線體伴護蛋白熱逆境蛋白質運輸mitochondrial chaperonesheat stressprotein importthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/272227/1/ntu-104-R02b22023-1.pdf