單價陽離子刺激葉綠體蛋白質輸入之分析與探討

Abstract

在細胞內，蛋白質的過膜運輸為一基本而重要的機制。葉綠體為一具雙層膜的植物細胞胞器，我們已知大部分的葉綠體蛋白質是由細胞核內基因所轉錄，於細胞質中合成聚胜肽(polypeptide)後，再送入葉綠體內執行其功能。由細胞核內基因轉錄轉譯而來的葉綠體前驅蛋白質(precursor protein)，在其N端帶有一訊息胜肽(transit peptide)，此訊息胜肽能夠被葉綠體表面上的運輸複合體(translocon complex)所辨認並透過這些複合體被送到葉綠體內。座落在葉綠體外膜的運輸複合體稱為Toc (translocon at the outer membrane of chloroplasts)蛋白，座落在葉綠體內膜的運輸複合體稱為Tic (translocon at the inner membrane of chloroplasts)蛋白。近十五年來，許多個葉綠體膜上的Toc，Tic蛋白已被發現，但複合體成員間的調控以及蛋白質如何透過運輸複合體進入葉綠體的細節仍不清楚。 在這篇論文中，我的研究結果發現，前驅蛋白質輸入葉綠體時，環境中若額外加入氯化鈉(NaCl)至一特定濃度，能夠提高蛋白質輸入進葉綠體的速度。若鹽類的組成改變為氯化鉀(KCl)或是醋酸鈉(NaOAc)、醋酸鉀(KOAc)，仍可以觀察到蛋白質輸入速度的提升，但當鹽類陽離子組成改變為二價的氯化鎂(MgCl2)、氯化鈣(CaCl2)時，則無法看見相同的效果；證明單價陽離子如鈉、鉀等是提高蛋白質輸入速度的主要原因。另外，即使將前驅蛋白質在輸入葉綠體之前以尿素(urea)先行解構(unfolding)，使其在以近乎直鏈態而非折疊態的狀況下輸入葉綠體，單價陽離子仍然能夠有效的提升輸入速度；說明了所觀察到的輸入速度提升的現象，並非肇究於單價陽離子對前驅蛋白質的解構。更進一步探討單價陽離子在蛋白質輸入過程中的不同階段所造成的影響時發現，單價陽離子很有可能是改變葉綠體在轉移(translocation)步驟中的ATP使用效率：在葉綠體ATP濃度只足夠支援前驅蛋白質黏接(binding)時，單價陽離子就能夠促使葉綠體僅使用如此低濃度的ATP來推動前驅蛋白質穿過內膜進入到葉綠體基質(stroma)中被切割成成熟蛋白質(mature protein)。

Protein translocation across membranes is a fundamental cellular process. The import of proteins into chloroplasts is mediated by translocon components located in the chloroplast outer (the TOC proteins) and inner (the TIC proteins) envelope membranes. Although many translocon components have been identified, the molecular mechanism of the translocation process is not yet clearly understood. Here I found a general stimulation of chloroplast protein translocation caused by NaCl. During import, addition of NaCl in the millimolar level could stimulate the rate of protein import into chloroplasts. The stimulation was not caused by increased osmotic pressure. Furthermore, my data showed that the stimulatory effect was due to monovalent cations like Na+ and K+, not the anion Cl-. NaCl further enhanced the import rate of urea denatured-precursor proteins, suggesting that NaCl stimulation of import rate was not due to the unfolding of precursor proteins. Moreover, NaCl resulted in protein translocation into the stroma at a much lower ATP concentration that normally only supports precursor binding to the chloroplast envelope. These data suggest that NaCl has altered the ATP consumption efficiency during protein translocation.