- 學位論文
葉綠體內膜stop-transfer訊號之胺基酸組成與膜間隙蛋白Tic22的運送路徑
Amino acid composition of chloroplast inner membrane stop-transfer signals and import pathway of intermembrane-space protein Tic22
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摘要
葉綠體具有三個膜系—外膜(outer membrane)、內膜(inner membrane)及類囊膜(thylakoid membrane)，並隔出三個空間，內外膜間隙(intermembrane space)、內膜內部的基質(stroma)與類囊體腔(thylakoid lumen)。蛋白質依據功能不同，會被運送到不同的區域，已知有兩種途徑可將蛋白質送至內膜上，分別是stop-transfer，及post-import。先前的研究僅指出，途徑的選擇是由蛋白質的穿膜區域(transmembrane domain, TMD)所決定，但具體上，兩種TMD的差異為何尚不明確。遂此，我們選取了14個會穿越內膜的蛋白，5個屬於stop-transfer，9屬於個post-import，分析其TMD的胺基酸組成，統計後發現在stop-transfer TMD中具有較多大的胺基酸，post-import TMD中則有較多小的胺基酸，故推測胺基酸大小，是一會影響選擇插入內膜途徑的因素。為測試此一假說，選取了以stop-transfer插入內膜的蛋白質TGD2，實驗結果發現將TGD2 TMD上的tryptophan(W)換成alanine(A) 或是glycine(G) 後，TGD2無法完全停在內膜上，將W 突變成phenylalanine (F)，則無影響。這些結果支持了我們的假說，TMD的胺基酸大小，是決定蛋白質送到葉綠體路徑的因素之一。 已知要進入葉綠體內部的蛋白質是透過內外膜上的運輸機組TOC 與TIC complexes，而外膜蛋白就只需要TOC complex，而位在膜間隙的蛋白質Tic22是如何進入到葉綠體，是否需要TOC complex，則至今仍有爭議。在加強了Tic22前驅蛋白 (prTic22) 進入到葉綠體的效率後，首先我們透過time course及能量的控制，來了解prTic22 進入葉綠體的最佳條件，再利用Tic22與RBCS進入葉綠體的競爭，及prTic22 進入到translocon complex 缺失的突變株所分離的葉綠體中，來研究prTic22 進入葉綠體的路徑。在與RBCS競爭的實驗中發現prTic22會與prRBCS競爭，顯示兩者路徑有所重疊。而進行突變株之進入葉綠體實驗時，發現只有在TOC complex的突變株(toc33、toc75)中，觀察到prTic22 進入葉綠體之效率下降；在TIC complex的tic20突變株中，Tic22進葉綠體的效率不受影響，甚至tic236突變株中有上升的情況。這些結果顯示prTic22應是透過TOC complex穿越外膜，到達膜間隙，無需使用到TIC complex。
並列摘要
Chloroplasts are composed of three independent membrane systems, including the outer membranes (OM), inner membranes (IM) and thylakoid membranes. These three membranes enclose three soluble areas, the intermembrane space, the stroma and the thylakoid lumen. Proteins need to be delivered to the correct compartment in order to be functional. For membrane proteins insertion into IM, two import pathways have been reported, the ‘‘stop-transfer’’ and the ‘‘post-import’’ pathways. It has been shown that the transmembrane domain (TMD) of each IM protein plays a critical role as the pathway determinant. However what features within TMD endow pathway selection is not known. Analysis of TMDs and surrounding amino acids from nine proteins of the post-import pathway and five proteins of the stop-transfer pathway, we found that there are more large amino acids in TMD of protein using the stop-transfer pathway while smaller amino acids are enriched in the post-import group. Thus, we hypothesize that one of determinants for IM insertion pathway selection is the amino acid size in TMD. We tested our hypothesis using TGD2, a protein using the stop-transfer pathway. After site-directed mutagenesis in TMDs and import assays using isolated pea chloroplasts, we found that TGD2 partly lost its ability to stop at IM after mutating a tryptophan (W) at the N terminus of its TMD into alanine (A) or glycine (G) in TMD, while mutating the W to phenylalanine (F) has no effect. These data suggest that N terminal amino acid sizes are important for TMD of chloroplast inner membrane proteins to function as a stop-transfer signal. Protein import into internal compartments of chloroplasts requires the TOC and TIC translocon complexes on the outer and inner membranes. Protein insertion into the OM only needs the TOC complex. Much less is known about how proteins are imported into the intermembrane space. For example, whether the import of Tic22, the best known intermembrane space protein, needs the TOC complex is still in debates. After enhancing the chloroplast import efficiency of Tic22 perprotein (prTic22), I performed import time course and ATP concentration experiments to characterize the import requirement of prTic22. I further performed import competition experiments using prRBCS and prTic22. My result showed that prTic22 import was competed by prRBCS, indicating that their import pathways at least partially overlap. Finally using chloroplasts isolated from translocon complex mutants, I showed that import of prTic22 was decreased in toc33 and toc75 mutant chloroplasts, was no changed in tic20 mutant chloroplasts and was increased in tic236 mutant chloroplasts. We concluded that prTic22 uses the TOC complex for crossing the OM to arrive at the intermembrance space, and its import does not require the TIC complex.
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