Identification of translocon subcomplexes associated with chloroplast protein import intermediates

• Main Authors: Kuan-Yu Chen, 陳冠毓
• Other Authors: Hsou-min Li
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/57596069834621869336

碩士 === 國立臺灣大學 === 植物科學研究所 === 93 === Most chloroplast proteins are encoded by the nucleus, synthesized in the cytosol and post-translationally imported into chloroplasts. These nucleus-encoded proteins are synthesized as precursors with cleavable N-terminal transit peptides, and are transported into chloroplasts by a set of translocon complex located in the chloroplast outer and inner envelope membranes. Translocon components are termed Toc (translocons at the outer envelope membrane of chloroplasts) and Tic (translocons at the inner envelope membrane of chloroplasts) proteins. In the past ten years, researchers have made great progresses in identification of the Tic / Toc components. They have identified more than 10 Tic/Toc components. However, we still do not understand how these components work, interact, and cooperate. We also do not know the sequential steps of import into chloroplasts. Therefore, I tried to import radio-labeled precursor proteins into pea chloroplasts and identify intermediate steps in the import process. I used a chemical crosslinker to fix the intermediate complexes, and then used sucrose density gradient centrifugation and blue native polyacrylamide gel electrophoresis (BN-PAGE) to separate and identify these intermediate complexes. I found 4 groups of complexes in total. First, I found a TicToc supercomplex containing all of the Tic、Toc components. Its molecular mass is at least 2 mDa. Then I found a lighter population of precursor-protein-containing complexes which can be chased to TicToc. Combined with BN-PAGE, I found this lighter population contained a ~700-kDa complex and an ~800-kDa complex. In further experiments, I found that the 700-kDa complex contained two populations of complexes. One population contained Toc75 and Toc34, and the other population contained Toc75, Toc34 and Toc159, which is a complete Toc complex. I also identified a TicToc subcomplex of 800 kDa which contained Toc75, Toc34, Toc159, Tic110 and Hsp93 but not Tic40. My results suggest that these complexes represent early intermediate steps in the import process and are general intermediates for all precursors.