structure and function of protein transport machineries in chloroplasts 左冰峰 杨洋 06/11/7chloroplastsThe chloroplasts is an ognanelle of prokaryotic origin that is situated in an eukaryotic cellular environment .chloroplastsSix subcompartments : An inner envelope membrane An outer envelope membrane Intermembrane space Stroma Thylakiod membrane Thylakiod lumenprotein transport mechanisms The two stypes : Import of nuclear-encoded proteins into the chloroplast stroma through envelope membraneprotein transport into thylakiod lumenacross the thylakoid membraneImport of proteins into the chloroplast stroma Process: .前体蛋白与叶绿体外表面结合: 前体蛋白N端的 转运肽(transit peptides)和叶绿体外表面蛋白转运复 合体上受体的相互作用;需少量ATP. .转运的早期阶段:在GTP或低浓度的ATP存在下, 前体蛋白与蛋白转运复合体(translocation complexes)紧密结合. .转运末期:前体蛋白穿膜转运进入叶绿体基质,在 分子伴侣(cytosolic chaperone)的协助下加工成 为成熟的蛋白.transit peptides (转运肽)General features : a high content in hydroxylated residues; an almost complete lack of acidic residues ; an overall hydrophilic nature.a number of transit peptides phosphorylation in the cytosol could support their association with guidance complexes.transit peptides (转运肽) Structure: transit peptide contains three parts: N端含非极性氨基酸残基参与前体蛋白与受体 的结合。中间段富含羟基和带正电荷的氨基酸残基参与 前体蛋白的精确定位。C端形成双极性的折叠 与转运后转运肽准确 切割有关。transit peptides (转运肽)The general founction: target the protein to chloroplastit provides an “address”that locatizes the polypeptide to one of the subcompartments. After reaching the organelle stroma, they are removed by a stromal processing peptidase(SPP)cytosolic chaperone 14-3-3 proteins and Hsp70在蛋白转运前,对前体蛋白进行解折叠,使其从 杂的空间结构变成线型结构,以便于进行穿膜 输;进入叶绿体的蛋白质,又在监护蛋白的作用下 重新恢复其空间构象并实施其功能.translocation complexes they are oligomeric protein complexes .they are divided into Toc complex and Tic complex.It is initiated by energy-independent binding of the precursor protein to surface exposed receptors followed by GTP- andATP dependent partialtranslocation across the outer envelope membrane. translocation complexes Toc complexs: the Toc complex which was first isolated from pea chloroplasts consists of three proteins that are designated according to their molecular weight as Toc34, Toc75, and toc159 .Import of proteins into the chloroplast stroma Toc34 and Toc159 are receptor proteins that are responsible for the initial binding of the precursor proteins, binding and hydrolysis of GTP, showing highest affinity for precursors in the GTP-form .Toc159 is embedded within the outer envelope with a COOH-terminal membrane anchor domain (M-domain), while both the NH2-terminal acidic domain (A-domain) and the centralGTP binding domain (G-domain) are exposed to the cytosol. Import of proteins into the chloroplast stroma Toc34 is likewise embedded within the membrane with a COOH-terminal anchor. Remarkably, its hydrophilic cytosolic domain shows significant homology to the G-domain of Toc159 extending beyond the G-motifs conserved in all GTP-binding proteins . Toc34 is the initial receptor that transfers the precursor protein to Toc159 by a heterodimerization step. Toc159 was furthermore shown to operate as GTP-driven motor in the translocation process Import of proteins into the chloroplast stroma Toc75 is a polytopic membrane protein with presumably 16 amphiphilic b-sheets which form a cation-selective ion channel within the membrane. Toc75 interacts with both Toc34 and Toc159 and possesses a lowaffinity precursor binding site which strongly suggests that it provides the major protein translocation channel in the outer envelope membrane.Import of proteins into the chloroplast stroma Toc64 Toc12 Toc132 Toc90 Toc33 Toc120