Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase[W]
Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase[W]
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豌豆双精氨酸蛋白转位酶核心亚基的信号肽结合和寡聚物接触位点图谱[W]
DOI：
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
K. Cline
中科院分区：
文献类型：
--
作者：
Xianyue Ma;K. Cline
Tat 系统独特地将完全折叠的蛋白质运输穿过类囊体膜。使用分离的豌豆叶绿体将多跨度 Tat 组件 cpTatC 的功能域解剖到 cpTatC 的三维模型上。这些结果提供了对折叠蛋白转运的深入了解以及膜蛋白结构功能研究的新方法。类囊体和细菌膜的双精氨酸易位（Tat）系统使用质子梯度作为唯一的能量来源运输折叠的蛋白质。 Tat 底物具有疏水性信号肽，带有必需的双精氨酸 (RR) 识别基序。多跨度 cpTatC 在 Tat 操作中起着核心作用：它结合信号肽，指导易位酶组装，并可能促进易位。开发了豌豆 (Pisum sativum) 叶绿体的体外测定法，以进行 cpTatC 功能的诱变和分析。丙氨酸扫描诱变鉴定出底物结合和受体复合物组装有缺陷的突变体。 N 末端 (S1) 和第一基质环 (S2) 的突变导致信号肽识别的特定缺陷。底物和导入的 cpTatC 之间的 Cys 匹配证实 S1 和 S2 直接且特异性地结合信号肽的 RR 近端区域。 cpTatC 的四个近腔区域的突变在受体复合物组装方面存在缺陷。共纯化和 Cys 匹配分析表明，几个管腔近端区域可能对 cpTatC-cpTatC 相互作用很重要。令人惊讶的是，相邻 cpTatC 的 RR 结合域引导强 cpTatC-cpTatC 交联。这表明多价受体复合物上的结合位点聚集，并解释了 Tat 转运交联多聚体的能力。与信号肽结合位点交联的底物蛋白的运输初步鉴定突变体在易位步骤中受损。
The Tat system uniquely transports fully folded proteins across the thylakoid membrane. Functional domains of the multispanning Tat component cpTatC were dissected with isolated pea chloroplasts onto a three-dimensional model of cpTatC. The results provide insight into folded protein transport and a novel assay for structure-function studies of a membrane protein. Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC–cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.
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影响因子：
11.4
作者：
CLINE, K;HENRY, R;YUAN, JG
通讯作者：
YUAN, JG
DOI：
10.1016/j.bbamcr.2012.06.028
发表时间：
2013-02
影响因子：
5.1
作者：
Celedon, Jose M.;Cline, Kenneth
通讯作者：
Cline, Kenneth
影响因子：
3.5
作者：
HORTON, RM;HUNT, HD;PEASE, LR
通讯作者：
PEASE, LR
DOI：
10.1111/j.1365-313x.2009.03816.x
发表时间：
2009-06
期刊：
The Plant journal : for cell and molecular biology
影响因子：
--
作者：
Martin JR;Harwood JH;McCaffery M;Fernandez DE;Cline K
通讯作者：
Cline K
影响因子：
9.5
作者：
Cline, Kenneth;Dabney-Smith, Carole
通讯作者：
Dabney-Smith, Carole