Marty Naomi J, Rajalingam Dakshinamurthy, Kight Alicia D, Lewis Nathaniel E, Fologea Daniel, Kumar Thallapuranam Krishnaswamy Suresh, Henry Ralph L, Goforth Robyn L
Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas 72701, USA.
J Biol Chem. 2009 May 29;284(22):14891-903. doi: 10.1074/jbc.M900775200. Epub 2009 Mar 17.
The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane.
叶绿体信号识别颗粒（cpSRP）及其受体（cpFtsY）在类囊体生物合成中发挥作用，将整合膜蛋白靶向运输到类囊体。与真核生物中的胞质SRP受体不同，cpFtsY分布在类囊体膜和可溶性基质之间。基于序列比对，在大肠杆菌FtsY中鉴定出的一个膜结合基序似乎在cpFtsY中保守，但该基序是否负责cpFtsY的膜结合功能尚未通过实验证实。我们的研究表明，cpFtsY中一个小的N端区域稳定了对cpFtsY在cpSRP依赖性蛋白靶向中的功能至关重要的膜相互作用。这个膜结合基序对于将cpFtsY和融合的乘客蛋白引导到类囊体既是必要的也是充分的。我们的结果表明，cpFtsY的膜结合基序在功能上可能被来自大肠杆菌的相应区域取代，证实了膜结合基序在细胞器和原核生物同源物中是保守的。此外，cpFtsY与脂质结合的能力与脂质体诱导的GTP水解刺激相关。削弱cpFtsY中膜结合基序的突变导致更高的GTP水解速率，这表明在没有双层膜的情况下，完整的膜结合区域提供负调控。此外，对N端区域和大肠杆菌SRP受体中类似区域的NMR和CD结构研究揭示了脂质结合时二级结构的构象变化。这些研究表明，cpFtsY膜结合基序在调节膜上cpSRP受体功能的分子内通讯中起关键作用。
