Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement
Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement
复制标题
DOI：
10.1104/pp.108.123075
复制
发表时间：
2008-10-01
期刊：
影响因子：
7.4
通讯作者：
Wada, Masamitsu
中科院分区：
文献类型：
--
作者：
Oikawa, Kazusato;Yamasato, Akihiro;Wada, Masamitsu
叶绿体改变了它们在细胞内的分布，以响应光照强度。在此之前，我们分离到了拟南芥的叶绿体异常位置1(Chup1)突变体。该突变体在正常的叶绿体重新定位运动中存在缺陷，在栅栏叶肉细胞底部出现叶绿体聚集现象。分离的基因编码一种带有肌动蛋白结合基序的蛋白质。在这里，我们使用生化分析来确定全长CHUP1在叶绿体外膜上的亚细胞定位。在叶肉细胞叶绿体最外层检测到的CHUP1-绿色荧光蛋白(GFP)融合，补充了CHUP1的表型，但主要定位于细胞质的GFP-CHUP1没有。CHUP1与GFP融合的N端疏水区(NtHR-GFP)的过表达诱导了类Chup1的表型，表明对叶绿体的重新定位具有显性-负性效应。在叶绿体外膜PROTEIN7(OEP7)-GFP转化体中也发现了类似的模式，含有OEP7的蛋白质取代了NtHR，补充了突变体的表型。在CHUP1突变背景下对表达截短CHUP1的转基因拟南芥植株的生理分析和细胞骨架抑制实验表明，CHUP1的卷曲卷曲区域将叶绿体牢固地锚定在质膜上，这与卷曲GFP在质膜上的定位一致。因此，CHUP1定位于叶绿体上，N端插入叶绿体外膜，C端面向胞浆，是CHUP1功能所必需的，CHUP1的卷曲区域阻止叶绿体聚集，参与叶绿体的定位运动。
Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7(OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.