A previous screening of Arabidopsis thaliana for mutants exhibiting dysfunctional chloroplast protein transport identified the chloroplast import apparatus (cia) gene. The cia2 mutant has a pale green phenotype and reduced rate of protein import into chloroplasts, but leaf shape and size are similar to wild-type plants of the same developmental stage. Microarray analysis showed that nuclear CIA2 protein enhances expression of the Toc75, Toc33, CPN10 and cpRPs genes, thereby up-regulating protein import and synthesis efficiency in chloroplasts. CIA2-like (CIL) shares 65% sequence identity to CIA2, suggesting that CIL and CIA2 are homologous proteins in Arabidopsis. Here, we further assess the protein interactions and sequence features of CIA2 and CIL. Subcellular localizations of truncated CIA2 protein fragments in our onion transient assay demonstrate that CIA2 contains two nuclear localization signals (NLS) located at amino acids (aa) 62-65 and 291-308, whereas CIL has only one NLS at aa 47-50. We screened a yeast two-hybrid (Y2H) Arabidopsis cDNA library to search for putative CIA2-interacting proteins and identified 12 nuclear proteins, including itself, CIL, and flowering-control proteins (such as CO, NF-YB1, NF-YC1, NF-YC9 and ABI3). Additional Y2H experiments demonstrate that CIA2 and CIL mainly interact with flowering-control proteins via their N-termini, but preferentially form homo- or hetero-dimers through their C-termini. Moreover, sequence alignment showed that the N-terminal sequences of CIA2, CIL and NF-YA are highly conserved. Therefore, NF-YA in the NF-Y complex could be substituted by CIA2 or CIL. We show that Arabidopsis CIA2 and CIL can interact with CO and NF-Y complex, so not only may they contribute to regulate chloroplast function but also to modulate flower development.

中文翻译：

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拟南芥CIA2和CIL蛋白的序列分析和蛋白相互作用。

先前对拟南芥中表现出叶绿体蛋白转运功能异常的突变体的筛选确定了叶绿体输入装置（CIA）基因。cia2突变体具有浅绿色的表型，并且减少了蛋白质向叶绿体中的导入速度，但是叶片的形状和大小与处于相同发育阶段的野生型植物相似。微阵列分析表明，核CIA2蛋白增强了Toc75，Toc33，CPN10和cpRPs基因的表达，从而上调了叶绿体中蛋白质的导入和合成效率。类CIA2（CIL）与CIA2具有65％的序列同一性，表明CIL和CIA2是拟南芥中的同源蛋白。在这里，我们进一步评估了CIA2和CIL的蛋白质相互作用和序列特征。在我们的洋葱瞬时分析中，截短的CIA2蛋白片段的亚细胞定位表明CIA2包含位于氨基酸（aa）62-65和291-308的两个核定位信号（NLS），而CIL在aa 47-50处只有一个NLS。我们筛选了酵母两杂交（Y2H）拟南芥cDNA文库，以寻找与CIA2相互作用的推定蛋白质，并鉴定了12种核蛋白，包括其自身，CIL和开花控制蛋白（例如CO，NF-YB1，NF-YC1， NF-YC9和ABI3）。额外的Y2H实验表明，CIA2和CIL主要通过其N末端与开花控制蛋白相互作用，但优先通过其C末端形成同型或异型二聚体。此外，序列比对显示CIA2，CIL和NF-YA的N末端序列是高度保守的。因此，NF-Y复合物中的NF-YA可以被CIA2或CIL取代。我们显示拟南芥CIA2和CIL可以与CO和NF-Y复杂相互作用，因此它们不仅可以调节叶绿体功能，而且可以调节花的发育。