Lisitsky I, Schuster G
Department of Biology, Technion-Israel Institute of Technology, Haifa.
Nucleic Acids Res. 1995 Jul 11;23(13):2506-11. doi: 10.1093/nar/23.13.2506.
An RNA-binding protein of 28 kDa (28RNP) was previously isolated from spinach chloroplasts and found to be required for 3' end-processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic- and glycine-rich amino terminal domain. Upon analysis of the RNA-binding properties of the 'native' 28RNP in comparison to the recombinant bacterial expressed protein, differences were detected in the affinity to some chloroplastic 3' end RNAs. It was suggested that post-translational modification can modulate the affinity of the 28RNP in the chloroplast to different RNAs. In order to determine if phosphorylation accounts for this post-translational modification, we examined if the 28RNP is a phosphoprotein and if it can serve as a substrate for protein kinases. It was found that the 28RNP was phosphorylated when intact chloroplasts were metabolically labeled with [32P] orthophosphate, and that recombinant 28RNP served as an excellent substrate in vitro for protein kinase isolated from spinach chloroplasts or recombinant alpha subunit of maize casein kinase II. The 28RNP was apparently phosphorylated at one site located in the acidic domain at the N-terminus of the protein. Site-directed mutagenesis of the serines in that region revealed that the phosphorylation of the protein was eliminated when serine number 22 from the N-terminus was changed to tryptophan. RNA-binding analysis of the phosphorylated 28RNP revealed that the affinity of the phosphorylated protein was reduced approximately 3-4-fold in comparison to the non-phosphorylated protein. Therefore, phosphorylation of the 28RNP modulates its affinity to RNA and may play a significant role in its biological function in the chloroplast.
一种28千道尔顿的RNA结合蛋白（28RNP）先前从菠菜叶绿体中分离出来，发现它是叶绿体mRNA 3'末端加工所必需的。28RNP的氨基酸序列显示有两个约80个氨基酸的RNA结合结构域，以及一个富含酸性和甘氨酸的氨基末端结构域。在分析“天然”28RNP与重组细菌表达蛋白的RNA结合特性时，检测到对某些叶绿体3'末端RNA的亲和力存在差异。有人提出，翻译后修饰可以调节叶绿体中28RNP对不同RNA的亲和力。为了确定磷酸化是否是这种翻译后修饰的原因，我们研究了28RNP是否是一种磷蛋白以及它是否可以作为蛋白激酶的底物。结果发现，当完整的叶绿体用[32P]正磷酸盐进行代谢标记时，28RNP被磷酸化，并且重组28RNP在体外是从菠菜叶绿体中分离的蛋白激酶或玉米酪蛋白激酶II重组α亚基的优良底物。28RNP显然在位于该蛋白N末端酸性结构域的一个位点被磷酸化。对该区域丝氨酸进行定点诱变表明，当N末端第22位丝氨酸变为色氨酸时，该蛋白的磷酸化被消除。对磷酸化28RNP的RNA结合分析表明，与未磷酸化的蛋白相比，磷酸化蛋白的亲和力降低了约3-4倍。因此，28RNP的磷酸化调节其对RNA的亲和力，并可能在其在叶绿体中的生物学功能中发挥重要作用。
