Phosphorylation of a chloroplast RNA-binding protein changes its affinity to RNA.
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.
Lisitsky
I
I
Department of Biology, Technion-Israel Institute of Technology, Haifa.
Schuster
G
G
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000737:chemistry*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018724:Spinacia oleracea; D013329:Structure-Activity Relationship
D010710:Phosphates; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins