Abstract
Bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases differ in their structural and catalytic properties and do not share common antigenic determinants. Polyadenylated mRNAs, prepared from young bean leaves, have been translated in vitro in a rabbit reticulocyte lysate cell-free system. The newly synthesized polypeptides have been submitted to immunoadsorption on protein A-Sepharose in the presence of the antibodies raised against the chloroplastic or the cytoplasmic leucyl-tRNA synthetase. The specificity of the immunoadsorption has been checked by competition experiments involving the pure enzymes. Bean chloroplastic leucyl-tRNA synthetase is synthesized in vitro from a polyadenylated mRNA as a precursor polypeptide of 130 kDa, which is somewhat larger than the mature enzyme of 120 kDa. Bean cytoplasmic leucyl-tRNA synthetase is synthesized in vitro as a polypeptide which has the size of the mature monomer (130 kDa). Processing of the precursor polypeptide of the chloroplastic leucyl-tRNA synthetase, yielding the mature enzyme, has been obtained by performing the in vitro translation in the presence of canine pancreatic microsomal membranes. These results suggest that in vivo bean chloroplastic leucyl-tRNA synthetase could be synthesized in the cytoplasm as a precursor which would be transported into the chloroplasts.
Highlights
From the Znstitut de BioloEie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique,Uniuersite Louis Pasteur, 15 rue Descartes,67084 Strasbourg, France
The newly synthesized polypeptides have been submitted to immunoadsorption on protein A-Sepharose in the presence of the antibodies raised against the chloroplastic or the cytoplasmic leucyl-tRNA synthetase
These results are in favor of a cytoplasmic synthesis for the chloroplastic aminoacyl-tRNA synthetases,which would be imported into the organelles (1,7, 8)
Summary
A PRECURSORPOLYPEPTIDEFOR THE CHLOROPLAST (Received for publication, October 15, 1986). The newly synthesized polypeptides have been submitted to immunoadsorption on protein A-Sepharose in the presence of the antibodies raised against the chloroplastic or the cytoplasmic leucyl-tRNA synthetase. The only data available are results obtained in the case o f Euglena,usingselective inhibitors of protein or RNA synthesisor bleached mutants. These results are in favor of a cytoplasmic synthesis for the chloroplastic aminoacyl-tRNA synthetases,which would be imported into the organelles (1,7, 8). We present here the first study on thien vitro biosynthesis and processing of a chloroplastic aminoacyl-tRNA synthetase, namely bean (Phaseolus vulgaris) chloroplastic leucyl-tRNA synthetase, and of its cytoplasmic counterpart. Our results suggest that in vivo bean chloroplastic leucyl-tRNA synthetase could be synthesized in thecytoplasm as a larger precursor and transported into thechloroplasts. MRNA as a precursor polypeptide of 130 kDa, which is somewhat larger than the mature enzyme of 120
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