Immunological characterization and chloroplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thaliana.
In order to study the tryptophan biosynthetic enzymes of the plant Arabidopsis thaliana, polyclonal antibodies were raised against five of the tryptophan biosynthetic pathway proteins: anthranilate synthase alpha subunit, phosphoribosylanthranilate transferase, phosphoribosylanthranilate isomerase, and the tryptophan synthase alpha and beta subunits. Immunoblot analysis of Arabidopsis leaf protein extracts revealed that the antibodies identify the corresponding proteins that are enriched in Arabidopsis chloroplast fractions. Precursors of phosphoribosylanthranilate isomerase and tryptophan synthase alpha subunit were synthesized by in vitro translation. The precursors were efficiently imported and processed by isolated spinach chloroplasts, and the cleavage sites within the precursors were determined. These results provide the first direct evidence that the tryptophan biosynthetic enzymes from Arabidopsis are synthesized as higher molecular weight precursors and then imported into chloroplasts and processed into their mature forms.
Zhao
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Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853-1801.
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D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D000877:Anthranilate Phosphoribosyltransferase / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D000878:Anthranilate Synthase / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology*; D002238:Carbohydrate Epimerases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000032:analysis / Q000096:biosynthesis; D015151:Immunoblotting; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D014364:Tryptophan / Q000096:biosynthesis*; D014367:Tryptophan Synthase / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*
D004792:Enzyme Precursors; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D000877:Anthranilate Phosphoribosyltransferase; D005982:Glutathione Transferase; D000878:Anthranilate Synthase; D014367:Tryptophan Synthase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase