title: De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.
abstract: Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.
authors.affiliation: Abteilung Pflanzenphysiologie, Fachbereich Biologie, Technische Universität Kaiserslautern, D-67663 Kaiserslautern, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Witz
authors.forename: Benjamin
authors.initials: B
authors.lastname: Jung
authors.forename: Sarah
authors.initials: S
authors.lastname: Fürst
authors.forename: Torsten
authors.initials: T
authors.lastname: Möhlmann
mesh_terms: D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000187:drug effects; D001937:Brassica / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D033702:Nucleobase Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000096:biosynthesis*; D018087:Plastids / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism; D011743:Pyrimidines / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014498:Uracil / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C572959:NCS1 protein, Arabidopsis; D033702:Nucleobase Transport Proteins; D009711:Nucleotides; D011743:Pyrimidines; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D027981:Symporters; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D014498:Uracil; D006147:Guanine; D000225:Adenine; C030986:pyrimidine