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

• Published in: The Plant cell Vol. 24; no. 4; pp. 1549 - 1559
• Main Authors: Witz, Sandra, Jung, Benjamin, Fürst, Sarah, Möhlmann, Torsten
• Format: Journal Article
• Language: English
• Published: United States American Society of Plant Biologists 01.04.2012
• Subjects: adenine; Adenine - metabolism; Amino Acid Sequence; Arabidopsis; Arabidopsis - drug effects; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis Proteins; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; biochemical pathways; biosynthesis; Biosynthetic Pathways; Biosynthetic Pathways - drug effects; Brassica; Brassica - metabolism; Brassica oleracea; buds; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carbonyl Cyanide m-Chlorophenyl Hydrazone - pharmacology; Cauliflower; chemistry; drug effects; E coli; enzymatic reactions; Enzymes; Escherichia coli; Gene Expression Regulation, Plant; Gene Expression Regulation, Plant - drug effects; genetics; green fluorescent protein; Green Fluorescent Proteins; Green Fluorescent Proteins - metabolism; guanine; Guanine - metabolism; Intracellular Membranes; Intracellular Membranes - drug effects; Intracellular Membranes - metabolism; metabolism; mitochondria; Models, Biological; Molecular Sequence Data; mutants; Nucleobase Transport Proteins; Nucleobase Transport Proteins - chemistry; Nucleobase Transport Proteins - genetics; Nucleobase Transport Proteins - metabolism; Nucleobases; Nucleosides; Nucleotides; Nucleotides - biosynthesis; pharmacology; Plant cells; Plastids; Plastids - drug effects; Plastids - metabolism; Protein Transport; Protein Transport - drug effects; Proteins; protons; Protoplasts; Protoplasts - drug effects; Protoplasts - metabolism; pyrimidine nucleotides; Pyrimidines; Pyrimidines - biosynthesis; Recombinant Fusion Proteins; Recombinant Fusion Proteins - metabolism; RNA, Messenger; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Alignment; Sodium Chloride; Sodium Chloride - pharmacology; Substrate Specificity; Substrate Specificity - drug effects; Symporters; Symporters - chemistry; Symporters - genetics; Symporters - metabolism; Time Factors; transporters; uracil; Uracil - metabolism
• ISSN: 1040-4651; 1532-298X; 1532-298X
• DOI: 10.1105/tpc.112.096743

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.