Characterization of the Arabidopsis Brittle1 transport protein and impact of reduced activity on plant metabolism
2008
SummaryThe Arabidopsis genome contains a gene (Atbt1) encoding a highly hydrophobic membrane protein of themitochondrial carrier family, with six predicted transmembrane domains, and showing substantial structuralsimilarity to Brittle1 proteins from maize and potato. We demonstrate that AtBT1 transports AMP, ADP andATP (but not ADP-glucose), shows a unidirectional mode of transport, and locates to the plastidial membraneand not to the ER as previously proposed. Analysis using an Atbt1 promoter–GUS construct revealedsubstantial gene expression in rapidly growing root tips and maturating or germinating pollen. Survival ofhomozygous Atbt1::T-DNAmutants is very limited, and those that do survive produce non-fertile seeds. Theseobservations indicate that no other carrier protein or metabolic mechanism can compensate for the loss of thistransporter. Atbt1RNAi dosage mutants show substantially retarded growth, adenylate levels similar to thoseof wild-type plants, increased glutamine contents and unchanged starch levels. Interestingly, the growthretardation of Atbt1 RNAi mutant plants was circumvented by adenosine feeding, and was accompanied byincreased adenylate levels. Further observations showed the presence of a functional nucleotide salvagepathway in Atbt1 RNAi mutants. In summary, our data indicate that AtBT1 is a plastidial nucleotide uniportcarrier protein that is strictly required to export newly synthesized adenylates into the cytosol.Keywords: nucleotide uniporter, brittle proteins, mitochondrial carrier, nucleotide biosynthesis, Arabidopsis.IntroductionNucleotides represent metabolites of enormous importancefor all living cells. They are required for RNA and DNA syn-thesis, for accumulation of storage products such as pro-teins, lipids and carbohydrates, serve as enzyme co-factors,and fulfil important roles in cell signaling (Alberts et al.,1994; Jeter et al., 2004). Adenylates are of special impor-tance because ATP is required for nearly all anabolic reac-tions (Lehninger et al., 1994), making this compound theuniversal energy supply of all organisms.In plants, regeneration of ATP at high rates occurs inchloroplasts during photo-phosphorylation, and in mito-chondria during oxidative phosphorylation. Accordingly,adenylates have to be transported across many internal cellmembranes to in order to be able to supply energy for thevast number of metabolic reactions. Various nucleotidetransport proteins have been identified at the biochemicaland molecular level. The mitochondrial ADP/ATP carrier(AAC) mediates ATP transport into the cytosol (Haferkampet al., 2002; Klingenberg, 1989), and belongs to the mito-chondrial carrier family (MCF), all of which function ashomodimers, with each monomer having six transmem-brane domains (Haferkamp, 2007). The peroxisomal ATP/AMP exchanger ANT from yeast is also a member of theMCF, and three homologs are encoded in the yeast genome(Palmieri et al., 2002; Schwacke et al., 2003). A further MCF-type ATP/ADP antiporter AtER-ANT1 has recently beenidentified as a plant-specific carrier located in the ERmembrane (Leroch et al., 2008). Corresponding knockoutmutants show strongly impaired growth and development,and show defects in accumulation of ER-related storagelipids and proteins (Leroch et al., 2008). Finally, plastidialATP/ADP transporters (nucleotide transporter, NTT) catalyzeATP/ADP antiport (Tjaden et al., 1998) and mediate ATPuptake into chloroplasts (Reinhold et al., 2007; Reiser et al.,
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