Identification and Characterization of ADNT1, a Novel Mitochondrial Adenine Nucleotide Transporter from Arabidopsis

Abstract

Despite the fundamental importance and high level of compartmentation of mitochondrial nucleotide metabolism in plants, our knowledge concerning the transport of nucleotides across intracellular membranes remains far from complete. Study of a previously uncharacterized Arabidopsis (Arabidopsis thaliana) gene (At4g01100) revealed it to be a novel adenine nucleotide transporter, designated ADNT1, belonging to the mitochondrial carrier family. The ADNT1 gene shows broad expression at the organ level. Green fluorescent protein-based cell biological analysis demonstrated targeting of ADNT1 to mitochondria. While analysis of the expression of b-glucuronidase fusion proteins suggested that it was expressed across a broad range of tissue types, it was most highly expressed in root tips. Direct transport assays with recombinant and reconstituted ADNT1 were utilized to demonstrate that this protein displays a relatively narrow substrate specificity largely confined to adenylates and their closest analogs. ATP uptake was markedly inhibited by the presence of other adenylates and general inhibitors of mitochondrial transport but not by bongkrekate or carboxyatractyloside, inhibitors of the previously characterized ADP/ATP carrier. Furthermore, the kinetics are substantially different from those of this carrier, with ADNT1 preferring AMP to ADP. Finally, isolation and characterization of a T-DNA insertional knockout mutant of ADNT1, alongside complementation and antisense approaches, demonstrated that although deficiency of this transporter did not seem to greatly alter photosynthetic metabolism, it did result in reduced root growth and respiration. These findings are discussed in the context of a potential function for ADNT1 in the provision of the energy required to support growth in heterotrophic plant tissues.

The Arabidopsis Thylakoid ADP/ATP Carrier TAAC Has an Additional Role in Supplying Plastidic Phosphoadenosine 5′-Phosphosulfate to the Cytosol

“…It may be noted that Arabidopsis also contains orthologs of animal PAPS transporters; however, these proteins do not share sequence similarities with MCF family members (see Supplemental Figure 5 online), are not localized to the plastid envelope (see Supplemental Figure 4 online), and corresponding mutant plants are not affected in GS contents (see Supplemental Figure 3 online). MCF members are not confined to mitochondria but are also found in other cell compartments, namely, chloroplasts, peroxisomes, the endoplasmic reticulum, or the plasma membrane (Bedhomme et al, 2005;Bouvier et al, 2006;Thuswaldner et al, 2007;Kirchberger et al, 2008;Leroch et al, 2008;Linka et al, 2008;Palmieri et al, 2008Palmieri et al, , 2009Rieder and Neuhaus, 2011;Bernhardt et al, 2012). They mediate the transport of various substrates, including nucleotides, mainly in an antiport manner (Palmieri et al, 2011).…”

“…Both proteins, which are named according to their amino acid composition at the putative substrate binding site "KVAG," belong to the drug/ metabolite transporter superfamily, which also includes the nucleotide-sugar transporter family (Knappe et al, 2003). TAAC, the thylakoid ADP/ATP carrier (At5g01500), exhibits distinct homology with carriers transporting diverse adenine nucleotides and PAP or APS and, thus, also represents an interesting PAPS transporter candidate (Palmieri et al, 2008;Fiermonte et al, 2009).…”

“…We performed a detailed biochemical investigation of a similarly truncated TAAC in the liposomal system to test its ability to transport PAPS, PAP, and structurally related molecules. Functional reconstitution was performed according to the protocol used for characterization of the adenylate carrier ADNT1 from Arabidopsis (Palmieri et al, 2008). This involved expression of the transporter in E. coli cells, enrichment and solubilization of inclusion bodies, and their functional refolding in presence of lipid/detergent micelles (Heimpel et al, 2001).…”

Combined Large-Scale Phenotyping and Transcriptomics in Maize Reveals a Robust Growth Regulatory Network

“…The ribosomal genes in this first subnetwork were also highly connected to GRMZM2G067877, a homolog of an Arabidopsis mitochondrial adenine nucleotide transporter (ADNT1) that plays a role in energy supply in heterotrophic tissues. This protein catalyzes the exchange between cytosolic AMP and intramitochondrial ATP, and plants with decreased ADNT1 expression show an inhibition of root growth (Palmieri et al, 2008). A third gene that was coexpressed with the ribosomal genes in subnetwork 1 is GRMZM2G389768, a putative COLD SHOCK DOMAIN (CSP) gene.…”

Glucose Deprivation Converts Poly(ADP-ribose) Polymerase-1 Hyperactivation into a Transient Energy-producing Process

“…This might occur either because of a possible existence of a mitochondrial PARP-1 or because of mitochondrial NAD efflux driven by reduced cytoplasmic content. Whereas the presence of PARP-1 within mitochondria is a highly controversial issue (14,(33)(34)(35), NAD fluxes across the mitochondrial membranes have been reported (36), as well as bidirectional mitochondrial NAD transporters in plants (37) and yeasts (38). A recent contribution, however, is at odds with the hypothesis that mitochondrial NAD content changes during PARP-1 hyperactivity (20).…”