Molecular identification and functional characterization of Arabidopsis thaliana mitochondrial and chloroplastic NAD+ carrier proteins.

The Arabidopsis thaliana L. genome contains 58 membrane proteins belonging to the mitochondrial carrier family. Two mitochondrial carrier family members, here named AtNDT1 and AtNDT2, exhibit high structural similarities to the mitochondrial nicotinamide adenine dinucleotide (NAD(+)) carrier ScNDT1 from bakers' yeast. Expression of AtNDT1 or AtNDT2 restores mitochondrial NAD(+) transport activity in a yeast mutant lacking ScNDT. Localization studies with green fluorescent protein fusion proteins provided evidence that AtNDT1 resides in chloroplasts, whereas only AtNDT2 locates to mitochondria. Heterologous expression in Escherichia coli followed by purification, reconstitution in proteoliposomes, and uptake experiments revealed that both carriers exhibit a submillimolar affinity for NAD(+) and transport this compound in a counter-exchange mode. Among various substrates ADP and AMP are the most efficient counter-exchange substrates for NAD(+). Atndt1- and Atndt2-promoter-GUS plants demonstrate that both genes are strongly expressed in developing tissues and in particular in highly metabolically active cells. The presence of both carriers is discussed with respect to the subcellular localization of de novo NAD(+) biosynthesis in plants and with respect to both the NAD(+)-dependent metabolic pathways and the redox balance of chloroplasts and mitochondria.

Characterization of the Arabidopsis Brittle1 transport protein and impact of reduced activity on plant metabolism.

The Arabidopsis genome contains a gene (Atbt1) encoding a highly hydrophobic membrane protein of the mitochondrial carrier family, with six predicted transmembrane domains, and showing substantial structural similarity to Brittle1 proteins from maize and potato. We demonstrate that AtBT1 transports AMP, ADP and ATP (but not ADP-glucose), shows a unidirectional mode of transport, and locates to the plastidial membrane and not to the ER as previously proposed. Analysis using an Atbt1 promoter-GUS construct revealed substantial gene expression in rapidly growing root tips and maturating or germinating pollen. Survival of homozygous Atbt1::T-DNA mutants is very limited, and those that do survive produce non-fertile seeds. These observations indicate that no other carrier protein or metabolic mechanism can compensate for the loss of this transporter. Atbt1 RNAi dosage mutants show substantially retarded growth, adenylate levels similar to those of wild-type plants, increased glutamine contents and unchanged starch levels. Interestingly, the growth retardation of Atbt1 RNAi mutant plants was circumvented by adenosine feeding, and was accompanied by increased adenylate levels. Further observations showed the presence of a functional nucleotide salvage pathway in Atbt1 RNAi mutants. In summary, our data indicate that AtBT1 is a plastidial nucleotide uniport carrier protein that is strictly required to export newly synthesized adenylates into the cytosol.

Identification of a novel adenine nucleotide transporter in the endoplasmic reticulum of Arabidopsis.

Many metabolic reactions in the endoplasmic reticulum (ER) require high levels of energy in the form of ATP, which is important for cell viability. Here, we report on an adenine nucleotide transporter residing in the ER membranes of Arabidopsis thaliana (ER-ANT1). Functional integration of ER-ANT1 in the cytoplasmic membrane of intact Escherichia coli cells reveals a high specificity for an ATP/ADP antiport. Immunodetection in transgenic ER-ANT1-C-MYC-tag Arabidopsis plants and immunogold labeling of wild-type pollen grain tissue using a peptide-specific antiserum reveal the localization of this carrier in ER membranes. Transgenic ER-ANT1-promoter-beta-glucuronidase Arabidopsis lines show high expression in ER-active tissues (i.e., pollen, seeds, root tips, apical meristems, or vascular bundles). Two independent ER-ANT1 Arabidopsis knockout lines indicate a high physiological relevance of ER-ANT1 for ATP transport into the plant ER (e.g., disruption of ER-ANT1 results in a drastic retardation of plant growth and impaired root and seed development). In these ER-ANT1 knockout lines, the expression levels of several genes encoding ER proteins that are dependent on a sufficient ATP supply (i.e., BiP [for luminal binding protein] chaperones, calreticulin chaperones, Ca2+-dependent protein kinase, and SEC61) are substantially decreased.

Molecular and biochemical analysis of the plastidic ADP-glucose transporter (ZmBT1) from Zea mays.

Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous synthesis of ZmBT1 in Escherichia coli cells leads to the functional integration of ZmBT1 into the bacterial cytoplasmic membrane. ZmBT1 transports ADP-Glc in counterexchange with ADP with apparent affinities of about 850 and 465 mum, respectively. Recently, a complete ferredoxin/thioredoxin system has been identified in cereal amyloplasts and BT1 has been proposed as a potential Trx target protein (Balmer, Y., Vensel, W. H., Cai, N., Manieri, W., Schurmann, P., Hurkman, W. J., and Buchanan, B. B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 2988-2993). Interestingly, we revealed that the transport activity of ZmBT1 is reversibly regulated by redox reagents such as diamide and dithiothreitol. The expression of ZmBT1 is restricted to endosperm tissues during starch synthesis, whereas a recently identified BT1 maize homologue, the ZmBT1-2, exhibits a ubiquitous expression pattern in hetero- and autotrophic tissues indicating different physiological roles for both maize BT1 isoforms. BT1 homologues are present in both mono- and dicotyledonous plants. Phylogenetic analyses classify the BT1 family into two phylogenetically and biochemically distinct groups. The first group comprises BT1 orthologues restricted to cereals where they mediate the ADP-Glc transport into cereal endosperm storage plastids during starch synthesis. The second group occurs in mono- and dicotyledonous plants and is most probably involved in the export of adenine nucleotides synthesized inside plastids.

Characterization and developmentally regulated localization of the mitochondrial carrier protein homologue MCP6 from Trypanosoma brucei.

Proteins of the mitochondrial carrier family (MCF) are located mainly in the inner mitochondrial membrane and mediate the transport of a large range of metabolic intermediates. The genome of Trypanosoma brucei harbors 29 genes encoding different MCF proteins. We describe here the characterization of MCP6, a novel T. brucei MCF protein. Sequence comparison and phylogenetic reconstruction revealed that MCP6 is closely related to different mitochondrial ADP/ATP and calcium-dependent solute carriers, including the ATP-Mg/Pi carrier of Homo sapiens. However, MCP6 lacks essential amino acids and sequence motifs conserved in these metabolite transporters, and functional reconstitution and transport assays with E. coli suggested that this protein indeed does not function as an ADP/ATP or ATP-Mg/Pi carrier. The subcellular localization of MCP6 is developmentally regulated: in bloodstream-form trypanosomes, the protein is predominantly glycosomal, whereas in the procyclic form, it is found mainly in the mitochondria. Depletion of MCP6 in procyclic trypanosomes resulted in growth inhibition, an increased cell size, aberrant numbers of nuclei and kinetoplasts, and abnormal kinetoplast morphology, suggesting that depletion of MCP6 inhibits division of the kinetoplast.

Identification and characterization of a novel plastidic adenine nucleotide uniporter from Solanum tuberosum.

Homologs of BT1 (the Brittle1 protein) are found to be phylogenetically related to the mitochondrial carrier family and appear to occur in both mono- and dicotyledonous plants. Whereas BT1 from cereals is probably involved in the transport of ADP-glucose, which is essential for starch metabolism in endosperm plastids, BT1 from a noncereal plant, Solanum tuberosum (StBT1), catalyzes an adenine nucleotide uniport when functionally integrated into the bacterial cytoplasmic membrane. Import studies into intact Escherichia coli cells harboring StBT1 revealed a narrow substrate spectrum with similar affinities for AMP, ADP, and ATP of about 300-400 mum. Transiently expressed StBT1-green fluorescent protein fusion protein in tobacco leaf protoplasts showed a plastidic localization of the StBT1. In vitro synthesized radioactively labeled StBT1 was targeted to the envelope membranes of isolated spinach chloroplasts. Furthermore, we showed by real time reverse transcription-PCR a ubiquitous expression pattern of the StBT1 in autotrophic and heterotrophic potato tissues. We therefore propose that StBT1 is a plastidic adenine nucleotide uniporter used to provide the cytosol and other compartments with adenine nucleotides exclusively synthesized inside plastids.