Charged amino-acid residues in transmembrane domains of the plastidic ATP/ADP transporter from Arabidopsis are important for transport efficiency, substrate specificity, and counter exchange properties

Abstract

Structure±function relationships of the plastidic ATP/ADP transporter from Arabidopsis thaliana have been determined using site-directed mutants at positions K155, E245, E385, and K527. These charged residues are found within highly conserved domains of homologous transport proteins from plants and bacteria and are located in predicted transmembrane regions. Mutants of K155 to K155E, K155R, or K155Q reduced ATP transport to values between 4 and 16% of wild-type uptake, whereas ADP transport was always less then 3% of the wild-type value. Site-directed mutations in which glutamate at positions 245 or 385 was replaced with lysine, abolished transport. However, conservative (E245D, E385D) or neutral (E245Q, E385Q) replacement at these two positions allowed transport. The fourth reciprocal exchange, K527E, also abolished uptake of both adenylates. K527R and K527Q were unable to transport ATP, but ADP transport remained at 35 and 27%, respectively, of the wild-type activity. There was a 70-fold decreased apparent affinity of K527R for ATP, but only a twofold decrease for ADP. The efflux of ATP, but not ADP, was also greatly reduced in K527R. These observations show strikingly that K527 plays a role in substrate specificity that is manifest in both the influx and efflux components of this antiporter.Keywords: ATP/ADP transporter; structure/function relationship; site-specific mutations; plastid; Arabidopsis thaliana.Transport of solutes across cellular membranes, whether catalyzed by channels or carriers, is necessary for complex metabolism. Although adenine nucleotides, the universal cellular energy currency, fulfil an irreplaceable function, they are viewed as part of the intracellular compartment and often are not transported across biological membranes. However, there are three exceptions to this generalization in which transport of ATP and ADP appropriately occurs: in obligate intracellular bacteria, in mitochondria, and in plastids.In heterotrophic eukaryotic cells, most ATP is synthesized during oxidative phosphorylation and accumulates in the mitochondrial matrix, making it necessary to export this intermediate into the cytosol to fuel anabolic reactions. This transport is mediated by the mitochondrial ADP/ATP carrier (AAC), one of the best-characterized membrane carrier proteins [1±7]. AAC is a member of a large group of transporters comprising the mitochondrial carrier family [3]. These carriers share the same evolutionary ancestor, exhibit an internal sequence triplication of about 100 amino acids, and in the functional state probably form a homodimer from the monomer that has six transmembrane domains [8±10].A second type of adenylate transporter resides in the inner envelope membrane of plant plastids [11±14]. This transporter displays a high structural similarity to the third type of ATP/ADP transporter, that from rickettsiae [15] and chlamydiae [16], obligate intracellular bacteria that grow within eukaryotic cells. Neither plastidic nor bacterial types show homology to the mitochondrial ADP/ATP carrier...

Characterization of an ATP Translocase Identified in the Destructive Plant Pathogen “ Candidatus Liberibacter asiaticus”

“…NttA showed structural (Fig. 1) and biochemical properties similar to those of translocases from other obligate intracellular organisms, including the conservation of all four amino acids identified as important for substrate specificity in the plastidic ATP/ADP transporter from Arabidopsis (27). Considering that both NttA and the Arabidopsis translocase show specificity for ATP and that ADP acts as a competitive inhibitor for both translocase reactions (Fig.…”

Discovery of Novel Conserved Peptide Domains by Ortholog Comparison within Plant Multi-Protein Families

“…The identification of peptide stretches that are either particularly conserved or that exhibit pronounced sequence diversification may provide experimental leads for subsequent functional analyses by targeted sitedirected mutational approaches (e.g. Trentmann et al, 2000;Shigaki et al, 2002;Elliott et al, 2005). Another potential application for multiple sequence alignments of protein isoforms is the allocation of putative functionally relevant peptide motifs.…”

Nucleotide Parasitism by Simkania negevensis ( Chlamydiae )

“…All four SnNTT proteins contain the conserved domain PF03219 of the PFAM TLC family, which comprises all known NTT proteins (17). SnNTT1 to SnNTT3 possess all of the charged amino acid residues reported to be critical for the function of the plastidic ATP/ADP transporter AtNTT1 from Arabidopsis thaliana (70): the residues K155, E245, E385, and K527 (referring to AtNTT1 numbering), of which only K527 is conserved in SnNTT4.…”

“…S2 and Table S3 in the supplemental material). Similar to that transporter, SnNTT4 lacks three of the conserved amino acids known to be critical for nucleotide transport in ATP/ADP translocases (K155, E245, and E385 in A. thaliana NTT1 numbering [70]). This might indicate that SnNTT4 does not act as a typical nucleotide transporter but, similar to PamNTT4, as an NAD ϩ transporter.…”