Identification, Expression, and Functional Analyses of a Thylakoid ATP/ADP Carrier from Arabidopsis

Abstract

TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.Chloroplasts perform oxygenic photosynthesis in algae and plants and have evolved by endosymbiosis from cyanobacteria. Chloroplasts have two distinct membrane systems, the double envelope surrounding the organelle and an internal membrane system named thylakoids. The envelope membrane represents the interface between the cytoplasm and chloroplast stroma, whereas the thylakoid membrane separates the stroma and the lumenal space. Altogether ϳ800 membrane proteins have been identified by proteomics in the envelope and thylakoid membranes of Arabidopsis thaliana (for reviews, see Refs. 1 and 2). As expected, the main function for the identified envelope proteins was transport of ions and metabolites, whereas photosynthesis was attributed to most of the identified thylakoid proteins. The major protein complexes in thylakoids are photosystems (PS) 4 I and II, the cytochrome b 6 f complex, and the proton-translocating ATP synthase. These photosynthetic complexes contain not only proteins but also pigments and other cofactors. Their assembly, activity, and removal require a large number of auxiliary, regulatory, and transport proteins (3, 4). Many biochemical reports pointed to the existence of transport activities in the thylakoid membrane, such as calcium transport (5), copper transport (6), anion channels (7), cation channels (8, 9), and nucleotide transport (10). Only the thylakoid copper transporter was identified at the genetic level in Arabidopsis (11). No hydrophobic proteins related to the above-mentioned transport activities were identified in the previous proteomic works on Arabidopsis thylakoid membranes (for a review, see Ref.2). Therefore, genetic strategies are required for identification and elucidation of their role in optimal function of the thylakoid.ATP is produced during the light-dependent photosynthetic reactions on the stromal side of the thylakoid membrane. Besides its utilization during CO 2 fixation in the stroma, ATP drives many energy-dependent processes in thylakoids, including protein phosphorylation, folding, import, and degradation.

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

“…uptake into nonpreloaded vesicles); therefore, ADP represents an inferior substrate. This is generally consistent with the results obtained in intact E. coli cells also exhibiting higher rates for ATP than for ADP import (Thuswaldner et al, 2007).…”

“…The identified substrate preferences, particularly the capacity for PAP and PAPS transport, suggest that PAPST1 not only acts as an ADP/ATP exchanger in thylakoids, as shown previously (Thuswaldner et al, 2007), but might also play a role in the transport of PAPS across the envelope membrane. The protein possesses an N-terminal extension (transit peptide) predicted to act as a plastid targeting signal (http://aramemnon.…”

“…Here, we report the identification and characterization of a plastidic PAPS transporter from Arabidopsis, PAPST1 (At5g01500), that was originally characterized as an ADP/ATP carrier of the thylakoid membrane, named TAAC (Thuswaldner et al, 2007). Our data demonstrate that TAAC/PAPST1 is additionally present in envelope membranes of chloroplasts and of thylakoid-free plastids in heterotrophic tissues.…”

“…For this, intact Escherichia coli cells were used that expressed the recombinant protein without the N-terminal plastidic targeting sequence (Thuswaldner et al, 2007). 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.…”

“…It was previously documented that the recombinant TAAC is specific for ATP and ADP because very high concentrations (50-fold excess) of these adenine nucleotides, but not of ADP-Glc, AMP, GTP, or GDP, competed with the radioactively labeled ATP (50 µM) for uptake (Thuswaldner et al, 2007). To investigate the substrate spectrum of TAAC in more detail and to test whether it can transport PAPS, we conducted [a-32 P]ATP import studies in the presence of moderate (10-fold) excess of 20 different nonlabeled adenine and guanine nucleotides, adenine nucleotide derivatives, precursors, and cofactors.…”