Dataset Information

Oligomeric status and nucleotide binding properties of the plastid ATP/ADP transporter 1: toward a molecular understanding of the transport mechanism.

ABSTRACT: BACKGROUND: Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana. By biochemical and biophysical analyses, we identified the best detergent for solubilization and purification of functional proteins, LAPAO. Purified NTT1 was found to accumulate as two independent pools of well folded, stable monomers and dimers. ATP and ADP binding properties were determined, and Pi, a co-substrate of ADP, was confirmed to be essential for nucleotide steady-state transport. Nucleotide binding studies and analysis of NTT1 mutants lead us to suggest the existence of two distinct and probably inter-dependent binding sites. Finally, fusion and deletion experiments demonstrated that the C-terminus of NTT1 is not essential for multimerization, but probably plays a regulatory role, controlling the nucleotide exchange rate. CONCLUSIONS/SIGNIFICANCE: Taken together, these data provide a comprehensive molecular characterization of a chloroplast ATP/ADP transporter.

SUBMITTER: Deniaud A

PROVIDER: S-EPMC3306366 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Oligomeric status and nucleotide binding properties of the plastid ATP/ADP transporter 1: toward a molecular understanding of the transport mechanism.

Deniaud Aurélien A Panwar Pankaj P Frelet-Barrand Annie A Bernaudat Florent F Juillan-Binard Céline C Ebel Christine C Rolland Norbert N Pebay-Peyroula Eva E

PloS one 20120316 3

<h4>Background</h4>Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters.<h4>Methodology/principal findings</h4>In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana. By biochemical and biophy ...[more]

PMID: 22438876

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Project description:Energy parasitism by ATP/ADP transport proteins is an essential, common feature of intracellular bacteria such as chlamydiae and rickettsiae, which are major pathogens of humans. Although several ATP/ADP transport proteins have so far been characterized, some fundamental questions regarding their function remained unaddressed. In this study, we focused on the detailed biochemical analysis of a representative ATP/ADP transporter (PamNTT1), from the amoeba symbiont Protochlamydia amoebophila (UWE25) to further clarify the principle of energy exploitation. We succeeded in the purification of the first bacterial nucleotide transporter (NTT) and its functional reconstitution into artificial lipid vesicles. Reconstituted PamNTT1 revealed high import velocities for ATP and an unexpected and previously unobserved stimulating effect of the luminal ADP on nucleotide import affinities. Latter preference of the nucleotide hetero-exchange is independent of the membrane potential, and therefore, PamNTT1 not only structurally but also functionally differs from the well-characterized mitochondrial ADP/ATP carriers. Reconstituted PamNTT1 exhibits a bidirectional orientation in lipid vesicles, but interestingly, only carriers inserted with the N-terminus directed to the proteoliposomal interior are functional. The data presented here comprehensively explain the functional basis of how the intracellular P. amoebophila manages to exploit the energy pool of its host cell effectively by using the nucleotide transporter PamNTT1. This membrane protein mediates a preferred import of ATP, which is additionally stimulated by a high internal (bacterial) ADP/ATP ratio, and the orientation-dependent functionality of the transporter ensures that it is not working in a mode that is detrimental to P. amoebophila. Heterologous expression and purification of high amounts of PamNTT1 provides the basis for its crystallization and detailed structure/function analyses. Furthermore, functional reconstitution of this essential chlamydial protein paves the way for high-throughput uptake studies in order to screen for specific inhibitors potentially suitable as anti-chlamydial drugs.

Dataset Information

Oligomeric status and nucleotide binding properties of the plastid ATP/ADP transporter 1: toward a molecular understanding of the transport mechanism.

Publications

Oligomeric status and nucleotide binding properties of the plastid ATP/ADP transporter 1: toward a molecular understanding of the transport mechanism.

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