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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 A, Panwar P, Frelet-Barrand A, Bernaudat F, Juillan-Binard C, Ebel C, Rolland N, Pebay-Peyroula E - PLoS ONE (2012)

Bottom Line: 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.Taken together, these data provide a comprehensive molecular characterization of a chloroplast ATP/ADP transporter.

View Article: PubMed Central - PubMed

Affiliation: CEA, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France.

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.

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Related in: MedlinePlus

Analysis of the oligomeric status of NTT1 in various detergents.A, SEC elution profiles of NTT1 in LAPAO. Pure NTT1 first run (black line), second SEC run of elution peak 1 from the first run (10 mL, blue line), second SEC run of elution peak 2 from the first run (12 mL, green line) and pure NTT1 mixed with ADP, ATP and Pi (red line). Stokes radii of the standard proteins used to calibrate the column are indicated on top of the graph. B, analysis of sedimentation velocity of pure NTT1 in LAPAO. The upper part shows several time-points during sedimentation velocity experiments with the experimental points as markers and the fits of the data as lines. The central part highlights the residuals between experimental points and fits. The lower part presents the absorbance and interference c(S) distributions of NTT1 in LAPAO. C, SEC elution profile of pure NTT1 in different detergents: NTT1 in C6FTac (purple line), NTT1 in FC12 (black line), NTT1 in LAPAO (green line), NTT1 in amphipols (red line) and NTT1 in β-DDM (blue line). D, sedimentation velocity of NTT1 in different detergents. Absorbance c(S) distributions of NTT1 in amphipols (red line), NTT1 in β-DDM (blue line) and NTT1 in FC12 (black line).
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pone-0032325-g002: Analysis of the oligomeric status of NTT1 in various detergents.A, SEC elution profiles of NTT1 in LAPAO. Pure NTT1 first run (black line), second SEC run of elution peak 1 from the first run (10 mL, blue line), second SEC run of elution peak 2 from the first run (12 mL, green line) and pure NTT1 mixed with ADP, ATP and Pi (red line). Stokes radii of the standard proteins used to calibrate the column are indicated on top of the graph. B, analysis of sedimentation velocity of pure NTT1 in LAPAO. The upper part shows several time-points during sedimentation velocity experiments with the experimental points as markers and the fits of the data as lines. The central part highlights the residuals between experimental points and fits. The lower part presents the absorbance and interference c(S) distributions of NTT1 in LAPAO. C, SEC elution profile of pure NTT1 in different detergents: NTT1 in C6FTac (purple line), NTT1 in FC12 (black line), NTT1 in LAPAO (green line), NTT1 in amphipols (red line) and NTT1 in β-DDM (blue line). D, sedimentation velocity of NTT1 in different detergents. Absorbance c(S) distributions of NTT1 in amphipols (red line), NTT1 in β-DDM (blue line) and NTT1 in FC12 (black line).

Mentions: We previously reported that NTT1 is present as a mixture of oligomers in LAPAO solution [6]. Herein, the oligomeric status of purified NTT1 was further assessed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) in order to identify the type of oligomers. Purified protein solubilized in LAPAO was recovered in two main peaks at around 10 mL and 12 mL on an analytical superdex-200 column (Figure 2A). These two peaks correspond to species with Stokes radii of 6.9 and 5.2 nm, respectively. Other species were also present in the solution at higher molecular weights, as observed by the broadening of the dimer peak and by the presence of a small peak around the void volume of the column (Figure 2A and C). These peaks were variable between protein batches in terms of intensity and broadening.


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

Deniaud A, Panwar P, Frelet-Barrand A, Bernaudat F, Juillan-Binard C, Ebel C, Rolland N, Pebay-Peyroula E - PLoS ONE (2012)

Analysis of the oligomeric status of NTT1 in various detergents.A, SEC elution profiles of NTT1 in LAPAO. Pure NTT1 first run (black line), second SEC run of elution peak 1 from the first run (10 mL, blue line), second SEC run of elution peak 2 from the first run (12 mL, green line) and pure NTT1 mixed with ADP, ATP and Pi (red line). Stokes radii of the standard proteins used to calibrate the column are indicated on top of the graph. B, analysis of sedimentation velocity of pure NTT1 in LAPAO. The upper part shows several time-points during sedimentation velocity experiments with the experimental points as markers and the fits of the data as lines. The central part highlights the residuals between experimental points and fits. The lower part presents the absorbance and interference c(S) distributions of NTT1 in LAPAO. C, SEC elution profile of pure NTT1 in different detergents: NTT1 in C6FTac (purple line), NTT1 in FC12 (black line), NTT1 in LAPAO (green line), NTT1 in amphipols (red line) and NTT1 in β-DDM (blue line). D, sedimentation velocity of NTT1 in different detergents. Absorbance c(S) distributions of NTT1 in amphipols (red line), NTT1 in β-DDM (blue line) and NTT1 in FC12 (black line).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3306366&req=5

pone-0032325-g002: Analysis of the oligomeric status of NTT1 in various detergents.A, SEC elution profiles of NTT1 in LAPAO. Pure NTT1 first run (black line), second SEC run of elution peak 1 from the first run (10 mL, blue line), second SEC run of elution peak 2 from the first run (12 mL, green line) and pure NTT1 mixed with ADP, ATP and Pi (red line). Stokes radii of the standard proteins used to calibrate the column are indicated on top of the graph. B, analysis of sedimentation velocity of pure NTT1 in LAPAO. The upper part shows several time-points during sedimentation velocity experiments with the experimental points as markers and the fits of the data as lines. The central part highlights the residuals between experimental points and fits. The lower part presents the absorbance and interference c(S) distributions of NTT1 in LAPAO. C, SEC elution profile of pure NTT1 in different detergents: NTT1 in C6FTac (purple line), NTT1 in FC12 (black line), NTT1 in LAPAO (green line), NTT1 in amphipols (red line) and NTT1 in β-DDM (blue line). D, sedimentation velocity of NTT1 in different detergents. Absorbance c(S) distributions of NTT1 in amphipols (red line), NTT1 in β-DDM (blue line) and NTT1 in FC12 (black line).
Mentions: We previously reported that NTT1 is present as a mixture of oligomers in LAPAO solution [6]. Herein, the oligomeric status of purified NTT1 was further assessed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) in order to identify the type of oligomers. Purified protein solubilized in LAPAO was recovered in two main peaks at around 10 mL and 12 mL on an analytical superdex-200 column (Figure 2A). These two peaks correspond to species with Stokes radii of 6.9 and 5.2 nm, respectively. Other species were also present in the solution at higher molecular weights, as observed by the broadening of the dimer peak and by the presence of a small peak around the void volume of the column (Figure 2A and C). These peaks were variable between protein batches in terms of intensity and broadening.

Bottom Line: 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.Taken together, these data provide a comprehensive molecular characterization of a chloroplast ATP/ADP transporter.

View Article: PubMed Central - PubMed

Affiliation: CEA, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France.

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.

Show MeSH
Related in: MedlinePlus