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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.

Show MeSH
Purification of NTT1.SDS-PAGE analysis of the NTT1 purification procedure. MM, Molecular weight markers. FT, flow-through.
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pone-0032325-g001: Purification of NTT1.SDS-PAGE analysis of the NTT1 purification procedure. MM, Molecular weight markers. FT, flow-through.

Mentions: During import into the chloroplast, NTT1 becomes folded and functional only after cleavage of its N-terminal transit peptide. NTT1 was thus expressed in E. coli as a matured form (i.e. lacking this N-terminal part). The overexpressed protein inserted into the bacterial plasma membrane in a functional state, as previously shown using radioactive ATP import into bacteria [6] and ATP export measured by luminescence [6]. To further purify NTT1, a two-step affinity chromatography protocol was set up. The protein was first solubilized in the presence of LAPAO (Figure 1). Solubilized proteins were then loaded onto a Ni-NTA matrix, which is compatible with high detergent concentrations. Proteins eluted from the Ni-NTA matrix were further purified on a Strep-Tactin chromatography column (Figure 1). Using this protocol, highly pure NTT1 was obtained in sub-milligram amounts per liter of E. coli culture. The yield and purity of the purified NTT1 protein was markedly better than in previous studies using a different purification procedure [3]. The good purification yield is mainly due to the high expression levels reached using our combination of bacterial strain and constructs. In addition, LAPAO appears to solubilize NTT1 more efficiently from the bacterial membrane than β-DDM [6], which was the detergent used in other studies [3], [6].


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)

Purification of NTT1.SDS-PAGE analysis of the NTT1 purification procedure. MM, Molecular weight markers. FT, flow-through.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3306366&req=5

pone-0032325-g001: Purification of NTT1.SDS-PAGE analysis of the NTT1 purification procedure. MM, Molecular weight markers. FT, flow-through.
Mentions: During import into the chloroplast, NTT1 becomes folded and functional only after cleavage of its N-terminal transit peptide. NTT1 was thus expressed in E. coli as a matured form (i.e. lacking this N-terminal part). The overexpressed protein inserted into the bacterial plasma membrane in a functional state, as previously shown using radioactive ATP import into bacteria [6] and ATP export measured by luminescence [6]. To further purify NTT1, a two-step affinity chromatography protocol was set up. The protein was first solubilized in the presence of LAPAO (Figure 1). Solubilized proteins were then loaded onto a Ni-NTA matrix, which is compatible with high detergent concentrations. Proteins eluted from the Ni-NTA matrix were further purified on a Strep-Tactin chromatography column (Figure 1). Using this protocol, highly pure NTT1 was obtained in sub-milligram amounts per liter of E. coli culture. The yield and purity of the purified NTT1 protein was markedly better than in previous studies using a different purification procedure [3]. The good purification yield is mainly due to the high expression levels reached using our combination of bacterial strain and constructs. In addition, LAPAO appears to solubilize NTT1 more efficiently from the bacterial membrane than β-DDM [6], which was the detergent used in other studies [3], [6].

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