Oligomeric Status and Nucleotide Binding Properties of the Plastid ATP-ADP Transporter 1: Toward a Molecular Understanding of the Transport MechanismReport as inadecuate




Oligomeric Status and Nucleotide Binding Properties of the Plastid ATP-ADP Transporter 1: Toward a Molecular Understanding of the Transport Mechanism - Download this document for free, or read online. Document in PDF available to download.

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.



Author: Aurélien Deniaud, Pankaj Panwar, Annie Frelet-Barrand, Florent Bernaudat, Céline Juillan-Binard, Christine Ebel, Norbert Rollan

Source: http://plos.srce.hr/



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