Abstract
A unique aspect of protein transport into plastids is the coordinate involvement of two GTPases in the translocon of the outer chloroplast membrane (Toc). There are two subfamilies in Arabidopsis, the small GTPases (Toc33 and Toc34) and the large acidic GTPases (Toc90, Toc120, Toc132, and Toc159). In chloroplasts, Toc34 and Toc159 are implicated in precursor binding, yet mechanistic details are poorly understood. How the GTPase cycle is modulated by precursor binding is complex and in need of careful dissection. To this end, we have developed novel in vitro assays to quantitate nucleotide binding and hydrolysis of the Toc GTPases. Here we present the first systematic kinetic characterization of four Toc GTPases (cytosolic domains of atToc33, atToc34, psToc34, and the GTPase domain of atToc159) to permit their direct comparison. We report the KM, Vmax, and Ea values for GTP hydrolysis and the Kd value for nucleotide binding for each protein. We demonstrate that GTP hydrolysis by psToc34 is stimulated by chloroplast transit peptides; however, this activity is not stimulated by homodimerization and is abolished by the R133A mutation. Furthermore, we show peptide stimulation of hydrolytic rates are not because of accelerated nucleotide exchange, indicating that transit peptides function as GTPase-activating proteins and not guanine nucleotide exchange factors in modulating the activity of psToc34. Finally, by using the psToc34 structure, we have developed molecular models for atToc33, atToc34, and atToc159G. By combining these models with the measured enzymatic properties of the Toc GTPases, we provide new insights of how the chloroplast protein import cycle may be regulated.
Highlights
The course of evolution, in both cases, the vast majority of the endosymbiont genes was relocated to the host nucleus [1]
Proteins targeted from the cytosol to these mitochondrial and chloroplast translocators require additional “information” in the form of an N-terminal targeting sequence known as a presequence and transit peptide, respectively [1]
From sequence analysis of Arabidopsis, the the outer chloroplast membrane (Toc) GTPases fall into two gene families as follows: the small Toc GTPases (Toc33 and Toc34) [18] and the large acidic Toc GTPases (Toc90, Toc120, Toc132, and Toc159) [19, 20]
Summary
Expression and Purification of Toc GTPases—The expression clones for the Toc proteins were kindly provided by the following: psToc, Prof. With the known specific activity of the isotope, the isotope purity, the enzyme concentration, and the counting efficiency, the slope was transformed into a velocity expressed as nanomoles of GTP hydrolyzed/min/mol of Toc protein. Deconvolution was performed as described previously [40] using the CDPro software with IBase, a reference set of 37 soluble proteins, available on line From these data, the secondary structural features of psToc34(R133A) were determined. After replacement on the magnet for 5 min to ensure immobilization of the beads, 20 l of the elution buffer was removed for scintillation counting These 20-l samples were removed from each well in triplicate, and each competition reaction was performed in duplicate. These values were fit to Equation 3 using Graphpad Prism 4.0c with the log Kd value shared for global fitting and the amount of hot and cold nucleotide constrained according to the experimental design
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