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J. Biol. Chem., Vol. 282, Issue 15, 11410-11426, April 13, 2007

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In Vitro Comparative Kinetic Analysis of the Chloroplast Toc GTPases^*

L. Evan Reddick¹², Michael D. Vaughn¹, Sarah J. Wright, Ian M. Campbell³, and Barry D. Bruce^¶4

From the Department of Biochemistry, Cellular and Molecular Biology, the Botany Department, and ^¶Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee 37996

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 K_M, V_max, and E_a values for GTP hydrolysis and the K_d 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.

Received for publication, October 10, 2006 , and in revised form, January 22, 2007.

^* This work was supported by Grant MCB-0344601 from the Cell Biology Program at the National Science Foundation (to B. D. B.) and a National Science Foundation Research Opportunity Award (to B. D. B). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 and supplemental Table I.

¹ Both authors contributed equally to this manuscript.

² Supported in part by a Science Alliance Award from University of Tennessee, Knoxville.

³ University of Tennessee, Knoxville undergraduate supported in part by Hope, Volunteer, and Merit Scholarships.

⁴ To whom correspondence should be addressed: Biochemistry, Cellular and Molecular Biology Dept., University of Tennessee, Knoxville, TN. Tel.: 865-974-4082; Fax: 865-974-6306; E-mail: bbruce{at}utk.edu.

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