In vitro comparative kinetic analysis of the chloroplast Toc GTPases.
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.
Reddick
L Evan
LE
Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville 37996, USA.
Vaughn
Michael D
MD
Wright
Sarah J
SJ
Campbell
Ian M
IM
Bruce
Barry D
BD
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D002021:Buffers; D002736:Chloroplasts / Q000201:enzymology*; D004789:Enzyme Activation; D004795:Enzyme Stability / Q000187:drug effects; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D008670:Metals / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D013379:Substrate Specificity
D002021:Buffers; D007477:Ions; D008670:Metals; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases