Unusual nucleotide-binding properties of the chloroplast protein import receptor, atToc33

Arabidopsis Toc33 (atToc33) is a GTP-binding protein of the chloroplast outer envelope membrane. We studied its nucleotide-binding properties in vitro, and found that it binds GTP, GDP and XTP, with similar efficiencies, but not ATP. We further demonstrated that atToc33 has intrinsic GTPase activity. Mutations within the putative G4 motif of the atToc33 nucleotide-binding domain (D217N, D219N and E220Q) had no effect on nucleotide specificity or GTPase activity. Similarly, a mutation in the newly assigned G5 motif (E208Q) did not affect nucleotide specificity or GTPase activity. Furthermore, the D217N and D219N mutations did not affect atToc33 functionality in vivo. The data demonstrate that atToc33 belongs to a novel class of GTPases with unusual nucleotide-binding properties.