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Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0A. We probed the catalytic center with the transition state analogue GDP/AlF(x) using NMR and analytical ultracentrifugation. AlF(x) binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.

Transport of precursor proteins across chloroplast membranes involves the GTPases Toc33/34 and Toc159 at the outer chloroplast envelope. The small GTPase Toc33/34 can homodimerize, but the regulation of this interaction has remained elusive. We show that dimerization is independent of nucleotide loading state, based on crystal structures of dimeric Pisum sativum Toc34 and monomeric Arabidopsis thaliana Toc33. An arginine residue is--in the dimer--positioned to resemble a GAP arginine finger. However, GTPase activation by dimerization is sparse and active site features do not explain catalysis, suggesting that the homodimer requires an additional factor as coGAP. Access to the catalytic center and an unusual switch I movement in the dimeric structure support this finding. Potential binding sites for interactions within the Toc translocon or with precursor proteins can be derived from the structures.

The organization of eukaryotic cells into different membrane-enclosed compartments requires an ordered and regulated system for targeting and translocating proteins synthesized in the cytosol across organellar membranes. Protein translocation through integral membrane proteinaceous complexes shares common principles in different organelles, whereas molecular mechanisms and energy requirements are diverse. Translocation into mitochondria and plastids requires most proteins to cross two membranes, and translocation must be regulated to accommodate environmental or metabolic changes. In the last decade, the first ideas were formulated about the regulation of protein translocation into chloroplasts, thereby laying the foundation for this field. Here, we describe recent models for the regulation of translocation by precursor protein phosphorylation, receptor dimerization, redox sensing and calcium signaling. We suggest how these mechanisms might fit within the regulatory framework for the entry of proteins into chloroplasts.

Chloroplast function depends on the translocation of cytosolically synthesized precursor proteins into the organelle. The recognition and transfer of most precursor proteins across the outer membrane depend on a membrane inserted complex. Two receptor components of this complex, Toc34 and Toc159, are GTPases, which can be phosphorylated by kinases present in the hosting membrane. However, the physiological function of phosphorylation is not yet understood in detail. It is demonstrated that both receptors are phosphorylated within their G-domains. In vitro, the phosphorylation of Toc34 disrupts both homo- and heterodimerization of the G-domains as determined using a phospho-mimicking mutant. In endogenous membranes this mutation or phosphorylation of the wild-type receptor disturbs the association of Toc34, but not of Toc159 with the translocation pore. Therefore, phosphorylation serves as an inhibitor for the association of Toc34 with other components of the complex and phosphorylation can now be discussed as a mechanism to exchange different isoforms of Toc34 within this ensemble.

The precursor protein receptor at the chloroplast outer membrane atToc33 is a GTPase, which can be inactivated by phosphorylation in vitro, being arrested in the GDP loaded state. To assess the physiological function of phosphorylation, attoc33 knock out mutants were complemented with a mutated construct mimicking the constitutively phosphorylated state. Our data suggest that the reduced functionality of the mutant protein can be compensated by its upregulation. Chloroplast biogenesis and photosynthetic activity are impaired in the mutants during the early developmental stage, which is consistent with the requirement of atToc33 in young photosynthetic tissues.

The xanthine oxidase class of molybdenum enzyzmes requires a terminal sulfur ligand at the active site. It has been proposed that a special sulfurase catalyzes the insertion of this ligand thereby activating the enzymes. Previous analyses of mutants in plants indicated that the genetic locus aba3 is involved in this step leading to activation of the molybdenum enzymes aldehyde oxidase and xanthine dehydrogenase. Here we report the cloning of the aba3 gene from Arabidopsis thaliana and the biochemical characterization of the purified protein. ABA3 is a two-domain protein with a N-terminal NifS-like sulfurase domain and a C-terminal domain that might be involved in recognizing the target enzymes. Molecular analysis of three aba3 mutants identified mutations in both domains. ABA3 contains highly conserved binding motifs for pyridoxal phosphate and for a persulfide. The purified recombinant protein possesses a cysteine desulfurase activity, is yellow in color, and shows a NifS-like change in absorbance in the presence of L-cysteine. Pretreatment of ABA3 with a thiol-specific alkylating reagent inhibited its desulfurase activity. These data indicate a transsulfuration reaction similar to bacterial NifS. In a fully defined in vitro system, the purified protein was able to activate aldehyde oxidase by using L-cysteine as sulfur donor. Finally, we show that the expression of the aba3 gene is inducible by drought-stress.