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In vivo interaction between atToc33 and atToc159 GTP-binding domains demonstrated in a plant split-ubiquitin system
The GTPases atToc33 and atToc159 are pre-protein receptor components of the translocon complex at the outer chloroplast membrane in Arabidopsis. Despite their participation in the same complex in vivo, evidence for their interaction is still lacking. Here, a split-ubiquitin system is engineered for use in plants, and the in vivo interaction of the Toc GTPases in Arabidopsis and tobacco protoplasts is shown. Using the same method, the self-interaction of the peroxisomal membrane protein atPex11e is demonstrated. The finding suggests a more general suitability of the split-ubiquitin system as a plant in vivo interaction assay.
A plant split-ubiquitin system and TAP-tagging to study in vivo protein interactions in the chloroplast protein import machinery
Most chloroplast proteins are synthesized as precursor proteins in the cytosol. The import of these precursor proteins is mediated by molecular complexes located at the outer and inner membrane of the chloroplast. These complexes are called Toc (translocon at the outer envelope membrane) and Tic (translocon at the inner envelope membrane) respectively. In Arabidopsis, the Toc complex consists of three principle components: two homologous receptor GTPases, atToc159 and atToc33 and a protein-import channel: atToc75. During import, the two GTPases undergo complex interactions with precursor proteins and amongst themselves although precise mechanisms remain unknown. In vitro studies revealed that Toc159 and Toc33 interact with each other via the dimerization of their GTP-binding domain (G-domain). Moreover, the crystal structure of the pea Toc33 ortholog, psToc34 indicates that it can stably homodimerize via its G-domain. However, neither Toc159/Toc33 heterodimers nor Toc33 homodimerization have been demonstrated in planta. To get new insight into the in vivo interactions of Toc GTPases, we have developed a plant split-ubiquitin system. This method, originally developed for yeast, was adapted to study interactions between the Toc GTPases atToc159 and atToc33 in Arabidopsis and tobacco protoplasts. We also demonstrated that the peroxisomal membrane protein atPex11e, used initially as a model membrane protein in our system, self-interacts as does its yeast homolog. The plant split-ubiquitin system proves to be widely usable. Another approach of this thesis was to get more information on the import mechanism via the identification of interaction partners of the Toc GTPase atToc33. atToc33 and proteins associated were isolated from Arabidopsis plants, using the tandem affinity purification (TAP) tag. We proved that this technique is suitable to purify Toc33, which encourages us to purify Toc proteins and complexes at a larger scale.
A Toc159 Import Receptor Mutant, Defective in Hydrolysis of GTP, Supports Preprotein Import into Chloroplasts
The heterotrimeric Toc core complex of the chloroplast protein import apparatus contains two GTPases, Toc159 and Toc34, together with the protein-conducting channel Toc75. Toc159 and Toc34 are exposed at the chloroplast surface and function in preprotein recognition. Together, they have been shown to facilitate the import of photosynthetic proteins into chloroplasts in Arabidopsis. Consequently, the ppi2 mutant lacking at Toc159 has a non-photosynthetic albino phenotype. Previous mutations in the conserved G1 and G3 GTPase motifs abolished the function of Toc159 in vivo by disrupting targeting of the receptor to chloroplasts. Here, we demonstrate that a mutant in a conserved G1 lysine (atToc159 K868R) defective in GTP binding and hydrolysis can target and assemble into Toc complexes. We show that atToc159 K868R can support protein import into isolated chloroplasts, albeit at lower preprotein binding and import efficiencies compared with the wild-type receptor. Considering the absence of measurable GTPase activity in the K868R mutant, we conclude that GTP hydrolysis at atToc159 is not strictly required for preprotein translocation. The data also indicate that preprotein import requires at least one additional GTPase other than Toc159.
In vivo interaction between atToc33 and atToc159 GTP-binding domains demonstrated in a plant split-ubiquitin system
The GTPases atToc33 and atToc159 are pre-protein receptor components of the translocon complex at the outer chloroplast membrane in Arabidopsis. Despite their participation in the same complex in vivo;, evidence for their interaction is still lacking. Here, a split-ubiquitin system is engineered for use in plants, and the in vivo; interaction of the Toc GTPases in Arabidopsis and tobacco protoplasts is shown. Using the same method, the self-interaction of the peroxisomal membrane protein atPex11e is demonstrated. The finding suggests a more general suitability of the split-ubiquitin system as a plant in vivo; interaction assay.