Investigation of the role of Arabidopsis Tic40, a co-chaperone-like component of the chloroplast protein import machinery
Protein import into chloroplasts is mediated by translocons of the outer and inner envelope membranes, termed TOC and TIC, respectively. To investigate the in vivo roles of three TIC components, Tic40, Ticl 10 and Hsp93, Arabidopsis null mutants for each were identified and characterized. The tic40, ticl 10 and hsp93-Vmutants displayed distinct chlorotic phenotypes and aberrant chloroplast ultrastructure. Furthermore, in vitro protein import assays using mutant chloroplasts revealed that each mutant has a general protein import defect. Genetic interaction studies performed with these mutants also revealed that the proteins may cooperate functionally, supporting their proposed cooperation in a putative protein import motor. Two approaches were taken to further investigate the role of Tic40 in vivo. Firstly, a genetic screen was performed for suppressor of tic40 (stic) mutants that alleviate the chlorotic phenotype of tic40. Several stic mutants were identified, and their analysis revealed that they were the result of semi-dominant and recessive mutations. All the mutants analysed showed a slight improvement in thylakoid development, and an increase in leaf cell number and density, consistent with the possibility that the mutants display increased import efficiency. Mapping may reveal that the affected genes encode functional partners of Tic40, such as Ticl 10 and Hsp93. Secondly, the significance of homology between Tic40 and the human co-chaperone, Hip, was investigated. Tic40 deletion and Tic40:Hip fusion constructs were generated, and transformed into tic40 to assess for complementation. Interestingly, two deletion constructs that failed to complement tic40 had a dominant-negative effect, causing a phenotype more severe than that of tic40. The corresponding proteins are proposed to associate with the TIC and interfere with its activity. Remarkably, Tic40:Hip fusions revealed that although Tic40 and Hip are not functionally equivalent, the extreme C-terminus of Hip can fully replace the homologous region of Tic40. This finding provides the first experimental evidence indicating that Tic40 may function as a co-chaperone.