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Tic40 is a component of the protein import apparatus of the inner envelope of chloroplasts, but its role in the import mechanism has not been clearly defined. The C terminus of Tic40 shares weak similarity with the C-terminal Sti1 domains of the mammalian Hsp70-interacting protein (Hip) and Hsp70/Hsp90-organizing protein (Hop) co-chaperones. Additionally, Tic40 may possess a tetratricopeptide repeat (TPR) protein-protein interaction domain, another characteristic feature of Hip/Hop co-chaperones. To investigate the functional importance of different parts of the Tic40 protein and to determine whether the homology between Tic40 and co-chaperones is functionally significant, different Tic40 deletion and Tic40:Hip fusion constructs were generated and assessed for complementation activity in the Arabidopsis Tic40 knock-out mutant, tic40. Interestingly, all Tic40 deletion constructs failed to complement tic40, indicating that each part removed is essential for Tic40 function; these included a construct lacking the Sti1-like domain (DeltaSti1), a second lacking a central region, including the putative TPR domain (DeltaTPR), and a third lacking the predicted transmembrane anchor region. Moreover, the DeltaSti1 and DeltaTPR constructs caused strong dominant-negative, albino phenotypes in tic40 transformants, indicating that the truncated Tic40 proteins interfere with the residual chloroplast protein import that occurs in tic40 plants. Remarkably, the Tic40:Hip fusion constructs showed that the Sti1 domain of human Hip is functionally equivalent to the Sti1-like region of Tic40, strongly suggesting a co-chaperone role for the Tic40 protein. Supporting this notion, yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated the in vivo interaction of Tic40 with Tic110, a protein believed to recruit stromal chaperones to protein import sites.

Original publication

DOI

10.1074/jbc.M611545200

Type

Journal article

Journal

J Biol Chem

Publication Date

20/07/2007

Volume

282

Pages

21404 - 21414

Keywords

Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Arabidopsis Proteins, Carrier Proteins, Chloroplasts, Gene Deletion, Gene Expression Regulation, Plant, Genetic Complementation Test, Humans, Membrane Proteins, Microscopy, Fluorescence, Models, Biological, Molecular Chaperones, Molecular Sequence Data, Plants, Genetically Modified, Protein Structure, Tertiary, Species Specificity, Two-Hybrid System Techniques