Functional Similarity between the Chloroplast Translocon Component, Tic40, and the Human Co-chaperone, Hsp70-interacting Protein (Hip)

• Published in: The Journal of biological chemistry Vol. 282; no. 29; pp. 21404 - 21414
• Main Authors: Bédard, Jocelyn, Kubis, Sybille, Bimanadham, Sarat, Jarvis, Paul
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.07.2007; American Society for Biochemistry and Molecular Biology
• Subjects: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Arabidopsis Proteins - physiology; Carrier Proteins - physiology; Chloroplasts - metabolism; Gene Deletion; Gene Expression Regulation, Plant; Genetic Complementation Test; Humans; Membrane Proteins - physiology; Microscopy, Fluorescence; Models, Biological; Molecular Chaperones - physiology; Molecular Sequence Data; Plants, Genetically Modified; Protein Structure, Tertiary; Species Specificity; Two-Hybrid System Techniques
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M611545200

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 (ΔSti1), a second lacking a central region, including the putative TPR domain (ΔTPR), and a third lacking the predicted transmembrane anchor region. Moreover, the ΔSti1 and ΔTPR 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.