Arabidopsis ORANGE protein regulates plastid pre-protein import through interacting with Tic proteins

Chloroplast-targeted proteins are actively imported into chloroplasts via the machinery spanning the double-layered membranes of chloroplasts. While the key translocons at the outer (TOC) and inner (TIC) membranes of chloroplasts are defined, proteins that interact with the core components to facilitate pre-protein import are continuously being discovered. A DnaJ-like chaperone ORANGE (OR) protein is known to regulate carotenoid biosynthesis as well as plastid biogenesis and development. In this study, we found that OR physically interacts with several Tic proteins including Tic20, Tic40, and Tic110 in the classic TIC core complex of the chloroplast import machinery. Knocking out or and its homolog or-like greatly affects the import efficiency of some photosynthetic and non-photosynthetic pre-proteins. Consistent with the direct interactions of OR with Tic proteins, the binding efficiency assay revealed that the effect of OR occurs at translocation at the inner envelope membrane (i.e. at the TIC complex). OR is able to reduce the Tic40 protein turnover rate through its chaperone activity. Moreover, OR was found to interfere with the interaction between Tic40 and Tic110, and reduces the binding of pre-proteins to Tic110 in aiding their release for translocation and processing. Our findings suggest that OR plays a new and regulatory role in stabilizing key translocons and in facilitating the late stage of plastid pre-protein translocation to regulate plastid pre-protein import.