Chloroplasts are double membrane organelles involved in the vital process of photosynthesis in plants. The thylakoid, the site of photosynthesis, is an internal membrane system found in the chloroplast with an aqueous lumen. All of the proteins located in the thylakoid lumen are encoded by nuclear genes, synthesized in the cytosol, and transported across three membranes to reach their final destination. The chloroplast Twin Arginine Translocation (cpTat) system facilitates the transport of fully folded proteins across the thylakoid membrane with only the energy from the proton motive force. Prior research has shown that the cpTat complex is composed of three protein subunits: cpTatC, Hcf106, and Tha4. cpTatC is the largest protein subunit of the complex with six transmembrane domains and four stromal segments including the C- and N-termini. At rest, i.e., when transport is not occurring, cpTatC exists in a receptor complex with Hcf106 and Tha4. Research has shown interaction points between the stromal S1 and S2 domains of cpTatC and the signal peptide of precursor proteins, but due to the transient nature of precursor binding and translocation, research regarding the interactions of cpTatC and the precursor mature domain is lacking. To determine the pathway of precursor transport and its interactions with the receptor complex, we are studying contact points between the N-terminus of cpTatC and the precursor mature domain. Single-cysteine-substituted cpTatC variants and precursor variants of tOE17 were generated through site-directed mutagenesis. Disulfide cross-linking studies were used to show in vitro expressed and radio-labeled precursor interaction with cpTatC variants imported into isolated chloroplasts and integrated in the thylakoid and analyzed by SDS-PAGE and fluorography. Preliminary data show crosslinking between the precursors and the N-terminal region of cpTatC, suggesting that cpTatC contributes to precursor transport across the thylakoid membrane through a tight interaction to prevent backwards slippage.

Crosslinking interactions between stromal cpTatC and mature precursor

255th National Meeting (2018)

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