Chloroplasts are surrounded by a double membrane envelope and are responsible for photosynthesis in plants. The thylakoid, the site of photosynthesis, is an internal membrane system containing an aqueous lumen. Proteins located in the thylakoid lumen are encoded by nuclear genes and cytosolically synthesized, meaning each must cross all three membrane systems to get to their final destination for proper function. The chloroplast Twin Arginine Translocation (cpTat) system facilitates the transport of about half of the lumen proteins across the thylakoid membranes. Biochemical research has revealed information about the transport of proteins into the thylakoid lumen by the cpTat system. The cpTat system comprises three protein components: Tha4, Hcf106, and cpTatC. The cpTatC component is the largest protein of the translocase containing six transmembrane domains with stromally exposed N- and C-terminals. In the resting state, cpTatC usually exists in a heterodimeric receptor complex along with Hcf106. Prior research has identified a cyclical transport process of binding of the signal peptide of the precursor to the receptor, assembly with additional Tha4, transport in the presence of a protonmotive force and disassembly to reset the system. As part of determining the organization of the receptor complex upon precursor binding and translocation, we are interested in studying contact points between cpTatC and the mature domain to be transported. Previous research has shown that the precursor signal peptide interacts with cpTatC, and that the mature domain interacts with Tha4 and Hcf106. Different models of transport predict that cpTatC may or may not be involved in the formation of the actual point of passage. If cpTaC is involved in forming the point of precursor passage across the membrane, we expect the mature domain of the precursor may interact with either stromal or luminal faces of cpTatC. We generated single cysteine-substituted cpTatC variants as well as a precursor containing single cysteine substitutions in the mature domain to investigate interactions through disulfide bond formation. Disulfide cross-linking between the precursor and the integrated cpTatC was analyzed using SDS-PAGE and fluorography. If detected, the N-proximal region might also contribute in binding the precursor mature domain and likely plays a role in forming the point of passage during translocation.

Interaction points between stromally exposed CpTatC and precursor signal peptide

253rd National Meeting (2017)

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