New, C. P. | bioRxiv Science

bioRxiv2020

Insights into substrate-mediated assembly of the chloroplast TAT receptor complex

The Twin Arginine Transport (TAT) system translocates fully folded proteins across the thylakoid membrane in the chloroplast (cp) and the cytoplasmic membrane of bacteria. In chloroplasts, cpTAT transport is achieved by three components: Tha4, Hcf106, and cpTatC. Hcf106 and cpTatC function as the substrate recognition/binding complex while Tha4 is thought to play a significant role in forming the translocation pore. Recent studies challenged this idea by suggesting that cpTatC-Hcf106-Tha4 function together in the active translocase. Here, we have mapped the inter-subunit contacts of cpTatC-Hcf106 during the resting state and built a cpTatC-Hcf106 structural model based on our crosslinking data. In addition, we have identified a substrate-mediated reorganization of cpTatC-Hcf106 contact sites during active substrate translocation. The proximity of Tha4 to the cpTatC-Hcf106 complex was also identified. Our data suggest a model for cpTAT function in which the transmembrane helices of Hcf106 and Tha4 may each contact the fifth transmembrane helix of cpTatC while the insertion of the substrate signal peptide may rearrange the cpTatC-Hcf106-Tha4 complex and initiate the translocation event. One sentence summaryProtein subunits of the thylakoidal twin arginine transport complex function together during substrate recognition and translocase assembly.

plant biology↗

bioRxiv2018

Thylakoid-integrated recombinant Hcf106 participates in the chloroplast Twin Arginine Transport (cpTat) system

The chloroplast Twin arginine transport (cpTat) system distinguishes itself as a protein transport pathway by translocating fully-folded proteins, using the proton-motive force (PMF) as the sole source of energy. The cpTat pathway is evolutionarily conserved with the Tat pathway found in the plasma membrane of many prokaryotes. The cpTat (E. coli) system uses three proteins, Tha4 (TatA), Hcf106 (TatB), and cpTatC (TatC), to form a transient translocase allowing the passage of precursor proteins. Briefly, cpTatC and Hcf106, with Tha4, form the initial receptor complex responsible for precursor protein recognition and binding in an energy-independent manner, while a separate pool of Tha4 assembles with the precursor-bound receptor complex in the presence the PMF. Analysis by blue-native polyacrylamide gel electrophoresis (BN-PAGE) shows that the receptor complex, in the absence of precursor, migrates near 700 kDa and contains cpTatC and Hcf106 with little Tha4 remaining after detergent solubilization. To investigate the role that Hcf106 may play in receptor complex oligomerization and/or stability, systematic cysteine substitutions were made in positions from the N-terminal transmembrane domain to the end of the predicted amphipathic helix of the protein. BN-PAGE analysis allowed us to identify the locations of amino acids in Hcf106 that were critical for interacting with cpTatC. Oxidative cross-linking allowed us to map interactions of the transmembrane domain and amphipathic helix region of Hcf106. In addition, we showed that in vitro expressed, integrated Hcf106 can interact with the precursor signal peptide domain and imported cpTatC, strongly suggesting that a subpopulation of the integrated Hcf106 is participating in competent cpTat complexes.

plant biology↗