Biology subjects

Dabney-Smith, C.

Publications and source records attributed to Dabney-Smith, C..

3 recordsLinked to original sources

bioRxiv2024

The N-terminal Domain of cpTatC Protein Interacts with the Precursor Mature Domain in Chloroplast TAT Translocation

The chloroplast Twin Arginine Transport (cpTAT) protein translocation pathway is one of the thylakoid membranes two protein transport pathways for getting proteins into the lumen. The cpTAT system distinguishes itself by transporting fully folded proteins across the thylakoid, using the sole energy source of the proton motive force (PMF). The cpTAT pathway is evolutionarily conserved with the TAT pathway found in many bacteria and archaea. Although small differences exist, TAT systems in different organisms share homologous protein composition and similar molecular mechanisms. The cpTAT system comprises cpTatC, Hcf106, and Tha4 (the prokaryotic homologs of these proteins are TatC, TatB, and TatA, respectively). (cp)TatC is one of the essential proteins in the (cp)TAT system, as it is present in the receptor complex. The amino acid sequence alignment of cpTatC and TatC protein in archaea and E. coli has shown a unique N-terminal amino acid extension of 70-100 amino acids in mature cpTatC that is not present in prokaryotic TatC. However, the role of the amino-terminal extension in cpTatC function is still unknown. We present crosslinking evidence that the amino-terminal extension directly interacts with the precursor mature domain during TAT protein translocation and may serve to prime the transporter with bound precursor in the absence of the PMF. HighlightsO_LIThe cpTatC protein in the cpTAT pathway has a unique amino-terminal extension not found in bacterial or archaeal TatC proteins. C_LIO_LIThe N-terminal extension of cpTatC interacts with the precursor mature domain during TAT translocation. C_LIO_LIThe crosslinking data indicate that different regions within the N-terminal extension exhibit varying intensities when binding to the precursor. C_LI

plant biology↗

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↗