4.5 Article

New insights into the Tat protein transport cycle from characterizing the assembled Tat translocon

期刊

MOLECULAR MICROBIOLOGY
卷 118, 期 6, 页码 637-651

出版社

WILEY
DOI: 10.1111/mmi.14984

关键词

cell membrane; protein transport; protonmotive force; twin-arginine translocation system

资金

  1. Wellcome Trust Investigator Award [107929/Z/15/Z]
  2. Wellcome Trust [107929/Z/15/Z] Funding Source: Wellcome Trust

向作者/读者索取更多资源

This study reports the recovery of receptor complexes associated with elevated levels of TatA through detergent solubilization of membranes during active transport. By combining biochemical analysis and live cell fluorescence imaging, sub-steps in the Tat translocation cycle were resolved, and it was inferred that TatA assembly precedes the functional interaction with TatC. It was also observed that dissipation of the protonmotive force releases TatA oligomers from the assembled translocation site, suggesting that the TatA oligomer is assembled at the periphery of the receptor complex.
The twin-arginine protein translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of chloroplasts. The Tat translocation site is transiently assembled by the recruitment of multiple TatA proteins to a substrate-activated TatBC receptor complex in a process requiring the protonmotive force. The ephemeral nature of the Tat translocation site has so far precluded its isolation. We now report that detergent solubilization of membranes during active transport allows the recovery of receptor complexes that are associated with elevated levels of TatA. We apply this biochemical analysis in combination with live cell fluorescence imaging to Tat systems trapped in the assembled state. We resolve sub-steps in the Tat translocation cycle and infer that TatA assembly precedes the functional interaction of TatA with a polar cluster site on TatC. We observe that dissipation of the protonmotive force releases TatA oligomers from the assembled translocation site demonstrating that the stability of the TatA oligomer does not depend on binding to the receptor complex and implying that the TatA oligomer is assembled at the periphery of the receptor complex. This work provides new insight into the Tat transport cycle and advances efforts to isolate the active Tat translocon.

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