Kinetics of precursor interactions with the bacterial Tat translocase detected by real-time FRET.
The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time.
Department of Molecular and Cellular Medicine, College of Medicine, The Texas A&M Health Science Center, 1114 TAMU, College Station, Texas 77843, USA.
Neal
N
Whitaker
Umesh K
UK
Bageshwar
Siegfried M
SM
Musser
D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000378:metabolism; D008958:Models, Molecular; D009068:Movement; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; C473632:SufI protein, E coli; C408585:twin-arginine translocase complex, E coli