Journal: bioRxiv

Article Title: Glycolipid MPIase is essential for the TAT (Twin-Arginine Translocation) pathway

doi: 10.64898/2026.06.05.730299

Figure Lengend Snippet: (A) Membrane fusion of TatABC proteoliposomes with MPIase liposomes enabled the detection of the SufI translocation. INV, prepared from BL21/pT7-TatABC, were solubilized with OG, and then mixed with purified F 0 F 1 -ATPase. The proteoliposomes were then formed by removing the detergent. PL/DAG liposomes or PL/DAG/MPIase liposomes were fused to the reconstituted proteoliposomes. SufI translocation was performed co-translationally (left) or post-translationally (right) in the presence of these proteoliposomes. SufI(RR) (top left) or SufI(KK) (bottom left) was used as the substrate. One-ninth of the reaction mixture was analyzed as the translation standard (-) for the co-translational reactions. For the post-translational reactions, ATP was added as indicated. Translocation activities are shown at the bottom of the left panels. (B) Purification of F 0 F 1 -ATPase and the Tat components. F 0 F 1 -ATPase from a thermophilic Bacillus PS3 (left), and E. coli TatA-His (lane 1), TatB-His (lane 2), and TatC-His (lane 3) (right) were subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining. (C, D) Complete reconstitution of the TAT system using the purified components. (C) Purified TatA, TatB, and TatC (in a 1:1:1 ratio) and F 0 F 1 -ATPase were mixed with phospholipids. Then, detergent was removed to form proteoliposomes. The reconstituted proteoliposomes were then fused with PL/DAG liposomes or PL/DAG/MPIase liposomes. Translocation activity was assayed with SufI(RR) (left) or SufI(KK) (right). (D) Proteoliposomes were reconstituted with the indicated components, followed by TorA-GFP translocation. The TatA:TatB:TatC ratio was 10:1:1. CCCP (20 μM), a protonophore, was added to the sample on the left. TorA-GFP(RR) (top) or TorA-GFP(KK) (bottom) was used as the substrate. Translocation activities are shown at the bottom of each gel.

Article Snippet: The E. coli polar lipid extract (phospholipids), 18:1 NBD-PE, and dioleoylglycerol were obtained from Avanti Polar Lipids, Inc. n -Octyl-β-D-glucopyranoside (OG) and n -dodecyl-β-D-maltopyranoside (DDM) were obtained from Dojindo Laboratories.

Techniques: Membrane, Liposomes, Translocation Assay, Purification, SDS Page, Staining, Activity Assay