Figure 5.

The short membrane anchor of TatA can cause membrane defects. A and C, acridine orange quenching of TatBC IMVs (A) or DADE IMVs (C) with the indicated reconstituted in vitro translated TatA variants. B and D, comparison of relative abundance of the membrane-inserted TatA constructs in the vesicles used for the quenching experiments in A and C via Strep-Tactin Chromeo 488 labeling and fluorescence spectroscopy. E, quantification of the data shown in the traces below A and C, as derived from the recovery of fluorescence after CCCP addition. F, Tricine-SDS-PAGE/Western blot analysis of carbonate washes of membrane inserted TatA variants in TatBC IMVs (top) or DADE IMVs (bottom). All constructs were Strep-tagged. Western blots were developed using Strep-Tactin-HRP and ECL detection. M, vesicle membranes; P, pellet after carbonate wash; S, supernatant after carbonate wash. AU, arbitrary units.