Fig. 2: Pore formation requires both the middle and receptor-binding domains.
a Schematic representation of AIP56 variants and chimeric proteins. AIP56 domains are colored as in Fig. 1a; β-lactamase (Bla) and diphtheria receptor-binding (DTR) domain, gray. b Only AIP56L258-N497 and BlaL19-W286AIP56L258-N497 interacted with artificial black lipid membranes. Single-channel record of DiPhPC/n‐decane membranes after addition of the indicated proteins to the cis-side of the black lipid bilayer at a final concentration indicated in the figure. Measurements were performed with 50 mV (AIP56L258-N497) or 150 mV (BlaL19-W286AIP56L258-N497) at room temperature. Membrane activity was induced by acidification (pH 4.8–5.0; arrows) of the aqueous phase at the cis‐side of the chamber with exception for BlaL19-W286AIP56L258-N497, which formed stable pores at pH 6. The average single-channel conductance was about 16 pS for 110 steps. Each result shown is representative of at least three (n = 3) independent measurements. c Pharmacological inhibition of vacuolar ATPase pump does not affect cytosolic delivery of Bla by BlaL19-W286AIP56L258-N497. The cleaved/uncleaved CCF4-AM ratios were determined by quantifying the indicated number of microscopic fields per condition. Representative images used for quantification are in Supplementary Fig. 4d. Results shown represent one out of three (n = 3) independent experiments. Statistical significance was tested by One-way ANOVA and p values for individual comparisons were calculated by Tukey’s HSD test and indicated on top of the brackets. Data are presented as mean values ± SD. Actual p values from left-to-right: p < 0.0001, p < 0.0001. Data of the three independent experiments are provided in the Source data file. CCF4, Fluorescence Resonance Energy Transfer (FRET) substrate. ConcA concanamycin A.
