Figure 1.
Inhibition of SNARE-mediated fusion by IncA is topologically restricted.
The inhibitory capacity of IncA was assessed in three topological configurations–on the t-SNARE liposome, v-SNARE liposome, or a third liposome. 45 µL of t-SNARE liposome reconstituted with Stx7, Stx8, Vti1b were mixed with 5 µL of v-SNARE liposome reconstituted with VAMP8 and 10 µL of third liposome and NBD fluorescence was measured every two minutes for 2 hrs at 37°C. 10 µL of n-Dodecyl-β-D-maltoside (2.5% w/v stock concentration) was added at the end of the 2 hrs and data were normalized to the 100% fluorescence value as described [24]. For each condition, IncA was reconstituted into just one liposome population, and its inhibition tested. (A) The average percent inhibition of IncA in each indicated liposome is presented. Error bars represent one standard deviation from three independent experiments. IncA inhibits SNARE-mediated liposome fusion significantly better when co-reconstituted with SNARE proteins. (B) IncA fails to inhibit when reconstituted singly on the third liposome population. Black circles represent fusion in the absence of IncA, red squares in the presence of IncA. Inset illustrates position of IncA (red bar) relative to SNARE proteins (black bars). SDS-PAGE analysis reveals incorporation of proteins into liposomes. The positions of each protein and liposome population are denoted. The fusion graph is representative of three independent experiments. (C) IncA inhibits fusion when reconstituted with the t-SNAREs. The design of the experiment was identical to (B), except that IncA was reconstituted with the late endocytic t-SNARE complex on the t-SNARE liposome. The fusion graph is representative of three independent experiments. (D) IncA inhibits fusion when reconstituted with the v-SNARE. The design of the experiment was identical to (B), except that IncA was reconstituted with the late endocytic SNARE VAMP8 on the v-SNARE liposome. The fusion graph is representative of three independent experiments.
Figure 2.
The N-terminal tail region and transmembrane domain of IncA are dispensible for the inhibitory function of IncA.
(A) Schematic of full-length wildtype IncA. The N-terminal tail encompasses the first 34 amino acids. The transmembrane domain is depicted by the black area (amino acids 35–82). SNARE-like domain1 (SLD1) is located between amino-acids 107 and 145; SLD2 between amino-acids 210 and 273. (B) 45 µL of t-SNARE liposome reconstituted with Stx7, Stx8, Vti1b were mixed with 5 µL of v-SNARE liposome reconstituted with VAMP8 and an increasing concentration of IncA. NBD fluorescence was measured every 2 min for 2 hrs at 37°C. 10 µL of n-Dodecyl-β-D-maltoside (2.5% w/v stock concentration) was added to the reaction and data were normalized to the 100% fluorescence value as described [24]. Protein gel lanes show co-reconstitution of Δ34-IncA with VAMP8 on v-SNARE liposomes. Values next to protein gel lanes refer to the ratio of IncA mutant to VAMP8 for that particular fusion curve. (C) Fusions were performed as in (B) except that the N-terminal tail region and TMD of IncA were replaced with the TMD of the transferrin receptor (TfR) before reconstituting increasing concentration of this chimeric protein with VAMP8. For (B) and (C), black circles represent fusion in the absence of IncA mutant and gray squares represent fusion in the presence of increasing concentrations of IncA mutant relative to VAMP8. Results are representative of at least four independent experiments.
Figure 3.
SLD2 inhibits late endocytic SNARE-mediated fusion independently of SLD1.
(A) SLD1 is sufficient for SNARE inhibition. Increasing concentrations of IncA1–141 were reconstituted in v-SNARE liposomes with VAMP8 and mixed with t-SNARE liposomes in which Stx7, Stx8, and Vti1b were reconstituted. Fusion rates were calculated as described in the legend of Figure 2B. Protein gel lanes show co-reconstitution of VAMP8 and IncA1–141. (B) Phenylalanine and Aspartic acid mutations inactivate SLD1. Increasing concentrations of Phe/Asp-IncA1–141 were reconstituted in v-SNARE liposomes with VAMP8. v-SNARE liposomes were mixed with t-SNARE liposomes and data analyzed as in (A). (C) SLD2 independently inhibits SNARE-mediated membrane fusion. Increasing concentrations of Phe/Asp-IncA were reconstituted in v-SNARE liposomes with VAMP8 and v-SNARE liposomes were mixed with t-SNARE liposomes. Data were analyzed as in (A). In A, B, and C, black circles represent fusion in the absence of IncA and gray squares represent fusion using liposomes containing IncA construct. Results are representative of at least four independent experiments. (D, E, F) Far-UV CD wavelength scans of IncA constructs. Protein was dissolved in CD Buffer (200 mM NaF, 20 mM Tris, 10% glycerol, 1 mM DTT, pH7.4) to a final concentration of 10 µM and far-UV scans were taken in a 1 mm pathlength quartz cuvette (Starna) at 20°C. Data are the averages of five scans. For comparison, the CD signal of ΔTMD-IncA (which contains wildtype SLD1 and SLD2) is shown as a dashed curve. (D) SLD1 displays an α-helical structure. ΔTMD-IncA1–141 was dissolved in CD Buffer and analyzed as above. (E) Phenylalanine and Aspartic acid mutations interfere with the α-helical structure of SLD1. ΔTMD-Phe/Asp-IncA1–141 was dissolved in CD Buffer and analyzed as in (D). The loss of structure cannot be attributed to aggregation of the protein. Inset shows elution profile of protein (red line) superimposed on elution profile of blue dextran (blue line) to determine void volume. The protein elutes far from the blue dextran indicating a soluble species of protein. (F) Phe/Asp-IncA displays an α-helical structure. ΔTMD-Phe/Asp-IncA was dissolved in CD Buffer and analyzed as in (D).
Figure 4.
SLD2 is required for oligomerization but both SNARE-like domains are necessary for homotypic fusion.
(A) SLD2-containing IncA mutants co-elute with GST-TfR-IncA. 6xHis-tagged IncA mutants were co-expressed with GST-TfR-IncA in BL21(DE3) E. coli, and GST-containing complexes were purified over glutathione beads. Co-eluted IncA mutants were detected by western blot using anti-6xHis antibody. GST-TfR-IncA was visualized by Coomassie Blue staining. IncA mutants that contain SLD2 co-precipitated while the two truncated mutants lacking SLD2 did not. GST control shows basal levels of wildtype 6x-His-IncA binding. Results shown are typical of five independent experiments. (B) Transfection with wildtype- or Δ34-IncA leads to nonfusogenic inclusions. HeLa cells were transfected with plasmids expressing the indicated DsRed-IncA mutant or vector control (pDsRed-monomer-C1) and subsequently infected with C. trachomatis L2 for 24 hr. The location of each DsRed-IncA construct is shown on the left (red), while the inclusions are shown in the middle (blue, Hoechst staining). The right column shows the overlay. Arrows denote inclusions. Note the multiple inclusions in wildtype-IncA and Δ34-IncA transfected cells compared to the cells transfected with other IncA constructs. Scale bars represent 10 µm. Images are typical of four independent experiments. (C) Expression of either wildtype or Δ34-IncA inhibits subsequent inclusion development in HeLa cells. The number of inclusions/cell was determined by fluorescence microscopy. More than fifty infected cells per replicate per transfection were assessed for multiple inclusions. The number of infected cells carrying a single inclusion was divided by the total number of infected cells and expressed as a percent. Data are averages of four independent experiments. Error bars represent one standard deviation. Asterisks denote a significant difference compared to control-transfected cells (p<0.05).