Fig. 7: Extracellularly externalized Vap33 is cleaved by Mmp1/2 on plasma membrane.

a, c Representative images of Western blot experiments of supernatant (Sup) and whole cell lysates (WCL) of transfected S2 cells. In Fig. 7a, FLAG-Vap33^WT-HA was co-expressed with GFP, Mmp1, or Mmp2. In Fig.7c, FLAG-Vap33^WT-HA or FLAG-Vap33^ΔL118-F127-HA was co-expressed with GFP, Mmp1, or Mmp2. Antibody to FLAG detects bands at around 17 kDa on a Western blot, which corresponds to the molecular weight of the Vap33 MSP domain. Antibody to α-tubulin is shown below. The results were reproduced in two biologically independent experiments. b Representative images of Western blot experiments of supernatant (Sup) and whole cell lysates (WCL) of dsRNA-treated S2 cells expressing FLAG-Vap33^WT-HA. The graph shows fold change in secretion level of the MSP domain compared with control dsRNA. Statistical analysis was performed by the ordinary one-way ANOVA with Dunnett’s multiple comparison test (****P < 0.0001). Error bars represent the standard error of the mean (SEM) of 5 biologically independent experiments (n = 5). d, f, g Results of flow cytometry analysis of transfected S2 cells stained with antibodies to FLAG and HA. The pUASTattB empty plasmid was used for the control. The percentage of the extracellular FLAG⁺ cells to cells with FLAG and/or HA facing extracellularly is shown. Statistical analysis was performed by the ordinary one-way ANOVA or the unpaired t-test (****P < 0.0001, ***P < 0.001 (0.0003), **P < 0.01 (0.004), ns: not significant, two-sided). Error bars represent the SEM of 8 biologically independent experiments (n = 8). e Western blot experiments of supernatant (Sup) and whole cell lysates (WCL) of transfected S2 cells. Antibody to FLAG detects bands at around 17 kDa on a Western blot, which corresponds to the molecular weight of the Vap33 MSP domain. Antibody to α-tubulin is shown below. The results were reproduced in two biologically independent experiments. h Secretion model of Vap33. ER protein Vap33 is transported to plasma membrane via a BFA-resistant transport pathway that remains to be characterized. Vap33 inverts its topology to externalize its MSP domain. The MSP domain is then cleaved by Matrix metalloproteinase 1 and/or 2 (Mmp1/2). Source data are provided as a Source Data file.