Fig 7. The N-terminal region of IMC6 is required for binding to ILP1 at E209.
The experimental design mimics the one reported for IMC3 in Fig 6. (A) Alveolin only (128–290, IMC6A) and alveolin plus C terminus (128–444, IMC6AC) truncations tagged with a V5 epitope were visualized. Both truncations localize to the growing daughter IMC but also exhibit partial mislocalization in the maternal cytoplasm, which does not appear to be rescued with the addition of the C-terminal region. Red, mouse anti-V5 antibody; green, rabbit anti-Myc antibody. Scale bar represents 2 μm. (B) Western blot of whole cell lysate showing uncrosslinked ILP1 (blue arrow) and the original upshifted species (black asterisk) but no visible lower molecular weight band that would indicate crosslinking to the smaller size truncations. (C) ILP1-3xHA E209 denaturing IP enriches for ILP1 (first panel), but no upshift is observed in the anti-V5 blot (second panel), indicating that neither IMC6A nor IMC6AC is sufficient to crosslink to ILP1 at residue E209. The IMC6A band is obscured by antibody light chain (lc) cross-reactivity. The anti-Myc blot (third panel) confirms successful IP, as seen by the original ILP1 E209/IMC6 full-length upshift (black asterisk). Uncrosslinked proteins are denoted with blue arrows. (D) Anti-V5 denaturing IP enriching for the IMC6 truncations correspondingly lacks any ILP1-3xHA signal (first panel). Uncrosslinked proteins are denoted with blue arrows, and both heavy chains (hc) and lc are seen as extra signal in the anti-V5 blot. (E) An N terminus plus alveolin domain truncation of IMC6 (1–290, IMC6NA) appears to rescue localization like wild-type IMC6. Red, mouse anti-V5 antibody; green, rabbit anti-Myc antibody. (F) Anti-HA immunoblot of both whole cell lysate (first panel, red arrowhead) and after anti-HA denaturing IP (second panel, red arrowhead) reveals a smaller upshifted species (approximately 90 kDa) for the IMC6NA strain, suggesting that ILP1 E209 is crosslinking to this mutant. Anti-V5 blot confirms that IMC6NA is detected at this molecular weight size (third panel, red arrowhead), demonstrating the identity of an ILP1 E209/IMC6NA crosslink. Anti-Myc blot confirms successful co-IP of the original IMC6 upshift (fourth panel). Uncrosslinked ILP1 is denoted with blue arrows, and the original upshift corresponding to ILP1 crosslinked to full-length IMC6 is denoted with black asterisks. A, alveolin domain only; AC, alveolin domain and C-terminus; Azi, p-azidophenylalanine; co-IP, co-immunoprecipitation; HA, hemagglutinin; hc, heavy chain; ILP1, IMC localizing protein 1; IMC, inner membrane complex; IP, immunoprecipitation; lc, light chain; NA, N-terminus and alveolin domain; wt, wild-type.