FIG. 5.

Localization of GFP fusion proteins. All cells were grown at 37°C and images taken at t₃. Membranes were stained with FM 4-64. Scale bar, 1 μm. (A to E) GFP-SpoIID localization in: A, wild type (KP1124); B, spoIIP null (KP1128); C, GFP-SpoIID^E88A (KP1125); D, GFP-SpoIID^R106A (KP1142); E, GFP-SpoIID^K203A (KP1126); and F, GFP-SpoIID^D210A (KP1127). GFP-SpoIID shows some enrichment at the leading edge together with diffuse fluorescence throughout the mother cell membranes. The only strain showing significantly enhanced localization is GFP-SpoIID^E88A (C), which is almost exclusively localized to the septum (C, arrow) or at the edges of the septum (C, arrowhead), although no membrane migration is seen. Signal is also more diffuse in the spoIIP-null strain (B) than in other strains, suggesting that, as previously reported, SpoIID localization depends on SpoIIP (8). GFP-SpoIID^R106A (D) is diffuse in the cytoplasm, suggesting protein degradation. (G to L) GFP-SpoIIP localization in: G, wild type (KP1129); H, spoIID null (KP1137); I, SpoIID^E88A (KP1130); J, SpoIID^R106A (KP1141); K, SpoIID^K203A (KP1131); and L, SpoIID^D210A (KP1132). GFP-SpoIIP localizes to the leading edges of migrating membranes in the wild type (G, arrow), as a diffuse signal after fusion (G, arrowheads) and as a sharp central focus in the absence of SpoIID (H). GFP-SpoIIP localizes across the septum and to the edges of the septal disk in SpoIID^E88A (I), as a central focus in SpoIID^R106A (J) and SpoIID^K203A (K), and similarly to the wild type in SpoIID^D210A (L, arrow), although GFP-SpoIIP signal is sometimes uneven (L, double arrowhead) or lagging behind the leading edge in SpoIID^D210A (L, arrowhead). (M to R) Diagrams representing SpoIIP (green) and SpoIID (orange) localization at t₃ in: M, wild type; N, spoIID null; O, SpoIID^E88A; P, SpoIID^R106A; Q, SpoIID^K203A; and R, SpoIID^D210A. Panel Q summarizes the results in the strain expressing untagged SpoIID^K203A (which fails to accumulate) and not GFP-SpoIID^K203A.