Figure 5.

Role of the N-terminal Cdc31-binding sites of Sfi1 for the SPB assembly. (A) Y2H analysis of tryptophan and phenylalanine mutants affecting the Cdc31-binding sites in Sfi1 after X-Gal overlay. Fig. S4 A shows the same Cdc31 Y2H analysis supplemented with further mutants of Sfi1. (B) Drop test of the SFI1 mutants in an SFI1-shuffle strain. 10-fold serial dilutions of cells on yeast peptone dextrose (YPD) G418 and 5-FOA plates were incubated for 2.5 d at 37°C and for 4 d at 23°C. Empty (sfi1∆) indicates the sfi1Δ cells with the empty control plasmid pRS305K. Cartoons on the right illustrate which Cdc31-binding site (Cdc31-binding sites are marked with light green shades; mutation is in the site marked with red) was mutated. (C) sfi1^{W211A W271A} mutant phenotypes 3 h after temperature shift to 37°C or growth at 23°C. Left: Quantification of the cell cycle phases. Graph illustrates average of 3 independent experiments (n = 3) with >100 cells analyzed each time. Error bars are SD. Right: Exemplary fluorescence images. G₁, S/M, and anaphase are sfi1^{W211A W271A} cells grown at 23°C; mitotic arrest is an sfi1^{W211A W271A} cell incubated at 37°C. (D) Quantification of the Spc42-yeGFP signal of the cells in C. Graph shows one representative result, which was confirmed in 3 independent experiments with n > 100 per strain and experiment. Average and SD are given. P value (< 0.0001) was derived by using a two-tailed t test. (E) EM micrographs of sfi1^{W211A W271A} cells after 3 h at 23°C and 37°C are shown. See Fig. S4 C for additional EM images. Cartoons illustrate the SPB phenotype. (F and G) Quantification of the EM images for the SPB size and bridge length, respectively. For each cell type and temperature, n ≥ 17 for the SPB size and n ≥ 11 for the length of the bridge. Average and SD are given. P values (<0.0007, F; < 0.0001, G) were derived by using a two-tailed t test. B, bridge; cMTs, cytoplasmic microtubules; HB, half bridge; nMTs, nuclear microtubules; RFI, relative fluorescence intensity.