Identification of putative phosphosite(s) regulating the asymmetric localization of Bfa1.

<p>(A) Schematic diagram of full-length Bfa1 and Bfa1-D8. The position of Ser and Thr residues in Bfa1-D8 is shown. The arrows and shaded boxes represent two regions of imperfect direct repeats (DR1 and DR2) that are highly conserved among fungi (Kim <i>et al.</i>, 2008). The Ser and Thr residues that are mutated to Ala in Bfa1-11A are shown in blue. Ser residues that were mutated to Ala in this study are shown in red, and Thr are shown in green. (B,D) The localization of Bfa1-D8 mutants (B) and Bfa1 Ser mutants (D) at anaphase. α-factor synchronized cells were released at 35°C for 3 h, and observed by fluorescence microscopy. Arrows indicate Bfa1 localized to the SPB^m. Bar, 5 µm. (B) YSK2195, 2196, 2197, 2198 strains and (D) YSK2192, 2194, 2330, 2332, 2193 and 2334 were used. (C) <i>In vitro</i> kinase assay of Bfa1-D8 mutants by Cdc5. The experiment was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002450#pgen-1002450-g004" target="_blank">Figure 4C</a>. No γ-[³²P] labeling of substrates was observed from reactions with GST-Cdc5KD. (E) The quantification of (D). Anaphase cells with the indicated phenotype were counted (n = 200). The average of two independent counts is plotted with standard deviation. (F) Box plots comparing the fluorescence intensities of Bfa1-GFP at SPBs. <i>cdc15-2SPC42-RFPBFA1-GFP</i> (YSK2545) and <i>cdc15-2SPC42-RFP BFA1^4A-GFP</i> (YSK2561) cells were released from α-factor arrest for 3 h at 35°C. GFP fluorescence signals were analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002450#s4" target="_blank">Materials and Methods</a> (n = 30 for Bfa1 and 40 for Bfa1^4A). The line inside the box indicates the median. D, GFP signal at the SPB^d. M, GFP signal at the SPB^m. (G) The anaphase localization of Tem1-RFP in <i>BFA1</i> and <i>BFA1^4A</i> cells. <i>cdc15-2TEM1-RFPBFA1-GFP</i> (YSK2720) and <i>cdc15-2TEM1-RFP Bfa1^4A-GFP</i> (YSK2721) cells were synchronized with α-factor at 25°C, released at 35°C for 3 h, and observed by fluorescence microscopy. Arrows indicate Bfa1-GFP and Tem1-RFP localized to the SPB^m. Bar, 5 µm.