Fig. 5. Role of Bud3 in targeting Clb2 to the neck. (A,B) Localization of Clb2-GFP fusion protein expressed from the GAL1p promoter in 15DU haploid cells harboring a deletion of the BUD3 gene or expressing a truncated form of Bud3 (bud3¹²²¹). The double ring labeling observed in wild-type cells is almost completely abolished in bud3 cells although some cells (about 25%) show a faint single band at the bud neck junction. In contrast, the BUD3 gene is not required for proper cell cycle-regulated localization of Hof1 to the mother-bud neck. (C) Clb2 localization to the neck depends on septin integrity. Cells were grown in SC raffinose at 22°C. Half of the culture was shifted to the restrictive temperature (37°C) for 2 hours and then induced for 3 hours on YEPGal plates prewarmed at the same temperature. The proportion of cells exhibiting bud neck staining was estimated in large budded cells with separated nuclei by microscopic observation and counting. At least 150 cells were counted for each strain and condition. (D) Clb2-YFP colocalizes with Bud3 at the mother-bud neck. CLB2-YFP was overexpressed from the GAL1p in a 15DU diploid strain in which a CFP tag was appended to the C terminus of one copy of the BUD3 gene. The left hand column shows YFP, the center column shows CFP and the right hand column shows merged YFP/CFP signals.