Figure 7.

Mutations in the Cdc42 binding region or the polybasic region of Sec3 affect its targeting to the bud tip in the presence of Lat B. (A) Yeast cells expressing SEC3 or various sec3 mutants under the SEC3 promoter as the only copy of Sec3 were GFP tagged and arrested at G₀ phase. Cells were then released into fresh medium with 100 μM Lat B (left) or DMSO (right) at 25°C for 90 min. Cells were fixed for fluorescence microscopy. Although the wild-type Sec3 formed a clear patch in the presumptive buds when treated with Lat B, the mutant sec3 proteins were dispersed to different extents. (B) Quantification of the percentages of Sec3-GFP cells that were polarized, partially polarized, or depolarized after treatment with Lat B (n = 300). Partially polarized was scored when the GFP signals were localized at one end of the cell but appeared as multiple patches. (C) Polarization of exo70-38 protein depends on actin. Cells expressing Exo70-GFP or exo70-38–GFP were arrested at G₀ phase. These cells were released into fresh medium at 25°C for 90 min in the presence of 100 μM Lat B or DMSO and fixed for fluorescence microscopy. In cells treated with Lat B (left), the wild-type Exo70 still formed a patch in the presumptive budding site. exo70-38–GFP was, however, dispersed throughout the cells. As controls, both Exo70-GFP and exo70-38–GFP were well polarized in cells treated with DMSO. Bars, 5 μm.