Figure 7.

The Ypt31/32-Myo2-GTD interaction is important for the polarized intracellular localization of Myo2. (A) Ypt31/32 GTPases are required for polarized intracellular distribution of Myo2. The effect of the ypt31Δ/32ts mutation on the cellular distribution of Myo2 was determined using immunofluorescence (IF) deconvolution microscopy and anti-Myo2 antibody. Cells were grown to mid-log at permissive temperature (24°C), shifted to 37°C for 2 h, and fixed and processed for IF (see Materials and Methods). In wild-type cells (NSY125), Myo2 localizes to sites of cell growth. In ypt31Δ/32ts mutant cells (NSY348) at the restrictive temperature (37°C), Myo2 staining is diffuse. (B) Ypt31/32 interaction mutation in Myo2-GTD results in an impaired Myo2 intracellular localization. The effect of the myo2-Y1415E mutation on the localization of Myo2 was determined by IF microscopy using anti-Myo2 and antiYpt31/32 antibodies. Cells were fixed and processed for IF (see Materials and Methods). In wild-type cells (NSY1268; top), Myo2 staining is sharp and localizes to sites of cell growth and the Ypt31/32 puncta are also mostly polarized to the buds. In cells carrying the myo2-Y1415E mutation (NSY1269, bottom), which disrupts the interaction with Ypt31 and Ypt32, at the permissive and restrictive temperatures Myo2 staining is diffuse even though the Ypt31/32 pattern is still punctate. Results shown in this figure represent at least two experiments. Bar, 5 μm.