FIG.8.

Phenotypes of myo2-573 cells. (A) Actin cytoskeleton and mitochondrial distribution in myo2-573 cells. Wild-type cells (strain YPH499, MYO2) and myo2-573 cells (strain yTO015, myo2-573) producing mitochondrion-targeting GFP were cultured until early log phase at 25°C, fixed, and stained with rhodamine-phalloidin to visualize actin. GFP of mitochondria (bottom) and actin staining of the same cells (top) are shown. (B) Distribution of vacuoles in myo2-573 cells. Wild-type cells (strain YPH499, MYO2) and myo2-573 cells (strain yTO015, myo2-573) were cultured until early log phase at 25°C (25°C), shifted to 37°C, and incubated for 2 h (37°C). The cells were stained with FM4-64. Phase-contrast images (left) and vacuole staining (right) of each culture were shown. (C) Localization of Sec2p. Wild-type cells (strain YPH499, MYO2) and myo2-573 cells (strain yTO015, myo2-573), producing GFP-tagged Sec2p, were cultured until early log phase at 25°C, shifted to 37°C, and incubated for 2 h. GFP signals were detected at the tips of the small-budded cells. (D) Localization of Myo2-573p. The myo2-573 locus of the myo2-573 cells (strain yTO015 with the disruption of the TRP1 marker with HIS3) was replaced with myo2-573 fused to GFP for producing Myo2-573p tagged at the C terminus with GFP under the control of its own promoter (Myo2-573p-GFP [right]). The MYO2 locus of wild-type cells (strain YPH499) was replaced with the GFP-tagged version of MYO2 (Myo2-GFP [left]). GFP signals (right) and phase-contrast images (left) are shown. Bars, 5 μm. (E) Suppression of the myo2 defects by a high-dose suppressor of myo2-66. myo2-338 cells (strain yTO014, myo2-338) and myo2-573 cells (strain yTO015, myo2-573), with an indicated plasmid (vector, pYO326 for a control, MYO2; YCp50MYO2, a low-copy-number plasmid carrying MYO2; YPT11, pK008, a high-copy-number plasmid carrying YPT11; SMY1, pSMY1, a high-copy-number plasmid carrying SMY1) were streaked on an SC plate lacking uracil and incubated for 3 days at the indicated temperatures.