pGAL-Ssd1-9A-GFP localizes to P-bodies in otherwise unstressed cells and Ssd1-9D localizes to the cytoplasm, bud cortex, and bud neck. (A) ssd1Δ cells were transformed with plasmids encoding galactose-inducible Ssd1-9A-GFP and Dcp2-RFP or Lsm1-RFP (P-body markers). Cells were observed by spinning disk confocal microscopy after 1–2 h of galactose induction. Ssd1-9A-GFP colocalizes with Dcp2-RFP and Lsm1-RFP in every cell that has detectable Ssd1-9A-GFP. All Ssd1-9A-GFP puncta colocalize with P-body proteins (n = 15 cells). Over the same time course, galactose induction did not cause wild-type Ssd1-GFP or phosphomimetic Ssd1-9D-GFP (from GAL expression vectors) to associate with cytoplasmic puncta (not depicted). Galactose addition did not cause P-bodies to aggregate in ssd1Δ or wild-type cells (not depicted). Each image represents a single optical section. (B) ssd1Δ cells expressing Ssd1-9D-GFP (from plasmid FLE1090) were observed by fluorescence microscopy. The arrowhead points to enriched Ssd1 localization in small buds. Ssd1-9D was rarely detectable (<1% cells) in P-bodies of log phase cells, in contrast to GPD-driven wild-type Ssd1 (Fig. 2 B). Ssd1-9D-GFP (expressed via the GPD promoter) localizes to the cortex in ∼54% (n = 77) of small budded cells and the bud neck in ∼31% (n = 51) of large budded cells. We obtain similar results when Ssd1-9D-GFP is expressed under the control of its physiological promoter. Bars 2 µm.