Ssd1 localizes to P-bodies. (A) Ssd1-GFP localizes to cytoplasmic puncta upon Cbk1 kinase inhibition. cbk1-as cells expressing Ssd1-GFP (FLY3249) were treated with 1NA-PP1 for 45 min and analyzed by fluorescence microscopy. Approximately 70% of 1NA-PP1–treated cbk1-as cells contain cytoplasmic puncta (n = 144). The images represent single optical sections. (B) Moderately overexpressed Ssd1 localizes to cytoplasmic puncta (P-bodies) preferentially in mother cells. ssd1Δ cells (FLY2184) containing pGPD-Ssd1-GFP (FLE1019) were observed by fluorescence microscopy. Approximately 30–60% of budded cells contained Ssd1-GFP cytoplasmic puncta (as determined by analyzing three independent cultures, n > 250). Of these, 55% contained Ssd1 puncta exclusively in the mother (M), 23% contained Ssd1 mostly in the mother (M > D) and 22% contained Ssd1 equally distributed between mother and bud (M = D); n = 152. This asymmetrical Ssd1 puncta localization mirrors the localization of Cbk1 kinase to the growing bud (Weiss et al., 2002) and may reflect the higher efficiency of Cbk1-dependent Ssd1 phosphorylation in buds. (C) Ssd1-associated cytoplasmic puncta colocalize with the P-body protein Edc3 (>63% Ssd1 puncta colocalize with Edc3). Cells in Fig. 2 B were transformed with plasmid encoding Edc3-RFP (pRP1574). The images in B and C are single optical sections captured via spinning disk confocal microscopy. Bars, 2 µm.