FIGURE 3:

GFP-tagged Snc1 accumulates in the ER of ypt1-1 mutant cells. (A) Colocalization of Snc1-GFP with the ER marker Hmg1 in ypt1-1, but not in wild-type or ypt31∆/32ts mutant cells. Wild-type and ypt1-1 and ypt31∆/32ts mutant cells expressing Hmg1-mCherry from its endogenous locus and Snc1-GFP from a 2 μ plasmid were visualized by live-cell fluorescence microscopy. Left to right, DIC, GFP, mCherry, merge, percentage colocalization of Snc1-GFP puncta with Hmg1-mCherry (number of GFP puncta/number of cells visualized). Arrows point to GFP puncta that colocalize with mCherry; arrowheads point to GFP puncta that do not colocalize with mCherry. In wild-type and ypt31∆/32ts mutant cells, Hmg1-mCherry localizes to rings around nuclei (Huh et al., 2003), which do not overlap with Snc1-GFP puncta. In contrast, in ypt1-1 mutant cells, about half of the Snc1-GFP puncta colocalize with Hmg1. (B) Only half of the intracellular Snc1-GFP is in endosomes of ypt1-1 mutant cells. Endosomes of cells expressing Snc1-GFP, as in Figure 1A, were labeled with FM4-64 and chased for 5 min. Cells were visualized by live-cell microscopy for GFP and FM4-64. Left to right, DIC, GFP, FM4-64, merge, and percentage colocalization of GFP with FM4-64 (number of GFP puncta/number of cells visualized). Arrows point to GFP puncta that colocalize with FM4-64; arrowheads point to GFP puncta that do not colocalize with FM4-64. Whereas all intracellular Snc1-GFP puncta overlap with endosomes in wild-type and ypt31∆/32ts mutant cells, only half localize to endosomes in ypt1-1 mutant cells. (C) The nuclear pore marker Nup60-mCherry colocalizes with intracellular GFP-Snc1. Wild-type and ypt1-1 mutant cells expressing endogenously tagged Nup60-mCherry and GFP-Snc1 from a 2 μ plasmid were visualized by live-cell microscopy. Left to right, DIC, GFP, mCherry, merge, percentage of cells with intracellular aberrant Nup60-mCherry (N, number of cells visualized), and percentage colocalization (percentage of cells in which intracellular GFP-Snc1 colocalizes with Nup60-mCherry). Arrows point to GFP puncta that colocalize with mCherry. In wild-type cells, Nup60-mCherry localizes to rings around nuclei (Huh et al., 2003). Almost all ypt1-1 mutant cells (96%) contain aberrant structures other than the rings in which Nup60-mCherry colocalizes with GFP-Snc1. Bars, 5 μm. Results represent at least two independent experiments.