Pex14-GFP containing PPVs originate in the ER. (A) Fluorescence microscopy (FM) images of pex3atg1Δ cells expressing endogenously tagged Pex14-GFP and Sec63-mCherry expressed from a plasmid. Arrows indicate Pex14-GFP puncta that colocalize with the ER (a), do not colocalize with the ER (b), or associate with the ER (c). Stacks of five images with a step of size of 0.25 µm were taken and deconvolved; images from a single plane are shown. Bar, 3 µm. (B) Quantification of the experiment in A; colocalize = (a) + (c) and do not colocalize = (b). At least 50 Pex14-GFP punctae were counted for each strain (mean ± SE; n = 3; **, P < 0.05). (C) Immunogold EM images (I–III) of pex3atg1Δ cells expressing endogenously tagged Pex14-GFP using anti-GFP antibodies. Ia and IIa are cartoon depictions of I and II, respectively. Red arrowheads indicate the gold particle; green arrows, cell wall (CW); blue arrows, plasma membrane (PM); yellow arrows, ER. (D) Time-lapse FM images of pex3atg1Δ cells expressing ss-RFP-HDEL and GFP-Pex14 under the GAL1 promoter. Cells were precultured in a medium with 2% raffinose, induced with 2% galactose for 15 min, shifted back to a medium with only 2% raffinose, and cells were visualized after 15 min (time = 0). Stacks of 10 images with a step of size of 0.25 µm were taken and deconvolved. Images from the indicated plane (z) are shown. Bar, 1 µm. (E) Immuno-EM of cells from D. Cells were grown as in D for 60 min after shifting back to medium with only 2% raffinose. Red arrowheads indicate gold particle.