Fig. 2

Pah1p also forms puncta in sei1Δ cells. a Chromosomally tagged Pah1p with GFP (Pah1-GFP) in wt and sei1Δ cells (BY4741 background). Puncta were apparent in sei1Δ when cells were fixed with 2 % formaldehyde solution but not in living cells. b Pah1p-FLAG was expressed similarly in wt and sei1Δ cells (W303 background). Lanes 1–3 mid-log cells; lanes 4–6 stationary phase cells. Lanes 1,4 wt; lanes 2,5 FLAG knock-in behind PAH1 ORF in wt; lanes 3,6 FLAG knock-in behind PAH1 ORF in sei1Δ cells. Whole cell lysates were immunoblotted with anti-FLAG and anti-Zwf1p and imaged by infrared fluorescence detection method (Odyssey™ Western blots). Asterisks, non-specific bands. c Catalytically-dead Pah1p forms dots that colocalize with Opi1p-puncta. Wild-type or sei1Δ cells, both containing the Opi1-mCherry allele were transformed with plasmid encoding Pah1p(D398A D400A)-GFP. Living cells were imaged, illustrating colocalized markers (Opi1p-mCh and Pah1(D398A D400A)-GFP colored red and green, respectively, in the merged images). d Dots of Pah1p(D398A D400A)-GFP in sei1Δ Opi1-mCherry recover slowly after photobleaching. After bleaching the indicated punctum with laser, recovery is slow, as shown by time-lapse (10 s interval). e Quantification of fluorescence recovery. The intensities were normalized to the initial intensity of the each puncta before bleaching. Data represent 5 bleaching experiments, mean ± SEM