Visualizing Peroxisomes and Mitochondria

BY4742 yeast strain co-transformed with the plasmids encoding ScPEX11-GFP (WT (Boutouja et al., 2019 (link)) and L₃₅ to P mutant, with the endogenous promoter) and DsRed-SKL (as a peroxisomal reporter (Kuravi et al., 2006 (link))) were grown overnight (16 h) with shaking in an SD synthetic medium without uracil and histidine. Next day the precultures were diluted to 0.1 OD₆₀₀/mL and were incubated under shaking until the cell density reached 0.6–0.8 OD₆₀₀/mL. After incubation, 1–2 mL cultures were harvested and washed with water. For mitochondrial staining, 5 mL of yeast cells, grown to a density of 0.6–0.8 OD₆₀₀/mL, expressing the ScPEX11 constructs were stained with 150 nM MitoTracker™ Orange CMTMRos (Invitrogen) for 30 min with shaking in dark. Following incubation, 1–2 mL cultures were harvested and washed with water. All incubation steps were performed at 30°C. Yeast cells expressing various fluorescent proteins were directly visualized microscopically without fixation. Microscopy was performed with Carl Zeiss Microscope, using the Axiovision 4.6.3 software, and images were analyzed using Zen 3.2 (blue edition), a Carl Zeiss software. The DsRed-SKL, a peroxisomal reporter is pseudo-colored to magenta for visualization.