Fig. 1.

Dual luciferase assay for monitoring autophagy in budding yeast. A) Shuttle vectors pPM5 and pPM10 were designed with Renilla luciferase and firefly luciferase gene respectively under the fatty acid driven promoter for Peroxisomal thiolase gene (POT1). Renilla luciferase was cloned without any signaling sequence whereas firefly luciferase was tagged with three amino acid long Peroxisomal Targetting sequence (PTS-1), SKL at its N-terminal. This directs the firefly luciferase gene to the peroxisomes. The principle of the assay involves simultaneously turning on the expression of firefly and Renilla luciferase during peroxisome biogenesis and then following their degradation via autophagy under starvation conditions. B) Fluorescence microscopy showing localization of firefly luciferase with the peroxisomal resident protein (Pot1-GFP). Firefly luciferase with N-terminal signal peptide colocalized with the peroxisomal marker whereas firefly luciferase without the signal peptide remained cytosolic. C) Immunolocalization of Renilla luciferase in the cytosol. D) Degradation of firefly luciferase protein under autophagy inducing conditions (nitrogen starvation) in wild type and autophagy mutant (Δatg1) cells. E) Quantification of decay in firefly luciferase levels showing the degradation is autophagy dependent. F) and G) Dual luciferase assay for monitoring autophagy in wild type and Δatg1 strains respectively using firefly and Renilla luciferase as markers for following rates of selective and general autophagy. H) Conventional autophagy assays for degradation of peroxisomes in wild type and autophagy mutant using fluorescence microscopy. Wild type cells when moved to starvation conditions led to degradation of peroxisomes, shown here with the diffused GFP signal inside the vacuole. Autophagy mutant strain on the other hand did not show any diffused GFP inside the vacuole and intact peroxisomes were observed in the cytosol. I) Immunoblotting showing degradation of peroxisomal protein Pot1-GFP through autophagy. Free-GFP was observed in wild type cells but not in autophagy mutant where only the fusion protein was observed. J) Quantification of Immunoblot for Pot1-GFP processing assay (pexophagy assay).