Isolation of Yeast Organelles

For preparation of crude organelles, 3 to 6 liter cultures were grown in synthetic medium containing appropriate amino acids for selection to logarithmic phase (OD₆₀₀ = 0.8–1) at 30°C. Cells were harvested and suspended in 25 ml 100 mM Tris/HCl buffer (pH 9.4) per liter of starting culture containing 10 mM DTT. The suspension was incubated for 10 min at room temperature and centrifuged at 600 × g for 5 min. Cell pellets were suspended in 25 ml lyticase buffer (0.7 M sorbitol, 0.75xYP, 0.5% glucose, 10 mM Hepes/OH, 1 mM DTT (pH 7.4)) per liter of starting culture and lyticase (10⁵ units per liter starting culture) was added. Suspensions were incubated for 30 to 45 min at 30°C and the efficiency of spheroplast formation was determined by measuring the decline of OD₆₀₀ after suspension of samples in H₂O. Spheroplasts were washed 3 times with 25 ml 2xJR buffer (0.4 M sorbitol, 100 mM KOAc, 40 mM Hepes/OH (pH 7.4), 4 mM EDTA, 2 mM DTT) per liter starting culture, suspended in 5 ml 2xJR (containing a protease inhibitor cocktail: 1 mM 4-aminobenzamidinedihydrochloride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml N-tosyl-L-phenylalanine chloromethyl ketone, and 1 μg/ml pepstatin) per liter starting culture and frozen at −80°C. Cells were thawed in iced water and disrupted by 20 strokes with a Potter-Elvehjem homogenizer. Nuclei were removed from homogenates by centrifugation 2 times at 600 × g. For differential centrifugation, 1 mg post-nuclear supernatant (PNS) fraction was used. PNS (100 μl) fractions were centrifuged at 13k × g for 5 min. The resulting supernatant was centrifuged at 100k × g for 20 min. All fractions were analyzed by SDS-PAGE and immunoblot in amounts representing the initial volume.