Yeast lipid droplet isolation
Yeast membrane fractions highly enriched for lipid droplets were
prepared essentially as described in (Ding et al. , 2013), with
minor modifications. Briefly, 25 ml yeast strains harboring pGO36-Erg6
(ura3:LEU2 ) and either the pYES2 vector control or the
galactose-inducible pYES2-PE17-mRuby2 were grown to saturation at 30°C
in selective media. These cultures were then used to inoculate 800 ml
fresh selective medium containing 1 µM β-estradiol to induce the
expression of PE17 and grown for 16h at 30°C. Cells were harvested via
centrifugation and washed twice with 30 ml PBS buffer. Cell pellets were
suspended in 7 ml Buffer A (20 mM tricine, 250 mM sucrose, pH = 7.8)
containing EDTA-free protease inhibitor cocktail, and left on ice 20
min. Suspended cells were lysed in a OneShot cell disruptor (Constant
Systems) with three passes at 22000psi (~1500 bar), and
cell debris was cleared via centrifugation (3000 x g , 10 min). 10
ml resultant supernatants were transferred to a SW41 ultracentrifuge
tubes and overlayed with Buffer B (20 mM HEPES, 100 mM KCl, 2 mM
MgCl2, pH = 7.4). Tubes were centrifuged (10500 xg , 4°C, 1 h), then immediately recentrifuged (185000 x g ,
4°C, 1h). Lipid droplets were removed from the top layer, then
centrifuged (20000 x g , 4°C, 5 min) and washed 3 times in Buffer
B.