Fractionation and Analysis of Yeast Polyribosomes

Fractionation of polyribosomes was done essentially as described before (9 (link),10 (link)) using 10–50% (17 000 rpm., 18 h) and/or 10–30% (20 000 rpm., 18 h) sucrose gradients and a Beckman SW32.1 rotor. All procedures were performed at 4°C. Yeast cells from 50 ml of log phase culture were pelleted, treated for 10 min with 100 μg/ml cycloheximide and repelleted. Cell extracts were made by glass bead cell disruption (3–5 cycles of 1 min each), with intermittent cooling on ice. The following buffer was used: 100 mM KCl, 2.5 mM magnesium acetate, 20 mM HEPES·KOH, pH 7.4, 14.4 mM β-mercaptoethanol, 100 μg/ml cycloheximide. Cell debris was removed by centrifugation at 7000 rpm for 8 min and polyribosomes were resolved by sucrose density gradient centrifugation as indicated. Gradients were collected using the ISCO Programmable Density Gradient System with continuous monitoring at 254 nm using an ISCO UA-6 absorbance detector. Analysis of ratios of 80S monosomes to polyribosomes was done essentially as before (10 (link)). Fractionation of cell extracts using formaldehyde cross-linking was done as described by Nielsen et al. (18 (link)). For western blotting, proteins collected from sucrose gradient fractions were solubilized in sodium dodecyl sulfate (SDS) polyacrylamide sample buffer for 10 min at 95°C, chilled on ice for 5 min and loaded onto polyacrylamide gel.