Figure 4. Rie1 is associated with the... | Rockefeller University Press

Figure 4.

$Rie1 is associated with the translational machinery and positively regulates IME1. (A–C) Strains harboring RIE1-3V5 (B3114) were induced to sporulate at 30°C and samples were taken either without (A) or with (B) light crosslinking (1% formaldehyde, 15 min on ice). Lysates were fractionated on 10–50% sucrose density gradients with continuous monitoring at 260 nm. Rie1 and Pgk1 (monomeric control) protein levels were determined in each fraction by immunoblot. Positions of the 40S, 60S, 80S, and polysomal ribosome peaks are indicated. (C) Progression through pre-meiotic S-phase was determined by flow cytometry. Biological replicates = 3. (D) Haploid strains harboring pGAL-3HA-IME1, GAL4.ER, and either wild type RIE1 (B2722, blue) or rie1∆ (B2719, red) were diluted to 0.5 OD600 in SDC. IME1 was induced by the addition of β-estradiol at 0.5 h. Ime1 and Pgk1 (loading) protein levels were determined at the indicated times. Biological replicates = 3. Source data are available for this figure: SourceData F4.$

Rie1 is associated with the translational machinery and positively regulates IME1. (A–C) Strains harboring RIE1-3V5 (B3114) were induced to sporulate at 30°C and samples were taken either without (A) or with (B) light crosslinking (1% formaldehyde, 15 min on ice). Lysates were fractionated on 10–50% sucrose density gradients with continuous monitoring at 260 nm. Rie1 and Pgk1 (monomeric control) protein levels were determined in each fraction by immunoblot. Positions of the 40S, 60S, 80S, and polysomal ribosome peaks are indicated. (C) Progression through pre-meiotic S-phase was determined by flow cytometry. Biological replicates = 3. (D) Haploid strains harboring pGAL-3HA-IME1, GAL4.ER, and either wild type RIE1 (B2722, blue) or rie1∆ (B2719, red) were diluted to 0.5 OD₆₀₀ in SDC. IME1 was induced by the addition of β-estradiol at 0.5 h. Ime1 and Pgk1 (loading) protein levels were determined at the indicated times. Biological replicates = 3. Source data are available for this figure: SourceData F4.