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. Author manuscript; available in PMC: 2019 May 7.
Published in final edited form as: Dev Cell. 2018 May 7;45(3):392–405.e6. doi: 10.1016/j.devcel.2018.04.001

Figure 2. Phosphorylation-driven clearance of Rim4 assemblies is required for timely meiotic progression.

Figure 2

Left panel: wild type, right panel: RIM4-47A

(A) Diagrams of Rim4. The region mutated in the RIM4-47A allele is bracketed. This region harbors 47 serine and threonine residues mutated to alanine.

(B – D) RIM4-47A cells exhibit defective CLB3 translation, failure to clear SDS-resistant assemblies, and delayed meiotic progression. Strains harboring pGAL-NDT80, GAL4.ER, CLB3-3HA, and either RIM4-3V5 (wild type, B48) or RIM4-47A-3V5 (A38075) were induced to sporulate 30°C. After 6 ho urs, cells were released from the G2 arrest. (B) Rim4-3V5, Clb3-3HA, and Pgk1 (loading control) protein and CLB3 mRNA and rRNA (loading control) levels were determined at the indicated times following β-estradiol addition. (C) Rim4’s ability to form SDS-resistant amyloid-like assemblies was analyzed by SDD-AGE as in (Halfmann and Lindquist, 2008) with minor modifications (see experimental procedures). (D) The percentage (n = 100 cells for each time point) of metaphase I, anaphase I, metaphase II, and anaphase II cells was determined by tubulin IF and DAPI staining.