Figure 5. The Rim15 pathway is activated after the diauxic shift where it contributes to genome stability.
(A) The level of Igo1 phosphorylation in IGO1-myc (E4974) and igo1S64A-myc (E4975) cells growing either on glucose or glycerol-lactate was determined on Phos-tag gels. Cells were shifted for 2 to 8 hr to YEPD (Glucose) or YEPGL (Glycerol/lactate) and whole cell protein extract (10 µg) analysed by SDS-PAGE or Phos-tag-PAGE. Cells grew longer in YEPGL (8 hr) so to reach the same cell density as in 4 hr in YEPD. The fraction of phospho-S64-Igo1, shown below the gel, was quantitated as in Figure 2. Swi6, loading control. (B) Cells containing non-phosphorylatable Igo1 pass START at a larger size when grown in respiratory conditions. IGO1 and igo1-S64A cells grown in YEP-Glycerol/lactate were fixed, imaged and their size at budding determined using BudJ software. Mean cell volume ± SEM, n = 100 and n = 64, respectively; Student t test. (C) Chromosome loss in igo1,2∆ mutants. Cells of the indicated genotype (E4989, E4990, E5003, E5001) containing a 61 kb circular chromosome harbouring three ARSs (RCIII-3ARS) were grown for 6–8 generations without selection and then plated to determine the fraction of cells having lost RCIII (red colonies). Cells were grown either in SC-D (left) or in YEP-Lactate/Glycerol (right). Numbers indicate loss rate /cell • generation (Mean ± SEM, n = 3, 5–8000 colonies/sample). (D) Chromosome loss in ADH1-RIM15 cells. Cells of shown genotype (E4989, E5497, E5003, E5498) were grown in SC-D for 12–15 generations and plated on sectoring medium to score red colonies having lost the RCIII chromosome, as in (C).