Yeast genetics (strain construction, FRB system, pESCs, integration/replacement constructs)

Yeast strains were constructed in the S288C or W303 (SCF strains) background. A list of all yeast strains used in this study can be found in Table 3, a list of all vectors used for generation of novel yeast strains can be found in Table 2. Yeast strain generation and methods were performed by standard procedures.

The anchor-away approach for characterization of Ame1 in SCF mutants or wild-type strain was performed as described (Haruki et al., 2008) using the ribosomal RPL13-FKBP12 anchor. Final rapamycin concentration in plates or liquid media was 1 μg/ml. For protein stability assays, cells were treated with rapamycin for 180 min, followed by the addition of cycloheximide (CHX) with a final concentration of 50 µg/ml. Serial twofold dilutions of overnight cultures were prepared on 96-well plates in minimal medium starting from OD₆₀₀ of 0.4 for anchor-away approach or 0.5 for overexpression approach. The dilutions were spotted on YPD medium with and without rapamycin or on minimal medium with either glucose or raffinose + galactose (2% each) and grown at 30°C for 2–3 days. To confirm phenotypes observed in the serial dilution assays for Ame1 mutants, Ame1 hemizygous deletion strains were used to introduce Ame1 wild-type or phospho-mutants at an exogenous locus before haploid spores were produced. Pds1-13xMyc was integrated exogenously into haploid strains for cell cycle experiments. For overexpression of proteins, pESC two-micron plasmids were transformed into haploid wild-type or SCF mutant strains without integration into the genome and clone pools were used for further analyses. Selective pressure was used for maintenance of the plasmids. Expression of integrated proteins was checked for all created yeast strains by protein extraction from yeast (Kushnirov, 2000) and western blotting against the respective tags of individual proteins (see Key resources table Table 2).