Genetic suppressor screens

All screens were performed by growing independent cultures overnight in YNB + 2% glucose medium, followed by plating onto toxicity-inducing medium (YNB + 2% glucose and 2% galactose for GAL1/2, YNB + 2% glucose, and 2% fructose for yrKHK). For each independent culture, ∼10⁷ cells were spread onto a single plate then incubated for 3–4 d at 30°C. Suppression due to mutations affecting the toxicity-causing genes was ruled out as described under Results. For the two suppressors identified using classic genetic screens, both were backcrossed to produce a pool of suppressing and nonsuppressing segregants. Pools were combined then sequenced in an Illumina HiSeq 2500, aligned to a reference S. cerevisiae genome using SAMtools (Li et al., 2009 ), and potential variants were identified using freebayes (Garrison and Marth, 2012 ). All potential variants were manually examined in Integrative Genomics Viewer (Robinson et al., 2011 ; Thorvaldsdóttir et al., 2013 ).

A diploid strain homozygous for overexpressed GAL1 and GAL2, and for leu2Δ0, was used for the high-copy suppressor screen. This strain was transformed with the LEU2⁺, barcoded MoBY 2.0 plasmid library (a kind gift from the Charlie Boone lab, University of Toronto) (Magtanong et al., 2011 ). Approximately 32,000 independent transformants were pooled together, and ∼200,000 cells from this pool were plated onto each of 15 YNB + 2% glucose and 2% galactose plates. All suppressor colonies were then pooled together for plasmid extraction. Plasmid barcodes were amplified using PCR on the extracted population of plasmids. The barcodes were then sequenced in an Illumina HiSeq 2500, and then barcodes were mapped to genes as previously described (Gibney et al., 2013 ). The 10 most abundant genes were tested for suppression by independently cloning the identified genes into a different high-copy plasmid (p425GPD).