Yeast transformations

Yeast transformations were carried out using standard lithium acetate protocols, and the resulting strains are cataloged in Supplementary Table 2. Gene constructs derived from pYZ12-B and pYZ23 vectors were genomically integrated into the HIS3 locus and δ-sites (YARCdelta5), respectively. These vectors were first linearized with PmeI, followed by purification of the DNA fragments using the Qiagen PCR purification kit before using them for yeast transformation. Gene deletions were carried out by homologous recombination. DNA fragments containing antibiotic resistance cassettes flanked with Lox-P sites were amplified with PCR from pAG26³⁹ (containing the hygromycin resistance gene HygB-PT), pUG6⁴⁰ (containing the G418 resistance gene KanMX), or pAG36³⁹ (containing the nourseothricin resistance gene NAT1), using primers with 40 base pairs of homology to the promoter and terminator regions of the gene targeted for deletion. Antibiotic resistance markers were subsequently removed by expressing Cre recombinase from the pSH62 ({"type":"entrez-nucleotide","attrs":{"text":"AF298785","term_id":"11344881","term_text":"AF298785"}}AF298785) vector⁴¹. After transformation, cells were plated on synthetic complete (SC) drop out media depending on the autotrophy restored by the construct. In the case of antibiotic selection, cells were plated onto nonselective YPD plates for 16 hours, then replica plated onto YPD plates with 300 μg/mL hygromycin (purchased from Invitrogen), 200 μg/mL nourseothricin (purchased from WERNER BioAgents), or 200 μg/mL G418, purchased from Gibco by Life Technologies). Zeocin was used to select for δ-integration ranging from 800 to 1200 μg/mL (purchased from Thermo Fisher Scientific).

All strains with genomic integrations or gene deletions were genotyped with PCR to confirm their accuracy. We integrated constructs in the HIS3 locus or δ-sites to promote strain stability.