Yeast Strain Construction and Genetic Manipulation

Yeast strains used and constructed in this study are listed in Supplemental Table S1 and were grown at 30°C on YEPD (2% dextrose, 2% peptone, 1% yeast extract) except where noted. Transformations of PCR products and plasmids into yeast cells were carried out using the lithium acetate method according to Ito et al. (1983) (link). Yeast strains in which the ULM of Hsh155 is mutated (Hsh155^RW-DA) were constructed by CRISPR/Cas9-mediated editing of the endogenous HSH155 using a modified approach from that described (DiCarlo et al. 2013 (link)) (see below). The CUS2 ORF was deleted from start to stop codon by transformation and integration of a natNT2 PCR product with ends homologous to sequences flanking the CUS2 ORF and conferring resistance to 100 µg/mL nourseothricin (GoldBio) as described (Janke et al. 2004 (link)). Strains expressing C-terminal Lea1-TAP were generated by PCR of sequences encoding the TAP-tag and the kanMX4 selectable marker, transformation, and selection on plates supplemented with 200 µg/mL G418 (Sigma-Aldrich) as described in Janke et al. (2004) (link) and Rigaut et al. (1999) (link).