Generating Mutant Yeast Expression Plasmids

pYesDEST52-LHR1-HA was constructed as described elsewhere [25 (link)]. Site-directed mutagenesis was performed on pYesDEST52-LHR1-HA using the QuikChange Site-Directed Mutagenesis protocol (Agilent Technologies; Cat. # 200518); Table 1 below describes the primers used.
To generate yeast expression plasmids for the glycerol/lactate spot growth assay, pYesDEST52-yLHR1-HA WT, Y18A, H36A, Y80A or Y129A plasmids were amplified by PCR using gene specific primers (5-BglII-yLHR1: 5ʹ-GACCGCGAGATCTAAAAAAATGAACGAATT AGAAAGAAAG-3ʹ, 3-XhoI-yHA: 5ʹ-GGACTGACATCTCGAGTTAAGCATAATCA GGAACATCGTATGGGTA-3ʹ), digested with BglII and XhoI, and ligated into Yep352/PGK91-2 vector (Gift from Dr. Caroline C. Philpott).
To generate yeast expression plasmids for the Gallium (III) Protoporphyrin IX (GaPPIX) spot growth assay, the yeast codon-optimized LHR1 (pYesDEST52-yLHR1) that was previously described [25 (link)] was tagged at the c-terminus with the HA epitope using primers 5ʹ-CGTCGTATGGGTAACCTGCACAGTTTT CCTTTG-3ʹ and 5ʹ-TCCCAGACTACGCTTAATCTAGAGGGCCCTTC-3ʹ to generate pYesDEST52-yLHR1-HA. pYesDEST52-yLHR1-HA was then used to generate the mutants using the QuikChange Site-Directed Mutagenesis protocol; Table 2 below describes the primers used.