Yeast strains and plasmids

Yeast Strain BY4741 was used for all wildtype experiments. The Vma2 knockout was obtained from the Euroscarf (MATa) haploid yeast knockout collection. Yeast strains containing plasmids expressing fluorescent sensors were created using a lithium acetate transformation. Yeast strains were grown in 2% glucose minimal media supplemented with –Leucine 100X amino acid dropout in a shaking 30 C incubator.

All plasmid cloning was performed in BW25113 E. coli before transformation into yeast. E. coli codon optimized SEP was PCR amplified for insertion into indicated plasmids. Ratiometric pHluorin, pHuji, and mRuby2 were constructed as a gBlock (IDT) using a Saccharomyces cerevisiae codon optimization. Mitochondrial matrix targeting was achieved by fusing the Su9 targeting sequence to the N-terminus of SEP-mRuby. Sensors were cloned into a 2-micron or centromeric plasmid (see Supplementary Table). All plasmids created for this publication will be available for distribution through Addgene.