Screen Of Yeast Genes Related To The Subcellular Localization Of The Calcium Ion Regulator ScRch1

Calcium is an important metal ion and regulates many important cellular processes ineukaryotic cells. The disorder of calcium homeostasis can lead to many human diseases.Therefore, understanding cellular regulatory mechanisms of calcium homeostasis inSaccharomyces cerevisiae can provide important clues for human disease pathogenesis.YMR034c is a functional homolog of the C. albicans CaRCH1gene in Saccharomycescerevisiae, we named it as ScRCH1. A previous study suggests that YMR034c is associatedwith azole resistance, but there is no further report of other functions about YMR034c.Previous research in our laboratory shows that ScRch1localizes to the plasma membranein response to high levels of extracellular calcium and is involved in the regulation of thecytosolic calcium homeostasis. To find out genes involved in the subcellular localization ofScRch1, we built a plasmid pGFP33-ScRCH1which expresses the ScRch1-GFP fusionprotein. We transformed this plasmid into a total of4,534yeast diploid single-gene deletionstrains, and examined the subcellular localization of ScRch1-GFP using fluorescencemicroscope. We found that deletion mutants for94genes did not show the plasma membranelocalization for ScRch1in response to0.2mol/L CaCl2, while deletion mutants for33genesshowed the plasma membrane localization for ScRch1in the absence of0.2mol/L CaCl2.Of those127genes, we focused our further study on the genes related to celltransportation and protein folding as well as the genes coding proteins localized in thecytoplasm, the vacuole membrane and lipid particles. A total of13genes play a role incellular transport, transport structures and transport routes, while11genes encode transporterproteins and channel proteins in the vacuole. Fourteen of the127genes are involved incellular metabolisms, with2of them encoding esterase and lipidase and10of them encodingproteins involved in the synthesis of phosphoinositide, phospholipid and membrane lipid. Wemeasured the lipid droplet (LD) indexes of the127mutant strains identified. We found that23of them showed LD indexes higher than20%as compared to the wild type, and15of themshowed LD indexes lower than-20%of that in the wild type. In addition, we measured theaverage number of LD for each strain by fluorescence microscope, and found that the averagenumber of LDs was not correlated with its LD index in these strains. These results can help usto explore the cytoplasm membrane localization mechanism of ScRch1, and provide a basisfor understanding the regulation of ScRch1subcellular localization in yeast cells.