Genome-wide analysis of iron-dependent growth reveals a novel yeast gene required for vacuolar acidification.
We conducted a genome-wide screen in the budding yeast Saccharomyces cerevisiae of 4,792 homozygous diploid deletions to identify genes that function in iron metabolism. Strains unable to grow on iron-restricted medium contained deletions of genes that encode the structural components of the high affinity iron transport system (FET3, FTR1), the iron-sensing transcription factor AFT1 or genes required for the assembly of the transport system. We also identified genes that were not previously known to play a role in iron metabolism. Deletion of the gene CWH36 resulted in a severe growth defect on iron-limited medium, as well as increased sensitivity to Congo red and calcofluor white. Iron transport studies demonstrated that Deltacwh36 cells have an inability to copper load apoFet3p. Furthermore, Deltacwh36 cells demonstrated additional phenotypes including distorted vacuole morphology and altered kinetics of FM4-64 trafficking. We show that Deltacwh36 cells have a defect in vacuolar acidification through the use of the pH-sensitive dye LysoSensor Green DND-189. In Deltacwh36 cells, the vacuolar H+-ATPase is not assembled and there are reduced levels of at least one subunit of the V0 complex. The open reading frame responsible for the Deltacwh36 phenotypes is YCL005W-A. This gene contains two introns, has homologues in other Saccharomyces strains, and shows weak homology to a component of the vacuolar H+-ATPase found in organisms as diverse as insect and cow.
Division of Immunology and Cell Biology, Department of Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 84132, USA.
Sandra R
SR
Davis-Kaplan
Diane McVey
DM
Ward
Shelly L
SL
Shiflett
Jerry
J
Kaplan
D000595:Amino Acid Sequence; D002570:Ceruloplasmin / Q000235:genetics / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D017353:Gene Deletion; D005800:Genes, Fungal; D016681:Genome, Fungal; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007438:Introns; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D003300:Copper; D007501:Iron; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases