Abstract
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.
Highlights
The ability of yeast to grow on iron-limited medium requires the activity of the high affinity iron transport system
Yeast that have a functional high affinity iron transport system can grow on low iron medium, while mutations in either the structural components of the transport system or in the assembly of the transport system result in impaired growth
The validity of the screen was shown by the identification of deletion strains expected to grow poorly on low iron (Table I) including: deletion of the genes that encode the cell surface high affinity iron transport system (FET3 and FTR1), the iron-sensing transcription factor AFT1, genes required for copper assembly of the multicopper oxidase Fet3p (CTR1, CCC2, GEF1), and genes required for vesicular acidification (GEF2 known as VMA3/CUP5)
Summary
The ability of yeast to grow on iron-limited medium requires the activity of the high affinity iron transport system. This system is comprised of two plasma membrane proteins, the multicopper oxidase Fet3p and the transmembrane permease Ftr1p [1, 2]. Proper targeting of the Fet3p/Ftr1p complex to the cell surface requires genes involved in vesicular traffic. In the absence of proper copper homeostasis or appropriate vesicular trafficking, an inactive apoFet3/Ftr1p complex is translocated to the cell surface [7, 8], and cells are unable to grow on low iron medium. To identify genes required for proper targeting/expression of Fet3p, we took advantage of the low iron growth phenotype of cells lacking a functional Fet3p. We characterize a novel gene that when deleted leads to defective vacuolar acidification, failure to copper load apoFet, and defective iron transport
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