Genome-wide Analysis of Iron-dependent Growth Reveals a Novel Yeast Gene Required for Vacuolar Acidification

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. 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). The genes that encode these proteins are under the control of the iron-sensing transcription factor Aft1p (3). In the absence of iron, Aft1p translocates from the cytosol to the nucleus and induces the transcription of at least 15 different genes that comprise the iron-regulon. Fet3p and Ftr1p must be coordinately synthesized for both proteins to be properly targeted through the secretory apparatus to the cell surface (4, 5). The activity of the multicopper oxidase Fet3p requires copper and copper loading of apoFet3p is dependent upon the expression of genes involved in copper transport. Copper enters the cell via the cell surface copper transporters Ctr1p, Ctr3p, and can be transported into the post-Golgi compartment in which apoFet3p is loaded via the copper transporter Ccc2p (1, 2, 6). Proper targeting of the Fet3p/Ftr1p complex to the cell surface also 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. Using a genomic screen, which employed an arrayed collection of homozygous diploid deletion strains, we identified genes required for growth on iron-limited medium. In particular, we characterize a novel gene that when deleted leads to defective vacuolar acidification, failure to copper load apoFet3, and consequently defective iron transport. his3⌬1/his3⌬1, ura3⌬0/ura3⌬0, leu2⌬0/leu2⌬0, lys2⌬0/ϩ, met15⌬0/ϩ) from Research Genetics was employed for the genetic screen. A 96-pin replicator was used to transfer 1 l of a cell suspension from wells in a master plate to wells in a 96-well plate containing 100 l of YPD (1% yeast extract, 2% peptone (Difco) 1.0% agarose, and 2.0% dextrose)/well. Cells were grown to saturation in a 30°C incubator (usually 48 h). The volume in the well was then brought to ϳ250 l (outside wells lost more liquid ...

The Yeast Vacuolar Proton-translocating ATPase Contains a Subunit Homologous to the Manduca sexta and Bovine e Subunits That Is Essential for Function

“…Vacuoles isolated from this deletion strain had a very low V-ATPase activity, suggesting a role for this gene in V-ATPase function. 2 Similar observations were also made by Davis-Kaplan et al (11) starting from a genomic screen for yeast mutants defective in irondependent growth (11).…”

“…2 One mutant, cwh36⌬, exhibited a very strong Vma Ϫ phenotype and no vacuolar acidification. Similar results were reported recently by Davis-Kaplan et al (11) as the result of a genome-wide screen for mutants defective in iron-dependent growth. Here we provide evidence that the cwh36⌬ mutation disrupts an open reading frame encoding a yeast homolog of the M. sexta and bovine e subunits.…”

“…YCL005W-A has two introns at the indicated positions in the ORF (11,25). The cwh36⌬ mutation cleanly deletes the CWH36 ORF, from the start to the stop codon, and replaces the ORF with the KanMX marker.…”

Functional Genomics Analysis of theSaccharomyces cerevisiaeIron Responsive Transcription Factor Aft1 Reveals Iron-Independent Functions

“…Numerous studies have systematically screened the yeast deletion mutant arrays for growth sensitivity to ironlimiting conditions (Davis- Kaplan et al 2004;Dudley et al 2005;Lesuisse et al 2005;Jo et al 2009) or iron toxicity (Jo et al 2008). As there is low concordance between these screens, we decided to directly assess the growth of the 77 deletion mutants identified in the AFT1 SL and SDL genetic network for growth on low (2.5 mm FeS0 4 ) and high (500 mm and 1000 mm FeS0 4 ) iron media ( Figure 1 and Table 2 and Table 3).…”

“…All dot assay experiments were repeated using two or three different isolates of each strain. For iron-limited YPD plates, 90 mm bathophenanthrolinedisulfonic acid disodium salt hydrate (BPS, Sigma, B1375) was used with the addition of FeSO 4 (Sigma, F8048) as previously described (Davis- Kaplan et al 2004).…”