The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth

Greene, Jonathan; Brown, Nathaniel H.; DiDomenico, Beth; Kaplan, Jerry; Eide, David J.

doi:10.1007/bf00279896

Cited by 114 publications

(98 citation statements)

References 44 publications

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“…Analysis of mutants unable to grow on low iron led to the discovery of the structural components of the high affinity iron transport system as well as genes required for copper transport (29), chloride transport (30), and vesicular traffic (8,31). These genes were identified through genetic screens in which mutagenized cells were selected for their inability to grow on either low iron (18), glycerol-ethanol media (30) or for the induction of iron-regulated genes (32).…”

Section: Discussionmentioning

confidence: 99%

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

Davis‐Kaplan

Ward

Shiflett

et al. 2004

Journal of Biological Chemistry

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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 ...

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Section: Discussionmentioning

confidence: 99%

“…These genes were identified through genetic screens in which mutagenized cells were selected for their inability to grow on either low iron (18), glycerol-ethanol media (30) or for the induction of iron-regulated genes (32). We have extended these studies using a genome-wide analysis of a homozygous diploid deletion collection.…”

Section: Discussionmentioning

confidence: 99%

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

Davis‐Kaplan

Ward

Shiflett

et al. 2004

Journal of Biological Chemistry

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“…The functional complementation by AtClC-c and -d (12, 32) of growth phenotypes of a yeast strain deleted for the single yeast CLC Gef1 (33) suggested that these plant CLC proteins function in anion transport but could not reveal details of their biophysical properties. We report here the first functional expression of a plant CLC in animal cells.…”

Section: /Hmentioning

confidence: 99%

“…A more definitive assignment of their physiological roles, however, is not yet possible. In this respect, it is interesting to note that only AtClC-c and AtClC-d were reported to complement (12,32) growth phenotypes of a Saccharomyces cerevisiae strain deleted for the single yeast CLC (ScClC or Gef) (33). Whether this is related to the fact that AtClC-c and -d, just like ScClC, carry serine in their signature sequence and hence probably prefer chloride over nitrate remains unclear.…”

Section: /Hmentioning

confidence: 99%

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Bergsdorf

Zdebik

Jentsch

2009

Journal of Biological Chemistry

108

127

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CLC proteins are found in all phyla from bacteria to humans and either mediate electrogenic anion/proton exchange or function as chloride channels (1). In mammals, the roles of plasma membrane CLC Cl Ϫ channels include transepithelial transport (2-5) and control of muscle excitability (6), whereas vesicular CLC exchangers may facilitate endocytosis (7) and lysosomal function (8 -10) by electrically shunting vesicular proton pump currents (11). In the plant Arabidopsis thaliana, there are seven CLC isoforms (AtClC-a-AtClC-g) 2 (12-15), which may mostly reside in intracellular membranes. AtClC-a uses the pH gradient across the vacuolar membrane to transport the nutrient nitrate into that organelle (16). This secondary active transport requires a tightly coupled NO 3 Ϫ /H ϩ exchange. Astonishingly, however, mammalian ClC-4 and -5 and bacterial EcClC-1 (one of the two CLC isoforms in Escherichia coli) display tightly coupled Cl Ϫ /H ϩ exchange, but anion flux is largely uncoupled from H ϩ when NO 3 Ϫ is transported (17-21). The lack of appropriate expression systems for plant CLC transporters (12) has so far impeded structure-function analysis that may shed light on the ability of AtClC-a to perform efficient NO 3 Ϫ /H ϩ exchange. This dearth of data contrasts with the extensive mutagenesis work performed with CLC proteins from animals and bacteria.The crystal structure of bacterial CLC homologues (22, 23) and the investigation of mutants (17, 19 -21, 24 -29) have yielded important insights into their structure and function. CLC proteins form dimers with two largely independent permeation pathways (22,25,30,31). Each of the monomers displays two anion binding sites (22). A third binding site is observed when a certain key glutamate residue, which is located halfway in the permeation pathway of almost all CLC proteins, is mutated to alanine (23). Mutating this gating glutamate in CLC Cl Ϫ channels strongly affects or even completely suppresses single pore gating (23), whereas CLC exchangers are transformed by such mutations into pure anion conductances that are not coupled to proton transport (17,19,20). Another key glutamate, located at the cytoplasmic surface of the CLC monomer, seems to be a hallmark of CLC anion/proton exchangers. Mutating this proton glutamate to nontitratable amino acids uncouples anion transport from protons in the bacterial EcClC-1 protein (27) but seems to abolish transport altogether in mammalian . In those latter proteins, anion transport could be restored by additionally introducing an uncoupling mutation at the gating glutamate (21).The functional complementation by 32) 2 The abbreviations used are: AtCIC-n, member n of the CLC family of Cl Ϫ channels and transporters in the plant Arabidopsis thaliana; CIC-n, member n of the CLC family of chloride channel and transporters (in animals); pH i , intracellular pH; pH 0 , extracellular pH; I(NO 3 Ϫ ) and I(Cl Ϫ ), current in the presence of NO 3 Ϫ and Cl Ϫ , respectively, which in CLC exchangers also involves an H ϩ component; WT, wild-type...

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“…The expression cloning of the voltage-gated chloride channel C1C-0 from the electric organ of the marine ray Torpedo marmorata [1] led to the subsequent discovery of a large gene family of related proteins [2], which even includes members from Saccharomyces cerevisiae [3] and Escherichia coli [4]. In a single mammalian species, seven different C1C genes have been described so far [5][6][7][8][9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

ClC‐6 and ClC‐7 are two novel broadly expressed members of the CLC chloride channel family

1995

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We cloned two novel members of the CLC chloride channel family from rat and human brain. CIC-6 is a 97-kDa protein, and CIC-7 a 89-kDa protein roughly 45% identical with CIC-6. Together they define a new branch of this gene family. Both genes are very broadly expressed, e.g. in brain, testes, muscle and kidney. In mouse embryos, both genes are expressed as early as day 7. While the human gene for CIC-6 is located on human chromosome lp36 and shares this region with hCIC-Ka and hCIC-Kb, CIC-7 is on 16p13. CIC-6 has a highly conserved glycosylation site between transmembrane domains D8 and D9, while CIC-7 is the only known eukaryotic CIC protein which lacks this site. Hydropathy analysis indicates that domain D4 cannot serve as a transmembrane domain. Both CIC-6 and CIC-7 cannot be expressed as chloride channels in Xenopus oocytes, either singly or in combination.

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The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth

Cited by 114 publications

References 44 publications

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

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

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

ClC‐6 and ClC‐7 are two novel broadly expressed members of the CLC chloride channel family

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