Golgi Localization and Functionally Important Domains in the NH2 and COOH Terminus of the Yeast CLC Putative Chloride Channel Gef1p

Schwappach, Blanche; Stobrawa, Sandra M.; Hechenberger, Mirko; Steinmeyer, Klaus; Jentsch, Thomas J.

doi:10.1074/jbc.273.24.15110

Cited by 122 publications

(109 citation statements)

References 63 publications

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“…Both Gef1p and Ccc2p are required for copper loading of apoFet3p (26). Examination of wild-type and ⌬cwh36 cells transformed with a GFP-tagged Gef1p showed punctate fluorescence limited to small vesicles (data not shown), similar to published studies (25,27). This result suggests that the defect in copper loading of apoFet3p cannot be ascribed to the mislocalization of Gef1p.…”

Section: Fig 4 Iron Transport and Fet3p Immunofluorescence Studies supporting

confidence: 74%

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: Fig 4 Iron Transport and Fet3p Immunofluorescence Studies supporting

confidence: 74%

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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“…Multiple partial carboxyl-terminal truncations and deletions constitutively locked hClC-2 in a closed conformation, without any effect on surface membrane insertion (50). For CLC anion/ proton antiporters that normally reside in intracellular cell organelles crucial roles for correct trafficking and sorting have been assigned to the carboxyl terminus (52)(53)(54)(55). The chloride/ proton antiporter hClC-7 is the only other member of the CLC family with an obligatory subunit, Ostm1, which is needed for proper function and subcellular localization (56).…”

Section: Discussionmentioning

confidence: 99%

Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking

Stölting

Bungert-Plümke

Franzen

et al. 2015

Journal of Biological Chemistry

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Background: Carboxyl-terminal truncations of hClC-Kb cause Bartter syndrome. Results: hClC-Kb channels lacking the carboxyl terminus still associate with barttin, but are neither stabilized in the membrane nor activated. Conclusion: Truncated hClC-Kb channels are not regulated by barttin. Significance: Elucidating the role of the carboxyl terminus of hClC-Kb significantly improves our understanding of CLC-K regulation by barttin.

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“…In this circumstance, it is likely that eliminating CFTR has no effect on pH G , because other K ϩ and Cl Ϫ channels can provide sufficient counterion conductance to ensure that membrane potential in the Golgi is small and therefore has little effect on H ϩ pumps or leaks. Two other studies have provided evidence for the presence of Cl Ϫ channels in the Golgi: Nordeen et al (35) found an anion channel in enriched Golgi fractions, and Schwappach et al (46) found that Gef1p (the only yeast ClC Cl Ϫ channel) localizes to the Golgi. Additionally, elimination of Gef1p from yeast does result in altered cation homeostasis (17) but does not effect Kar2p secretion or glycosylation of invertase, both dependent on an acidic Golgi (46).…”

Section: Cftr Counterion Conductance and Membrane Potential In Detementioning

confidence: 99%

Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells

Chandy¹,

Grabe

Moore³

et al. 2001

American Journal of Physiology-Cell Physiology

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Work addressing whether cystic fibrosis transmembrane conductance regulator (CFTR) plays a role in regulating organelle pH has remained inconclusive. We engineered a pH-sensitive excitation ratiometric green fluorescent protein (pHERP) and targeted it to the Golgi with sialyltransferase (ST). As determined by ratiometric imaging of cells expressing ST-pHERP, Golgi pH (pHG) of HeLa cells was 6.4, while pHG of mutant (ΔF508) and wild-type CFTR-expressing (WT-CFTR) respiratory epithelia were 6.7–7.0. Comparison of genetically matched ΔF508 and WT-CFTR cells showed that the absence of CFTR statistically increased Golgi acidity by 0.2 pH units, though this small difference was unlikely to be physiologically important. Golgi pH was maintained by a H+ vacuolar (V)-ATPase countered by a H+leak, which was unaffected by CFTR. To estimate Golgi proton permeability ( P H+ ), we modeled transient changes in pHG induced by inhibiting the V-ATPase and by acidifying the cytosol. This analysis required knowing Golgi buffer capacity, which was pH dependent. Our in vivo estimate is that Golgi P H+ = 7.5 × 10−4 cm/s when pHG = 6.5, and surprisingly, P H+ decreased as pHG decreased.

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Golgi Localization and Functionally Important Domains in the NH2 and COOH Terminus of the Yeast CLC Putative Chloride Channel Gef1p

Cited by 122 publications

References 63 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

Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking

Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells

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