The yeast CLC protein counteracts vesicular acidification during iron starvation

Braun, Nikolai A.; Morgan, Bruce; Dick, Tobias P.; Schwappach, Blanche

doi:10.1242/jcs.068403

Cited by 45 publications

(61 citation statements)

References 59 publications

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“…These results have several implications. First, the pHluorin-HR-Gef1-containing compartment is only slightly acidic in wild-type cells, in agreement with previous measurements (18). Second, in contrast to the vacuole, this compartment becomes more alkaline following glucose readdition to glucose-deprived cells.…”

Section: Pma1p Remains At the Cell Surface In A Vph1⌬supporting

confidence: 91%

“…We used a new pHluorin-based reporter (pHluorin-HR-Gef1) reported by Braun et al (18) to assess pH responses in a subset of prevacuolar compartments. This reporter is targeted by fusion to Gef1, which is localized to the Golgi apparatus and other prevacuolar compartments (27,28).…”

Section: Pma1p Remains At the Cell Surface In A Vph1⌬mentioning

confidence: 99%

“…This reporter is targeted by fusion to Gef1, which is localized to the Golgi apparatus and other prevacuolar compartments (27,28). The pHluorin fusion is under control of the inducible MET25 promoter and complemented the growth defects of a gef1⌬ strain (18), suggesting that it was functional and that induced overexpression did not dramatically alter cellular pH homeostasis. This is consistent with previous functional studies with overexpressed wild-type GEF1 (28).…”

Section: Pma1p Remains At the Cell Surface In A Vph1⌬mentioning

confidence: 99%

“…5, the localization of pHluorin-HR-Gef1 is clearly distinct from the cytosol and the vacuole. Steady state pH measurements with this probe indicated that the compartments occupied maintained a slightly more acidic pH than the cytosol (18). We asked how the pH of these compartments responded to a brief glucose deprivation and readdition.…”

Section: Pma1p Remains At the Cell Surface In A Vph1⌬mentioning

confidence: 99%

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Consequences of Loss of Vph1 Protein-containing Vacuolar ATPases (V-ATPases) for Overall Cellular pH Homeostasis

Tarsio

Zheng

Smardon

et al. 2011

Journal of Biological Chemistry

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In yeast cells, subunit a of the vacuolar proton pump (VATPase) is encoded by two organelle-specific isoforms, VPH1 and STV1. V-ATPases containing Vph1 and Stv1 localize predominantly to the vacuole and the Golgi apparatus/endosomes, respectively. Ratiometric measurements of vacuolar pH confirm that loss of STV1 has little effect on vacuolar pH. Loss of VPH1 results in vacuolar alkalinization that is even more rapid and pronounced than in vma mutants, which lack all V-ATPase activity. Cytosolic pH responses to glucose addition in the vph1⌬ mutant are similar to those in vma mutants. The extended cytosolic acidification in these mutants arises from reduced activity of the plasma membrane proton pump, Pma1p. Pma1p is mislocalized in vma mutants but remains at the plasma membrane in both vph1⌬ and stv1⌬ mutants, suggesting multiple mechanisms for limiting Pma1 activity when organelle acidification is compromised. pH measurements in early prevacuolar compartments via a pHluorin fusion to the Golgi protein Gef1 demonstrate that pH responses of these compartments parallel cytosolic pH changes. Surprisingly, these compartments remain acidic even in the absence of V-ATPase function, possibly as a result of cytosolic acidification. These results emphasize that loss of a single subunit isoform may have effects far beyond the organelle where it resides.Vacuolar proton-translocating ATPases (V-ATPases) 3 acidify multiple organelles, including mammalian lysosomes, plant and fungal vacuoles, the Golgi apparatus, endosomes, and regulated secretory granules. Through their effects on organelle acidification, V-ATPases impact numerous cellular processes including protein sorting, macromolecular degradation, cytosolic pH and ion homeostasis, and nutrient storage and mobilization (1, 2). Consistent with these diverse roles, complete loss of V-ATPase function is lethal in most organisms. Fungi, however, can tolerate a complete loss of V-ATPase function, and Saccharomyces cerevisiae has emerged as a major model system for mechanistic studies of V-ATPases (3). Yeast mutants lacking V-ATPase activity (vma mutants) show a well defined set of Vma Ϫ growth phenotypes, including sensitivity to high extracellular pH, high Ca 2ϩ concentrations, and heavy metals (4).V-ATPases are highly conserved both at the level of individual subunit sequences and at an overall structural level. A complex of peripheral membrane subunits containing the sites of ATP hydrolysis, V 1 , is attached to an integral membrane complex, V o , containing the proton pore. In higher eukaryotes, many of the subunits are present as multiple isoforms, encoded as multiple genes and/or splice variants (5). These subunit isoforms exhibit tissue-specific expression and/or organelle-specific localization, and in some cases, impart different biochemical characteristics on V-ATPases, possibly tuning their activity to the requirements of different locales (2). Subunit a of the V o sector is present as multiple isoforms in many organisms. Humans have four different subunit a genes (...

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Section: Pma1p Remains At the Cell Surface In A Vph1⌬supporting

confidence: 91%

Section: Pma1p Remains At the Cell Surface In A Vph1⌬mentioning

confidence: 99%

Section: Pma1p Remains At the Cell Surface In A Vph1⌬mentioning

confidence: 99%

Section: Pma1p Remains At the Cell Surface In A Vph1⌬mentioning

confidence: 99%

See 2 more Smart Citations

Consequences of Loss of Vph1 Protein-containing Vacuolar ATPases (V-ATPases) for Overall Cellular pH Homeostasis

Tarsio

Zheng

Smardon

et al. 2011

Journal of Biological Chemistry

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show abstract

“…We applied SITS (4-acetamido-4=-isothiocyano-2,2=-disulfonic stilbene), an anion-channel inhibitor (51,52), which was purchased from Sigma and dissolved in water to a final stock concentration of 200 mM. To isolate protoplasts from N. benthamiana callus culture, 1 g cellulysin and 0.2 g macerase (Calbiochem) were used as described previously (53,54).…”

Section: Methodsmentioning

confidence: 99%

The GEF1 Proton-Chloride Exchanger Affects Tombusvirus Replication via Regulation of Copper Metabolism in Yeast

Sasvari

Kovalev

Nagy

2013

J Virol

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Replication of plus-strand RNA viruses [(؉)RNA viruses] is performed by viral replicases, whose function is affected by many cellular factors in infected cells. In this paper, we demonstrate a surprising role for Gef1p proton-chloride exchanger in replication of Tomato bushy stunt virus (TBSV) model (؉)RNA virus. A genetic approach revealed that Gef1p, which is the only proton-chloride exchanger in Saccharomyces cerevisiae, is required for TBSV replication in the yeast model host. We also show that the in vitro activity of the purified tombusvirus replicase from gef1⌬ yeast was low and that the in vitro assembly of the viral replicase in a cell extract was inhibited by the cytosolic fraction obtained from gef1⌬ yeast. Altogether, our data reveal that Gef1p modulates TBSV replication via regulating Cu 2؉ metabolism in the cell. This conclusion is supported by several lines of evidence, including the direct inhibitory effect of Cu 2؉ ions on the in vitro assembly of the viral replicase, on the activity of the viral RNA-dependent RNA polymerase, and an inhibitory effect of deletion of CCC2 copper pump on TBSV replication in yeast, while altered iron metabolism did not reduce TBSV replication. In addition, applying a chloride channel blocker impeded TBSV replication in Nicotiana benthamiana protoplasts or in whole plants. Overall, blocking Gef1p function seems to inhibit TBSV replication through altering Cu 2؉ ion metabolism in the cytosol, which then inhibits the normal functions of the viral replicase.

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Cell Biology and Physiology of CLC Chloride Channels and Transporters

Stauber

Weinert

Jentsch

2012

Comprehensive Physiology

134

138

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Proteins of the CLC gene family assemble to homo‐ or sometimes heterodimers and either function as Cl – channels or as Cl – /H + ‐exchangers. CLC proteins are present in all phyla. Detailed structural information is available from crystal structures of bacterial and algal CLCs. Mammals express nine CLC genes, four of which encode Cl – channels and five 2Cl – /H + ‐exchangers. Two accessory β‐subunits are known: (1) barttin and (2) Ostm1. ClC‐Ka and ClC‐Kb Cl – channels need barttin, whereas Ostm1 is required for the function of the lysosomal ClC‐7 2Cl – /H + ‐exchanger. ClC‐1, ‐2, ‐Ka and ‐Kb Cl – channels reside in the plasma membrane and function in the control of electrical excitability of muscles or neurons, in extra‐ and intracellular ion homeostasis, and in transepithelial transport. The mainly endosomal/lysosomal Cl – /H + ‐exchangers ClC‐3 to ClC‐7 may facilitate vesicular acidification by shunting currents of proton pumps and increase vesicular Cl – concentration. ClC‐3 is also present on synaptic vesicles, whereas ClC‐4 and ‐5 can reach the plasma membrane to some extent. ClC‐7/Ostm1 is coinserted with the vesicular H + ‐ATPase into the acid‐secreting ruffled border membrane of osteoclasts. Mice or humans lacking ClC‐7 or Ostm1 display osteopetrosis and lysosomal storage disease. Disruption of the endosomal ClC‐5 Cl – /H + ‐exchanger leads to proteinuria and Dent's disease. Mouse models in which ClC‐5 or ClC‐7 is converted to uncoupled Cl – conductors suggest an important role of vesicular Cl – accumulation in these pathologies. The important functions of CLC Cl – channels were also revealed by human diseases and mouse models, with phenotypes including myotonia, renal loss of salt and water, deafness, blindness, leukodystrophy, and male infertility. © 2012 American Physiological Society. Compr Physiol 2:1701‐1744, 2012.

show abstract

The yeast CLC protein counteracts vesicular acidification during iron starvation

Cited by 45 publications

References 59 publications

Consequences of Loss of Vph1 Protein-containing Vacuolar ATPases (V-ATPases) for Overall Cellular pH Homeostasis

Consequences of Loss of Vph1 Protein-containing Vacuolar ATPases (V-ATPases) for Overall Cellular pH Homeostasis

The GEF1 Proton-Chloride Exchanger Affects Tombusvirus Replication via Regulation of Copper Metabolism in Yeast

Cell Biology and Physiology of CLC Chloride Channels and Transporters

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