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1
Yamashiro, C. T., P. M. Kane, D. F. Wolczyk, R. A. Preston et T. H. Stevens. « Role of vacuolar acidification in protein sorting and zymogen activation : a genetic analysis of the yeast vacuolar proton-translocating ATPase ». Molecular and Cellular Biology 10, no 7 (juillet 1990) : 3737–49. http://dx.doi.org/10.1128/mcb.10.7.3737-3749.1990.
Texte intégralRésumé :
Vacuolar acidification has been proposed to play a key role in a number of cellular processes, including protein sorting, zymogen activation, and maintenance of intracellular pH. We investigated the significance of vacuolar acidification by cloning and mutagenizing the gene for the yeast vacuolar proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying a vat2 null allele were viable; however, these cells were severely defective for growth in medium buffered at neutral pH. Vacuoles isolated from cells bearing the vat2 null allele were completely devoid of vacuolar ATPase activity. The pH of the vacuolar lumen of cells bearing the vat2 mutation was 7.1, compared with the wild-type pH of 6.1, as determined by a flow cytometric pH assay. These results indicate that the vacuolar proton-translocating ATPase complex is essential for vacuolar acidification and that the low-pH state of the vacuole is crucial for normal growth. The vacuolar acidification-defective vat2 mutant exhibited normal zymogen activation but displayed a minor defect in vacuolar protein sorting.
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Yamashiro, C. T., P. M. Kane, D. F. Wolczyk, R. A. Preston et T. H. Stevens. « Role of vacuolar acidification in protein sorting and zymogen activation : a genetic analysis of the yeast vacuolar proton-translocating ATPase. » Molecular and Cellular Biology 10, no 7 (juillet 1990) : 3737–49. http://dx.doi.org/10.1128/mcb.10.7.3737.
Texte intégralRésumé :
Vacuolar acidification has been proposed to play a key role in a number of cellular processes, including protein sorting, zymogen activation, and maintenance of intracellular pH. We investigated the significance of vacuolar acidification by cloning and mutagenizing the gene for the yeast vacuolar proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying a vat2 null allele were viable; however, these cells were severely defective for growth in medium buffered at neutral pH. Vacuoles isolated from cells bearing the vat2 null allele were completely devoid of vacuolar ATPase activity. The pH of the vacuolar lumen of cells bearing the vat2 mutation was 7.1, compared with the wild-type pH of 6.1, as determined by a flow cytometric pH assay. These results indicate that the vacuolar proton-translocating ATPase complex is essential for vacuolar acidification and that the low-pH state of the vacuole is crucial for normal growth. The vacuolar acidification-defective vat2 mutant exhibited normal zymogen activation but displayed a minor defect in vacuolar protein sorting.
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Morano, K. A., et D. J. Klionsky. « Differential effects of compartment deacidification on the targeting of membrane and soluble proteins to the vacuole in yeast ». Journal of Cell Science 107, no 10 (1 octobre 1994) : 2813–24. http://dx.doi.org/10.1242/jcs.107.10.2813.
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Lysosomal/vacuolar protein targeting is dependent on compartment acidification. In yeast, sorting of soluble vacuolar proteins such as carboxypeptidase Y is sensitive to acute changes in vacuolar pH. In contrast, the vacuolar membrane protein alkaline phosphatase is missorted only under conditions of chronic deacidification. We have undertaken a temporal analysis to define further the relationship between compartment acidification and sorting of soluble and membrane vacuolar proteins. Depletion of either the Vma3p or Vma4p subunits of the yeast vacuolar ATPase over time resulted in loss of vacuolar ATPase activity and vacuolar acidification. A kinetic delay in processing of carboxypeptidase Y occurred concomitant with these physiological changes while transport of alkaline phosphatase remained unaffected. Carboxypeptidase S, another vacuolar hydrolase that transits through the secretory pathway as an integral membrane protein, displayed a pH sensitivity similar to that of soluble vacuolar proteins. These results indicate that compartment acidification is tightly coupled to efficient targeting of proteins to the vacuole and that there may be multiple distinct mechanisms for targeting of vacuolar membrane proteins.
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Chen, Shuliang, Maureen Tarsio, Patricia M. Kane et Miriam L. Greenberg. « Cardiolipin Mediates Cross-Talk between Mitochondria and the Vacuole ». Molecular Biology of the Cell 19, no 12 (décembre 2008) : 5047–58. http://dx.doi.org/10.1091/mbc.e08-05-0486.
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Cardiolipin (CL) is an anionic phospholipid with a dimeric structure predominantly localized in the mitochondrial inner membrane, where it is closely associated with mitochondrial function, biogenesis, and genome stability ( Daum, 1985 ; Janitor and Subik, 1993 ; Jiang et al., 2000 ; Schlame et al., 2000 ; Zhong et al., 2004 ). Previous studies have shown that yeast mutant cells lacking CL due to a disruption in CRD1, the structural gene encoding CL synthase, exhibit defective colony formation at elevated temperature even on glucose medium ( Jiang et al., 1999 ; Zhong et al., 2004 ), suggesting a role for CL in cellular processes apart from mitochondrial bioenergetics. In the current study, we present evidence that the crd1Δ mutant exhibits severe vacuolar defects, including swollen vacuole morphology and loss of vacuolar acidification, at 37°C. Moreover, vacuoles from crd1Δ show decreased vacuolar H+-ATPase activity and proton pumping, which may contribute to loss of vacuolar acidification. Deletion mutants in RTG2 and NHX1, which mediate vacuolar pH and ion homeostasis, rescue the defective colony formation phenotype of crd1Δ, strongly suggesting that the temperature sensitivity of crd1Δ is a consequence of the vacuolar defects. Our results demonstrate the existence of a novel mitochondria-vacuole signaling pathway mediated by CL synthesis.
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Raymond, C. K., I. Howald-Stevenson, C. A. Vater et T. H. Stevens. « Morphological classification of the yeast vacuolar protein sorting mutants : evidence for a prevacuolar compartment in class E vps mutants. » Molecular Biology of the Cell 3, no 12 (décembre 1992) : 1389–402. http://dx.doi.org/10.1091/mbc.3.12.1389.
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The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.
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Raymond, C. K., P. J. O'Hara, G. Eichinger, J. H. Rothman et T. H. Stevens. « Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle. » Journal of Cell Biology 111, no 3 (1 septembre 1990) : 877–92. http://dx.doi.org/10.1083/jcb.111.3.877.
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vps3 mutants of the yeast Saccharomyces cerevisiae are impaired in the sorting of newly synthesized soluble vacuolar proteins and in the acidification of the vacuole (Rothman, J. H., and T. H. Stevens. Cell. 47:1041-1051; Rothman, J. H., C. T. Yamashiro, C. K. Raymond, P. M. Kane, and T. H. Stevens. 1989. J. Cell Biol. 109:93-100). The VPS3 gene, which was cloned using a novel selection procedure, encodes a low abundance, hydrophilic protein of 117 kD that most likely resides in the cytoplasm. Yeast strains bearing a deletion of the VPS3 gene (vps3-delta 1) are viable, yet their growth rate is significantly reduced relative to wild-type cells. Temperature shift experiments with strains carrying a temperature conditional vps3 allele demonstrate that cells rapidly lose the capacity to sort the vacuolar protein carboxypeptidase Y upon loss of VPS3 function. Vacuolar morphology was examined in wild-type and vps3-delta 1 yeast strains by fluorescence microscopy. The vacuoles in wild-type yeast cells are morphologically complex, and they appear to be actively partitioned between mother cells and buds during an early phase of bud growth. Vacuolar morphology in vps3-delta 1 mutants is significantly altered from the wild-type pattern, and the vacuolar segregation process seen in wild-type strains is defective in these mutants. With the exception of a vacuolar acidification defect, the phenotypes of vps3-delta 1 strains are significantly different from those of mutants lacking the vacuolar proton-translocating ATPase. These data demonstrate that the acidification defect in vps3-delta 1 cells is not the primary cause of the pleiotropic defects in vacuolar function observed in these mutants.
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Rothman, J. H., C. T. Yamashiro, C. K. Raymond, P. M. Kane et T. H. Stevens. « Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins. » Journal of Cell Biology 109, no 1 (1 juillet 1989) : 93–100. http://dx.doi.org/10.1083/jcb.109.1.93.
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Organelle acidification plays a demonstrable role in intracellular protein processing, transport, and sorting in animal cells. We investigated the relationship between acidification and protein sorting in yeast by treating yeast cells with ammonium chloride and found that this lysosomotropic agent caused the mislocalization of a substantial fraction of the newly synthesized vacuolar (lysosomal) enzyme proteinase A (PrA) to the cell surface. We have also determined that a subset of the vpl mutants, which are deficient in sorting of vacuolar proteins (Rothman, J. H., and T. H. Stevens. 1986. Cell. 47:1041-1051; Rothman, J. H., I. Howald, and T. H. Stevens. EMBO [Eur. Mol. Biol. Organ.] J. In press), failed to accumulate the lysosomotropic fluorescent dye quinacrine within their vacuoles, mimicking the phenotype of wild-type cells treated with ammonium. The acidification defect of vpl3 and vpl6 mutants correlated with a marked deficiency in vacuolar ATPase activity, diminished levels of two immunoreactive subunits of the protontranslocating ATPase (H+-ATPase) in purified vacuolar membranes, and accumulation of the intracellular portion of PrA as the precursor species. Therefore, some of the VPL genes are required for the normal function of the yeast vacuolar H+-ATPase complex and may encode either subunits of the enzyme or components required for its assembly and targeting. Collectively, these findings implicate a critical role for acidification in vacuolar protein sorting and zymogen activation in yeast, and suggest that components of the yeast vacuolar acidification system may be identified by examining mutants defective in sorting of vacuolar proteins.
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Klionsky, D. J., H. Nelson, N. Nelson et D. S. Yaver. « Mutations in the yeast vacuolar ATPase result in the mislocalization of vacuolar proteins. » Journal of Experimental Biology 172, no 1 (1 novembre 1992) : 83–92. http://dx.doi.org/10.1242/jeb.172.1.83.
Texte intégralRésumé :
The vacuolar ATPase of the yeast Saccharomyces cerevisiae acidifies the vacuolar lumen and generates an electrochemical gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of genes (delta vat) encoding the A (69 x 10(3) M(r)), B (57 x 10(3) M(r)) or c (16 x 10(3) M(r)) subunits of the vacuolar ATPase accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. A kinetic analysis suggests that these precursor proteins accumulate in, and are secreted from, the Golgi complex or post-Golgi vesicles. In addition, subcellular fractionation shows that vacuolar hydrolase-invertase hybrid proteins are inefficiently localized to the vacuole in delta vat strains. This result suggests that the vat mutations cause a steady-state defect in vacuolar protein sorting. The vat mutations also affect the sorting of vacuolar membrane proteins. Precursor forms of alkaline phosphatase are accumulated in vat mutant cells, but to a lesser extent than is seen for the soluble vacuolar hydrolases. This finding, coupled with the insensitivity of alkaline phosphatase to the ATPase inhibitor bafilomycin A1, suggests that vacuolar membrane protein sorting is less sensitive to changes in lumenal pH when compared with the targeting of soluble vacuolar proteins. These results indicate that acidification of the vacuolar system is important for efficient sorting of soluble proteins to the vacuole.
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Banta, L. M., J. S. Robinson, D. J. Klionsky et S. D. Emr. « Organelle assembly in yeast : characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. » Journal of Cell Biology 107, no 4 (1 octobre 1988) : 1369–83. http://dx.doi.org/10.1083/jcb.107.4.1369.
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Yeast vacuole protein targeting (vpt) mutants exhibit defects in the sorting and processing of multiple vacuolar hydrolases. To evaluate the impact these vpt mutations have on the biogenesis and functioning of the lysosome-like vacuole, we have used light and electron microscopic techniques to analyze the vacuolar morphology in the mutants. These observations have permitted us to assign the vpt mutants to three distinct classes. The class A vpt mutants (26 complementation groups) contain 1-3 large vacuoles that are morphologically indistinguishable from those in the parental strain, suggesting that only a subset of the proteins destined for delivery to this compartment is mislocalized. One class A mutant (vpt13) is very sensitive to low pH and exhibits a defect in vacuole acidification. Consistent with a potential role for vacuolar pH in protein sorting, we found that bafilomycin A1, a specific inhibitor of the vacuolar ATPase, as well as the weak base ammonium acetate and the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, collapse the pH gradient across the vacuolar membrane and cause the missorting and secretion of two vacuolar hydrolases in wild-type cells. Mutants in the three class B vpt complementation groups exhibit a fragmented vacuole morphology. In these mutants, no large normal vacuoles are observed. Instead, many (20-40) smaller vacuole-like organelles accumulate. The class C vpt mutants, which constitute four complementation groups, exhibit extreme defects in vacuole biogenesis. The mutants lack any organelle resembling a normal vacuole but accumulate other organelles including vesicles, multilamellar membrane structures, and Golgi-related structures. Heterozygous class C zygotes reassemble normal vacuoles rapidly, indicating that some of the accumulated aberrant structures may be intermediates in vacuole formation. These class C mutants also exhibit sensitivity to osmotic stress, suggesting an osmoregulatory role for the vacuole. The vpt mutants should provide insights into the normal physiological role of the vacuole, as well as allowing identification of components required for vacuole protein sorting and/or vacuole assembly.
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Steele-Mortimer, Olivia, Maryse St-Louis, Martin Olivier et B. Brett Finlay. « Vacuole Acidification Is Not Required for Survival ofSalmonella enterica Serovar Typhimurium within Cultured Macrophages and Epithelial Cells ». Infection and Immunity 68, no 9 (1 septembre 2000) : 5401–4. http://dx.doi.org/10.1128/iai.68.9.5401-5404.2000.
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ABSTRACT Phagosome acidification is an important component of the microbicidal response by infected eukaryotic cells. Thus, intracellular pathogens that reside within phagosomes must either block phagosome acidification or be able to survive at low pH. In this work, we studied the effect of phagosomal acidification on the survival of intracellular Salmonella enterica serovar Typhimurium in different cell types. Bafilomycin A1, a specific inhibitor of the vacuolar proton-ATPases, was used to block acidification of salmonella-containing vacuoles. We found that in several epithelial cell lines, treatment with bafilomycin A1 had no effect on intracellular survival or replication. Furthermore, although acidification was essential for Salmonella intracellular survival in J774 cultured macrophages, as reported previously (13), it is not essential in other macrophage cell lines. These data suggest that vacuolar acidification may play a role in intracellular survival of salmonellae only under certain conditions and in specific cell types.
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Voynova, Natalia S., Carole Roubaty, Hector M. Vazquez, Shamroop K. Mallela, Christer S. Ejsing et Andreas Conzelmann. « Saccharomyces cerevisiae Is Dependent on Vesicular Traffic between the Golgi Apparatus and the Vacuole When Inositolphosphorylceramide Synthase Aur1 Is Inactivated ». Eukaryotic Cell 14, no 12 (2 octobre 2015) : 1203–16. http://dx.doi.org/10.1128/ec.00117-15.
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ABSTRACTInositolphosphorylceramide (IPC) and its mannosylated derivatives are the only complex sphingolipids of yeast. Their synthesis can be reduced by aureobasidin A (AbA), which specifically inhibits the IPC synthase Aur1. AbA reportedly, by diminishing IPC levels, causes endoplasmic reticulum (ER) stress, an increase in cytosolic calcium, reactive oxygen production, and mitochondrial damage leading to apoptosis. We found that when Aur1 is gradually depleted by transcriptional downregulation, the accumulation of ceramides becomes a major hindrance to cell survival. Overexpression of the alkaline ceramidaseYPC1rescues cells under this condition. We established hydroxylated C26fatty acids as a reliable hallmark of ceramide hydrolysis. Such hydrolysis occurs only whenYPC1is overexpressed. In contrast, overexpression ofYPC1has no beneficial effect when Aur1 is acutely repressed by AbA. A high-throughput genetic screen revealed that vesicle-mediated transport between Golgi apparatus, endosomes, and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and quinacrine uptake into vacuoles shows that AbA activates vacuolar acidification. The antioxidantN-acetylcysteine does not improve cell growth on AbA, indicating that reactive oxygen radicals induced by AbA play a minor role in its toxicity. AbA strongly induces the cell wall integrity pathway, but osmotic support does not improve the viability of wild-type cells on AbA. Altogether, the data support and refine current models of AbA-mediated cell death and add vacuolar protein transport and acidification as novel critical elements of stress resistance.
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Forgac, M. « Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases ». Journal of Experimental Biology 203, no 1 (1 janvier 2000) : 71–80. http://dx.doi.org/10.1242/jeb.203.1.71.
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The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells. This review is focused on our work on the V-ATPases of clathrin-coated vesicles and yeast vacuoles. The coated-vesicle V-ATPase undergoes trafficking to endosomes and synaptic vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively. The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane. The V-ATPases are multisubunit complexes composed of two functional domains. The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73–14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c' and c”) that is responsible for proton translocation. To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly. A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells. We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo. Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl(−) conductance that is necessary for vacuolar acidification.
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Myers, M., et M. Forgac. « Mechanism and Function of Vacuolar Acidification ». Physiology 8, no 1 (1 février 1993) : 24–29. http://dx.doi.org/10.1152/physiologyonline.1993.8.1.24.
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Vacuolar acidification plays an important role in such processes as receptor-mediated endocytosis, intracellular membrane traffic, and protein degradation. Vacuolar H+-adenosinetriphosphatases (ATPases) are responsible for this acidification.
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Bonangelino, C. J., N. L. Catlett et L. S. Weisman. « Vac7p, a novel vacuolar protein, is required for normal vacuole inheritance and morphology. » Molecular and Cellular Biology 17, no 12 (décembre 1997) : 6847–58. http://dx.doi.org/10.1128/mcb.17.12.6847.
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During cell division, the vacuole of Saccharomyces cerevisiae partitions between mother and daughter cells. A portion of the parental vacuole membrane moves into the bud, and ultimately membrane scission divides the vacuole into two separate structures. Here we characterize two yeast mutations causing defects in vacuole membrane scission, vac7-1 and vac14-1. A third mutant, afab1-2 strain, isolated in a nonrelated screen (A. Yamamoto et al., Mol. Biol. Cell 6:525-539, 1995) shares the vacuolar phenotypes of the vac7-1 and vac14-1 strains. Unlike the wild type, mutant vacuoles are not multilobed structures; in many cases, a single vacuole spans both the mother and bud, with a distinct gap in the mother-bud neck. Thus, even where the membranes are closely opposed, vacuole fission is arrested. Simply enlarging the vacuole does not produce this mutant phenotype. An additional common phenotype of these mutants is a defect in vacuole acidification; however, vacuole scission in most other vacuole acidification mutants is normal. An alteration in vacuole membrane lipids could account for both the vacuole membrane scission and acidification defects. Because a directed screen has not identified additional class III complementation groups, it is likely that all three genes are involved in a similar process. Interestingly, FAB1, was previously shown to encode a putative phosphatidylinositol-4-phosphate 5-kinase. Moreover, overexpression of FAB1 suppresses the vac14-1 mutation, which suggests that VAC14 and FAB1 act at a common step. VAC7 encodes a novel 128-kDa protein that is localized at the vacuole membrane. This location of Vac7p is consistent with its involvement in vacuole morphology and inheritance.
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Zhang, Chi, Adam Balutowski, Yilin Feng, Jorge D. Calderin et Rutilio A. Fratti. « High throughput analysis of vacuolar acidification ». Analytical Biochemistry 658 (décembre 2022) : 114927. http://dx.doi.org/10.1016/j.ab.2022.114927.
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Ruckenstuhl, Christoph, Christine Netzberger, Iryna Entfellner, Didac Carmona-Gutierrez, Thomas Kickenweiz, Slaven Stekovic, Christina Gleixner et al. « Autophagy extends lifespan via vacuolar acidification ». Microbial Cell 1, no 5 (5 mai 2014) : 160–62. http://dx.doi.org/10.15698/mic2014.05.147.
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Suriapranata, I., U. D. Epple, D. Bernreuther, M. Bredschneider, K. Sovarasteanu et M. Thumm. « The breakdown of autophagic vesicles inside the vacuole depends on Aut4p ». Journal of Cell Science 113, no 22 (15 novembre 2000) : 4025–33. http://dx.doi.org/10.1242/jcs.113.22.4025.
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Autophagy is a degradative transport pathway that delivers cytosolic proteins to the lysosome (vacuole). Cytosolic proteins appear inside the vacuole enclosed in autophagic vesicles. These autophagic vesicles are broken down in the vacuole together with their cytosolic content. The breakdown of vesicular transport intermediates is a unique feature of autophagy. We here identify Aut4p, a component essential for the disintegration of autophagic vesicles, inside the vacuole of S. cerevisiae cells. Aut4p is a putative integral membrane protein with limited homologies to permeases. Chromosomal deletion of AUT4 has no obvious influence on growth, vacuolar acidification and the activities of vacuolar proteinases. Like proteinase B-deficient cells, aut4-deleted cells show a partial reduction in total protein breakdown during nitrogen starvation. A biologically active fusion protein of Aut4p and the green fluorescent protein is visualized at the vacuolar membrane and in punctate structures attached to the vacuole.
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Kane, P. M. « Biogenesis of the yeast vacuolar H(+)-ATPase. » Journal of Experimental Biology 172, no 1 (1 novembre 1992) : 93–103. http://dx.doi.org/10.1242/jeb.172.1.93.
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Achieving an understanding of the biosynthesis, assembly and intracellular targeting of the vacuolar H(+)-ATPase is critical for understanding the distribution of acidic compartments and the regulation of organelle acidification. The assembly of the yeast vacuolar H(+)-ATPase requires the attachment of several cytoplasmically oriented, peripheral subunits (the V1 sector) to a complex of integral membrane subunits (the Vo sector) and thus is not easily described by the established mechanisms for transport of soluble or vacuolar membrane proteins to the vacuole. In order to examine the assembly of the enzyme complex, yeast mutants lacking one of the subunit genes have been constructed and the synthesis and assembly of the other subunits have been examined. In mutants lacking one subunit, the remaining ATPase subunits seem to be synthesized, but in many cases are either not assembled or not targeted to the vacuole. Immunofluorescence and subcellular fractionation experiments have revealed that deletion of one peripheral subunit prevents the other peripheral subunits, but not the integral membrane subunits, from reaching the vacuole. In contrast, the absence of one of the integral membrane subunits appears to prevent both the peripheral subunits and another integral subunit from reaching the vacuole and also results in reduced cellular levels of the other integral membrane subunit. These data suggest that transport of integral and peripheral membrane subunits to the vacuole may employ somewhat independent mechanisms and that some assembly of the V1 and Vo sectors may occur before the two sectors are joined. Current models for the assembly process and the implications for organelle acidification are discussed.
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Charoenbhakdi, Sirikarn, Thanittra Dokpikul, Thanawat Burphan, Todsapol Techo et Choowong Auesukaree. « Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses ». Applied and Environmental Microbiology 82, no 10 (18 mars 2016) : 3121–30. http://dx.doi.org/10.1128/aem.00376-16.
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ABSTRACTDuring fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H+-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2and Δvma3mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype ofvmamutants resulted from severe cytosolic acidification. Interestingly, thevmamutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol.IMPORTANCEThe yeastSaccharomyces cerevisiaehas been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic fermentation. The results revealed that straight-chain alcohols induced cytosolic and vacuolar acidification through their membrane-permeabilizing effects. Contrary to expectations, a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress, but not in the maintenance of intracellular pH, seems to be important for protecting yeast cells against ethanol stress. These findings will expand our understanding of the mechanisms of ethanol tolerance and provide promising clues for the development of ethanol-tolerant yeast strains.
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Smardon, Anne M., Heba I. Diab, Maureen Tarsio, Theodore T. Diakov, Negin Dehdar Nasab, Robert W. West et Patricia M. Kane. « The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast ». Molecular Biology of the Cell 25, no 3 (février 2014) : 356–67. http://dx.doi.org/10.1091/mbc.e13-05-0231.
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The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H+-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1∆ mutants exhibit a Vma− growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma− growth defect when combined with the rav1∆ mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma− growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1∆ mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1∆ and rav1∆vph1∆, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1∆ perturbed the normal localization of Stv1–green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma− phenotypes arise from defects in Vph1p-containing complexes caused by rav1∆, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor.
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OHKUMA, Shoji, Tomohiko SATO, Masayuki OKAMOTO, Hidekazu MATSUYA, Kunizo ARAI, Takao KATAOKA, Kazuo NAGAI et Harry H. WASSERMAN. « Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H+/Cl− symport ». Biochemical Journal 334, no 3 (15 septembre 1998) : 731–41. http://dx.doi.org/10.1042/bj3340731.
Texte intégralRésumé :
We reported previously [Kataoka, Muroi, Ohkuma, Waritani, Magae, Takatsuki, Kondo, Yamasaki and Nagai (1995) FEBS Lett. 359, 53–59] that prodigiosin 25-C (one of the red pigments of the prodigiosin group produced by micro-organisms like Streptomycesand Serratia) uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification and affected glycoprotein processing. In the present study we show that prodigiosin, metacycloprodigiosin and prodigiosin 25-C, all raise intralysosomal pH through inhibition of lysosomal acidification driven by vacuolar-type (V-)ATPase without inhibiting ATP hydrolysis in a dose-dependent manner with IC50 values of 30–120 pmol/mg of protein. The inhibition against lysosomal acidification was quick and reversible, showing kinetics of simple non-competitive (for ATP) inhibition. However, the prodigiosins neither raised the internal pH of isolated lysosomes nor showed ionophoric activity against H+ or K+ at concentrations where they strongly inhibited lysosomal acidification. They required Cl- for their acidification inhibitory activity even when driven in the presence of K+ and valinomycin, suggesting that their target is not anion (chloride) channel(s). In fact, the prodigiosins inhibited acidification of proteoliposomes devoid of anion channels that were reconstituted from lysosomal vacuolar-type (V-)ATPase and Escherichia coli phospholipids. However, they did not inhibit the formation of an inside-positive membrane potential driven by lysosomal V-ATPase. Instead, they caused quick reversal of acidified pH driven by lysosomal V-ATPase and, in acidic buffer, produced quick acidification of lysosomal pH, both only in the presence of Cl-. In addition, they induced swelling of liposomes and erythrocytes in iso-osmotic ammonium salt of chloride but not of gluconate, suggesting the promotion of Cl- entry by prodigiosins. These results suggest that prodigiosins facilitate the symport of H+ with Cl- (or exchange of OH- with Cl-) through lysosomal membranes, resulting in uncoupling of vacuolar H+-ATPase.
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Pérez-Castiñeira, José R., Agustín Hernández, Rocío Drake et Aurelio Serrano. « A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast ». Biochemical Journal 437, no 2 (28 juin 2011) : 269–78. http://dx.doi.org/10.1042/bj20110447.
Texte intégralRésumé :
V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.
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Kwon, Yun, Jinbo Shen, Myoung Hui Lee, Kyoung Rok Geem, Liwen Jiang et Inhwan Hwang. « AtCAP2 is crucial for lytic vacuole biogenesis during germination by positively regulating vacuolar protein trafficking ». Proceedings of the National Academy of Sciences 115, no 7 (29 janvier 2018) : E1675—E1683. http://dx.doi.org/10.1073/pnas.1717204115.
Texte intégralRésumé :
Protein trafficking is a fundamental mechanism of subcellular organization and contributes to organellar biogenesis. AtCAP2 is an Arabidopsis homolog of the Mesembryanthemum crystallinum calcium-dependent protein kinase 1 adaptor protein 2 (McCAP2), a member of the syntaxin superfamily. Here, we show that AtCAP2 plays an important role in the conversion to the lytic vacuole (LV) during early plant development. The AtCAP2 loss-of-function mutant atcap2-1 displayed delays in protein storage vacuole (PSV) protein degradation, PSV fusion, LV acidification, and biosynthesis of several vacuolar proteins during germination. At the mature stage, atcap2-1 plants accumulated vacuolar proteins in the prevacuolar compartment (PVC) instead of the LV. In wild-type plants, AtCAP2 localizes to the PVC as a peripheral membrane protein and in the PVC compartment recruits glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2) to the PVC. We propose that AtCAP2 contributes to LV biogenesis during early plant development by supporting the trafficking of specific proteins involved in the PSV-to-LV transition and LV acidification during early stages of plant development.
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Patenaude, Cassandra, Yongqiang Zhang, Brendan Cormack, Julia Köhler et Rajini Rao. « Essential Role for Vacuolar Acidification inCandida albicansVirulence ». Journal of Biological Chemistry 288, no 36 (24 juillet 2013) : 26256–64. http://dx.doi.org/10.1074/jbc.m113.494815.
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Martin-Orozco, Natalia, Nicolas Touret, Michael L. Zaharik, Edwin Park, Raoul Kopelman, Samuel Miller, B. Brett Finlay, Philippe Gros et Sergio Grinstein. « Visualization of Vacuolar Acidification-induced Transcription of Genes of Pathogens inside Macrophages ». Molecular Biology of the Cell 17, no 1 (janvier 2006) : 498–510. http://dx.doi.org/10.1091/mbc.e04-12-1096.
Texte intégralRésumé :
The objective of these studies was to analyze the role of the ionic environment of phagosomal vacuoles in the control of pathogens by macrophages. Digital imaging and flow cytometry were used to follow the induction of the phoP promoter of Salmonella enterica Typhimurium within live macrophages. Manipulating the Mg2+concentration within the Salmonella-containing vacuole (SCV) was without effect on the early induction of PhoPQ. Moreover, direct measurement of [Mg2+] within the SCV using nanosensor particles showed that, during this initial period of phoP activation, the concentration of the divalent cation is rapidly regulated and stabilizes around 1 mm. Extrusion of other divalent cations via the Nramp1 efflux pump was similarly ruled out as an important contributor to the activation of the regulon. By contrast, induction of PhoP was greatly attenuated when the pH gradient across the SCV membrane was dissipated. A second, more modest pH-independent component of PhoP induction was unmasked by inhibition of the vacuolar proton pump. This second component was eliminated by pretreatment of cells with IFNγ, even though the cytokine augmented the overall PhoP response. These findings demonstrate the existence of at least three separate activators of phoP transcription: resting and IFNγ-stimulated pH-sensitive components, plus a pH-independent component.
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Boutouja, Stiehm, Reidick, Mastalski, Brinkmeier, Magraoui et Platta. « Vac8 Controls Vacuolar Membrane Dynamics during Different Autophagy Pathways in Saccharomyces cerevisiae ». Cells 8, no 7 (30 juin 2019) : 661. http://dx.doi.org/10.3390/cells8070661.
Texte intégralRésumé :
The yeast vacuole is a vital organelle, which is required for the degradation of aberrant intracellular or extracellular substrates and the recycling of the resulting nutrients as newly available building blocks for the cellular metabolism. Like the plant vacuole or the mammalian lysosome, the yeast vacuole is the destination of biosynthetic trafficking pathways that transport the vacuolar enzymes required for its functions. Moreover, substrates destined for degradation, like extracellular endocytosed cargoes that are transported by endosomes/multivesicular bodies as well as intracellular substrates that are transported via different forms of autophagosomes, have the vacuole as destination. We found that non-selective bulk autophagy of cytosolic proteins as well as the selective autophagic degradation of peroxisomes (pexophagy) and ribosomes (ribophagy) was dependent on the armadillo repeat protein Vac8 in Saccharomyces cerevisiae. Moreover, we showed that pexophagy and ribophagy depended on the palmitoylation of Vac8. In contrast, we described that Vac8 was not involved in the acidification of the vacuole nor in the targeting and maturation of certain biosynthetic cargoes, like the aspartyl-protease Pep4 (PrA) and the carboxy-peptidase Y (CPY), indicating a role of Vac8 in the uptake of selected cargoes. In addition, we found that the hallmark phenotype of the vac8 strain, namely the characteristic appearance of fragmented and clustered vacuoles, depended on the growth conditions. This fusion defect observed in standard glucose medium can be complemented by the replacement with oleic acid or glycerol medium. This complementation of vacuolar morphology also partially restores the degradation of peroxisomes. In summary, we found that Vac8 controlled vacuolar morphology and activity in a context- and cargo-dependent manner.
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Wang, Jia-Gang, Chong Feng, Hai-Hong Liu, Qiang-Nan Feng, Sha Li et Yan Zhang. « AP1G mediates vacuolar acidification during synergid-controlled pollen tube reception ». Proceedings of the National Academy of Sciences 114, no 24 (30 mai 2017) : E4877—E4883. http://dx.doi.org/10.1073/pnas.1617967114.
Texte intégralRésumé :
Double fertilization in angiosperms requires the delivery of immotile sperm through pollen tubes, which enter embryo sacs to initiate synergid degeneration and to discharge. This fascinating process, called pollen tube reception, involves extensive communications between pollen tubes and synergids, within which few intracellular regulators involved have been revealed. Here, we report that vacuolar acidification in synergids mediated by AP1G and V-ATPases might be critical for pollen tube reception. Functional loss of AP1G or VHA-A, encoding the γ subunit of adaptor protein 1 or the shared component of two endomembrane V-ATPases, respectively, impaired synergid-controlled pollen tube reception and caused partial female sterility. AP1G works in parallel to the plasma membrane-associated receptor FERONIA in synergids, suggesting that synergid-mediated pollen tube reception requires proper sorting of vacuolar cargos by AP1G. Although AP1G did not mediate the targeting of V-ATPases, AP1G loss of function or the expression of AP1G-RNAi compromised vacuolar acidification mediated by V-ATPases, implying their genetic interaction. We propose that vacuolar acidification might represent a distinct cell-death mechanism specifically adopted by the plant phylum, which is critical for synergid degeneration during pollen tube reception.
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Manolson, M. F., D. Proteau et E. W. Jones. « Evidence for a conserved 95-120 kDa subunit associated with and essential for activity of V-ATPases. » Journal of Experimental Biology 172, no 1 (1 novembre 1992) : 105–12. http://dx.doi.org/10.1242/jeb.172.1.105.
Texte intégralRésumé :
Vacuoles purified from Saccharomyces cerevisiae bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. Furthermore, the vacuolar H(+)-ATPase (V-ATPase) nucleotide binding subunits were no longer associated with vacuolar membranes yet were present at wild-type levels in yeast whole-cell extracts. The VPH1 gene was cloned by screening a lambda gt11 expression library with antibodies directed against a 95 kDa vacuolar integral membrane protein and independently cloned by complementation of the vph1-1 mutation. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is required for vacuolar H(+)-ATPase assembly and vacuolar acidification but is not essential for cell viability or for targeting and maturation of vacuolar proteases. VPH1 encodes a predicted polypeptide of 840 amino acid residues (95.6 kDa) with putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with V-ATPase activity. Vph1p has 42% identity to the 116 kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, 42% identity to the TJ6 mouse immune suppressor factor, 42% identity to the Caenorhabditis elegans proton pump homologue and 54% identity to the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene.
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Swanson, Sarah J., et Russell L. Jones. « Gibberellic Acid Induces Vacuolar Acidification in Barley Aleurone ». Plant Cell 8, no 12 (décembre 1996) : 2211. http://dx.doi.org/10.2307/3870462.
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Feng, Y., et M. Forgac. « A novel mechanism for regulation of vacuolar acidification. » Journal of Biological Chemistry 267, no 28 (octobre 1992) : 19769–72. http://dx.doi.org/10.1016/s0021-9258(19)88619-2.
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Bray, Patrick G., Robert E. Howells et Stephen A. Ward. « Vacuolar acidification and chloroquine sensitivity in plasmodium falciparum ». Biochemical Pharmacology 43, no 6 (mars 1992) : 1219–27. http://dx.doi.org/10.1016/0006-2952(92)90495-5.
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Huynh, My-Hang, et Vern B. Carruthers. « Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress ». PLOS Pathogens 18, no 5 (5 mai 2022) : e1010139. http://dx.doi.org/10.1371/journal.ppat.1010139.
Texte intégralRésumé :
The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during ionomycin or zaprinast induced egress in both wild-type RH and Δplp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in Δplp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead to an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification.
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Kleinman, J. G. « Proton ATPases and urinary acidification. » Journal of the American Society of Nephrology 5, no 5 (novembre 1994) : S6. http://dx.doi.org/10.1681/asn.v55s6.
Texte intégralRésumé :
Acidification of the urine is mediated by vectorial H+ transport from cells at a number of sites in the kidney. A proton ATPase has been described that appears to mediate a significant proportion of this H+ transport. In particular, in proximal tubule and collecting duct, there is evidence both for the presence of transporter protein and for H+ transport with features that have been identified with it. This review highlights some of the unresolved questions regarding this transporter, specifically, its distribution and relationship to the vacuolar pump present in endocytotic vesicles, how physiologic control is asserted, and its role in pathophysiology. The review discusses in greater detail the issue of whether the vacuolar H+ ATPase is responsible for all of the urinary acidification and concludes that it probably is not. Specifically, compelling evidence for acidification at sites in the kidney that appear to lack this transporter is presented. In addition, the evidence for the presence in the kidney of a gastric-type H(+)-K+ ATPase is also reviewed. The evidence appears to be strong for a K(+)-stimulated ATPase that is sensitive to omeprazole and SCH 28080, the prototypical H(+)-K+ ATPase inhibitors; however, uncertainties remain because of problems of transport inhibition specificity and discordant results of molecular biologic studies.
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Oluwatosin, Yemisi E., et Patricia M. Kane. « Mutations in the Yeast KEX2 Gene Cause a Vma−-Like Phenotype : a Possible Role for the Kex2 Endoprotease in Vacuolar Acidification ». Molecular and Cellular Biology 18, no 3 (1 mars 1998) : 1534–43. http://dx.doi.org/10.1128/mcb.18.3.1534.
Texte intégralRésumé :
ABSTRACT Mutants of Saccharomyces cerevisiae that lack vacuolar proton-translocating ATPase (V-ATPase) activity show a well-defined set of Vma− (stands for vacuolar membrane ATPase activity) phenotypes that include pH-conditional growth, increased calcium sensitivity, and the inability to grow on nonfermentable carbon sources. By screening based on these phenotypes and the inability ofvma mutants to accumulate the lysosomotropic dye quinacrine in their vacuoles, five new vma complementation groups (vma41 to vma45) were identified. TheVMA45 gene was cloned by complementation of the pH-conditional growth of the vma45-1 mutant strain and shown to be allelic to the previously characterized KEX2gene, which encodes a serine endoprotease localized to the late Golgi compartment. Both vma45-1 mutants and kex2 null mutants exhibit the full range of Vma− growth phenotypes and show no vacuolar accumulation of quinacrine, indicating loss of vacuolar acidification in vivo. However, immunoprecipitation of the V-ATPase from both strains under nondenaturing conditions revealed no defect in assembly of the enzyme, vacuolar vesicles isolated from akex2 null mutant showed levels of V-ATPase activity and proton pumping comparable to those of wild-type cells, and the V-ATPase complex purified from kex2 null mutants was structurally indistinguishable from that of wild-type cells. The results suggest that kex2 mutations exert an inhibitory effect on the V-ATPase in the intact cell but that the ATPase is present in the mutant strains in a fully assembled state, potentially capable of full enzymatic activity. This is the first time a mutation of this type has been identified.
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Wada, Yoh, Yoshinori Ohsumi et Yasuhiro Anraku. « Chloride transport of yeast vacuolar membrane vesicles : a study of in vitro vacuolar acidification ». Biochimica et Biophysica Acta (BBA) - Bioenergetics 1101, no 3 (août 1992) : 296–302. http://dx.doi.org/10.1016/0005-2728(92)90085-g.
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RODRIGUES, Claudia O., David A. SCOTT et Roberto DOCAMPO. « Presence of a vacuolar H+-pyrophosphatase in promastigotes of Leishmania donovani and its localization to a different compartment from the vacuolar H+-ATPase ». Biochemical Journal 340, no 3 (8 juin 1999) : 759–66. http://dx.doi.org/10.1042/bj3400759.
Texte intégralRésumé :
Inorganic pyrophosphate promoted the acidification of an intracellular compartment in permeabilized promastigotes of Leishmania donovani, as measured by Acridine Orange uptake. The proton gradient generated by pyrophosphate was collapsed by addition of nigericin or NH4Cl. Pyrophosphate-driven proton translocation was stimulated by potassium ions, and inhibited by NaF, the pyrophosphate analogues imidodiphosphate and aminomethylenediphosphonate (AMDP), dicyclohexylcarbodiimide, and the thiol reagents p-hydroxymercuribenzoate and N-ethylmaleimide, all at concentrations similar to those that inhibit the plant vacuolar proton-pumping pyrophosphatase (H+-PPase). The proton translocation activity had a pH optimum in the range 7.0-7.5, and was unaffected by bafilomycin A1 (40 nM), concanamycin A (5 nM), sodium o-vanadate (500 μM) and KNO3 (200 mM). AMDP-sensitive pyrophosphate hydrolysis was also detected in promastigotes, and potassium ions also stimulated this activity. Sodium ions disrupted pH gradients established in the presence of ATP but not in the presence of pyrophosphate, and sequential addition of ATP and pyrophosphate resulted in partially additive Acridine Orange accumulation, suggesting that the vacuolar H+-PPase is in a different intracellular compartment from the vacuolar H+-ATPase and Na+/H+ exchanger of L. donovani promastigotes. Separation of promastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity but lacked ATPase activity and markers for mitochondria, glycosomes and lysosomes. The organelles in this fraction appeared by electron microscopy to consist of electron-dense vacuoles. In summary, these results indicate that, in contrast to plant vacuoles, vacuolar H+-PPase and vacuolar ATPase activities are present in different compartments in L. donovani promastigotes.
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Poltermann, Sophia, Monika Nguyen, Juliane Günther, Jürgen Wendland, Albert Härtl, Waldemar Künkel, Peter F. Zipfel et Raimund Eck. « The putative vacuolar ATPase subunit Vma7p of Candida albicans is involved in vacuole acidification, hyphal development and virulence ». Microbiology 151, no 5 (1 mai 2005) : 1645–55. http://dx.doi.org/10.1099/mic.0.27505-0.
Texte intégralRésumé :
The vacuolar H+-ATPase (V-ATPase) component Vma7p of the human-pathogenic yeast Candida albicans regulates hyphal growth induced by serum and Spider medium and is essential for virulence. In order to characterize the functions of the putative V-ATPase subunit Vma7p of C. albicans, null mutants were generated. The resulting mutants showed reduced vacuole acidification, which correlated with defective growth at alkaline pH. In addition, defects in degradation of intravacuolar putative endosomal structures were observed. vma7 null mutants were sensitive towards the presence of metal ions. It is concluded that the sequestration of toxic ions in the vacuole via a H+ gradient generated by the V-ATPase is affected. The vma7 null mutant strains were avirulent in a mouse model of systemic candidiasis. In addition, C. albicans vma7 null mutants and the null mutant strain of the Vma7p-interacting phosphatidylinositol 3-kinase Vps34p showed similar phenotypes. In summary, the V-ATPase subunit Vma7p is involved in vacuolar ion transport and this transport is required for hyphal growth and virulence of C. albicans.
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MAQUOI, Erik, Karine PEYROLLIER, Agnès NOËL, Jean-Michel FOIDART et Francis FRANKENNE. « Regulation of membrane-type 1 matrix metalloproteinase activity by vacuolar H+-ATPases ». Biochemical Journal 373, no 1 (1 juillet 2003) : 19–24. http://dx.doi.org/10.1042/bj20030170.
Texte intégralRésumé :
Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a key enzyme in normal development and malignant processes. The regulation of MT1-MMP activity on the cell surface is a complex process involving autocatalytic processing, tissue inhibitor of MMPs (TIMP) binding and constitutive internalization. However, the fate of internalized MT1-MMP is not known. Acidification of intracellular vacuolar compartments is essential for membrane trafficking, protein sorting and degradation. This acidification is controlled by vacuolar H+-ATPases, which can be selectively inhibited by bafilomycin-A1. Here, we treated human tumour cell lines expressing MT1-MMP with bafilomycin-A1, and analysed its effects on MT1-MMP activity, internalization and processing. We show that the activity of MT1-MMP on the cell surface is constitutively down-regulated through a vacuolar H+-ATPase-dependent degradation process. Blockade of this degradation caused the accumulation of TIMP-free active MT1-MMP molecules on the cell surface, although internalization was not affected. As a consequence of this impaired degradation, pro-MMP-2 activation was strongly enhanced. This study demonstrates that the catalytic activity of MT1-MMP on the cell surface is regulated through a vacuolar H+-ATPase-dependent degradation process.
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Singer-Krüger, B., H. Stenmark, A. Düsterhöft, P. Philippsen, J. S. Yoo, D. Gallwitz et M. Zerial. « Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast. » Journal of Cell Biology 125, no 2 (15 avril 1994) : 283–98. http://dx.doi.org/10.1083/jcb.125.2.283.
Texte intégralRésumé :
The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.
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Brune, Andreas, Mathias Müller, Lincoln Taiz, Pedro Gonzalez et Ed Etxeberria. « Vacuolar Acidification in Citrus Fruit : Comparison between Acid Lime (Citrus aurantifolia) and Sweet Lime (Citrus limmetioides) Juice Cells ». Journal of the American Society for Horticultural Science 127, no 2 (mars 2002) : 171–77. http://dx.doi.org/10.21273/jashs.127.2.171.
Texte intégralRésumé :
Vacuolar acidification was investigated in `Palestine' sweet (Citrus limmetioides Tanaka) and `Persian' acid lime [(Citrus aurantifolia (Christm.) Swingle] (vacuolar pHs of 5.0 and 2.1, respectively) using tonoplast vesicles isolated from juice cells. The ATPase activity of tonoplast-enriched vesicles from sweet limes was strongly inhibited by bafilomycin A1 and NO3-, but was unaffected by vanadate. In contrast, the ATPase activity in acid lime membranes was only slightly inhibited by bafilomycin A1 and NO3- and was strongly inhibited by high concentrations of vanadate. The vacuolar origin of the acid lime vesicles was confirmed by immunoblotting. After solubilization and partial purification of the two enzymes by gel filtration, their inhibitor profiles were largely unchanged. Based on equal ATPase activities, vesicles from sweet and acid limes were able to generate similar pH gradients. However, in tonoplast vesicles from sweet limes, the maximum ΔpH was reached four times faster than in those from acid limes. Addition of ethylenediamine tetraacetic acid (EDTA) to chelate Mg+2 after the maximal ΔpH was attained resulted in collapse of the pH gradient in vesicles from sweet limes, whereas no change in ΔpH was observed in vesicles from acid limes, indicating a less H+ permeable membrane. Vacuolar ATPases from both cultivars exhibited identical pH optima and showed similar Mg+2 dependence, but only the acid lime ATPase activity was inhibited by Ca+2. These data confirm that the vanadate-sensitive form of the V-ATPase found in lemon and acid limes is specific to hyperacidifying tissues rather than to citrus juice cells. Sweet lime vacuoles bear the classical V-ATPase also found in vegetative plant tissues.
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Johnson, L. S., K. W. Dunn, B. Pytowski et T. E. McGraw. « Endosome acidification and receptor trafficking : bafilomycin A1 slows receptor externalization by a mechanism involving the receptor's internalization motif. » Molecular Biology of the Cell 4, no 12 (décembre 1993) : 1251–66. http://dx.doi.org/10.1091/mbc.4.12.1251.
Texte intégralRésumé :
To examine the relationship between endosome acidification and receptor trafficking, transferrin receptor trafficking was characterized in Chinese hamster ovary cells in which endosome acidification was blocked by treatment with the specific inhibitor of the vacuolar H(+)-ATPase, bafilomycin A1. Elevating endosome pH slowed the receptor externalization rate to approximately one-half of control but did not affect receptor internalization kinetics. The slowed receptor externalization required the receptor's cytoplasmic domain and was largely eliminated by substitutions replacing either of two aromatic amino acids within the receptor's cytoplasmic YTRF internalization motif. These results confirm, using a specific inhibitor of the vacuolar proton pump, that proper endosome acidification is necessary to maintain rapid recycling of intracellular receptors back to the plasma membrane. Moreover, receptor return to the plasma membrane is slowed in the absence of proper endosome acidification by a signal-dependent mechanism involving the receptor's cytoplasmic tyrosine-containing internalization motif. These results, in conjunction with results from other studies, suggest that the mechanism for clustering receptors in plasma membrane clathrin-coated pits may be an example of a more general mechanism that determines the dynamic distribution of membrane proteins among various compartments with luminal acidification playing a crucial role in this process.
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Scholz-Starke, Joachim. « How may PI(3,5)P2 impact on vacuolar acidification ? » Channels 11, no 6 (3 août 2017) : 497–98. http://dx.doi.org/10.1080/19336950.2017.1354584.
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Jung, Joo-Yong, et Cory M. Robinson. « Interleukin-27 inhibits phagosomal acidification by blocking vacuolar ATPases ». Cytokine 62, no 2 (mai 2013) : 202–5. http://dx.doi.org/10.1016/j.cyto.2013.03.010.
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Futai, M., T. Oka, G. Sun-Wada, Y. Moriyama, H. Kanazawa et Y. Wada. « Luminal acidification of diverse organelles by V-ATPase in animal cells ». Journal of Experimental Biology 203, no 1 (1 janvier 2000) : 107–16. http://dx.doi.org/10.1242/jeb.203.1.107.
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Eukaryotic cells contain organelles bounded by a single membrane in the cytoplasm. These organelles have differentiated to carry out various functions in the pathways of endocytosis and exocytosis. Their lumina are acidic, with pH ranging from 4.5 to 6.5. This article describes recent studies on these animal cell organelles focusing on (1) the primary proton pump (vacuolar-type H(+)-ATPase) and (2) the functions of the organelle luminal acidity. We also discuss similarities and differences between vacuolar-type H(+)-ATPase and F-type ATPase. Our own studies and interests are emphasized.
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Munn, A. L., et H. Riezman. « Endocytosis is required for the growth of vacuolar H(+)-ATPase-defective yeast : identification of six new END genes. » Journal of Cell Biology 127, no 2 (15 octobre 1994) : 373–86. http://dx.doi.org/10.1083/jcb.127.2.373.
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Yeast mutants that are defective in acidification of the lysosome-like vacuole are able to grow at pH 5.5, but not at pH 7. Here, we present evidence that endocytosis is required for this low pH-dependent growth and use this observation to develop a screen for mutants defective in endocytosis. By isolating mutants that cannot grow when they lack the 60-kD vacuolar ATPase subunit (encoded by the VAT2 gene), we isolated a number of vat2-synthetic lethal (Vsl-) mutant strains. Seven of the Vsl- mutants are defective in endocytosis. Four of these mutant strains (end8-1, end9-1, end10-1, and end11-1) show altered uptake of the endocytosed ligand, alpha-factor, and three (end12-1, end12-2, and end13-1) are probably defective in transfer of internalized material to the vacuole. Most of the mutations also confer a strong Ts- growth defect. The mutants defective in uptake of alpha-factor sort newly synthesized vacuolar proteins correctly, while those which may be defective in subsequent transport steps secrete at least a fraction of the newly synthesized soluble vacuolar proteins. The mutations that result in a defect in alpha-factor uptake are not allelic to any of the genes previously shown to encode endocytic functions.
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Kataoka, Takao, Makoto Muroi, Shoji Ohkuma, Takaki Waritani, Junji Magae, Akira Takatsuki, Shunzo Kondo, Makari Yamasaki et Kazuo Nagai. « Prodigiosin 25-C uncouples vacuolar type H+ -ATPase, inhibits vacuolar acidification and affects glycoprotein processing ». FEBS Letters 359, no 1 (6 février 1995) : 53–59. http://dx.doi.org/10.1016/0014-5793(94)01446-8.
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Perzov, Natalie, Vered Padler-Karavani, Hannah Nelson et Nathan Nelson. « Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis ». Journal of Experimental Biology 205, no 9 (1 mai 2002) : 1209–19. http://dx.doi.org/10.1242/jeb.205.9.1209.
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SUMMARYSubunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in contrast to its other subunits, is encoded by two genes VPH1 and STV1. While disruption of any other gene encoding the V-ATPase subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p can grow at this pH. We used a polyclonal antibody to yeast Stv1p and a commercially available monoclonal antibody to Vph1p for analysis of yeast membranes by sucrose gradient fractionation, and two different vital dyes to characterize the phenotype of vph1 ▵ and stv1 ▵mutants as compared to the double mutant and the wild-type cells. Immunological assays of sucrose gradient fractions revealed that the amount of Stv1p was elevated in the vph1 ▵ strain, and that vacuoles purified by this method with no detectable endosomal contamination contain an assembled V-ATPase complex, but with much lower activity than the wild type. These results suggest that Stv1p compensates for the loss of Vph1p in the vph1 ▵ strain. LysoSensor Green DND-189 was used as a pH sensor to demonstrate unexpected changes in vacuolar acidification in stv1▵ as the Vph1p-containing V-ATPase complex is commonly considered to acidify the vacuoles. In the vph1 ▵ strain, the dye revealed slight but definite acidification of the vacuole as well. The lipophilic dye FM4-64 was used as an endocytic marker. We show that the null V-ATPase mutants, as well as the vph1 ▵ one, markedly slow down endocytosis of the dye.
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Huang, Chunjuan, et Amy Chang. « pH-dependent Cargo Sorting from the Golgi ». Journal of Biological Chemistry 286, no 12 (14 janvier 2011) : 10058–65. http://dx.doi.org/10.1074/jbc.m110.197889.
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The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.
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de Castro, Patrícia Alves, Marcela Savoldi, Diego Bonatto, Mário Henrique Barros, Maria Helena S. Goldman, Andresa A. Berretta et Gustavo Henrique Goldman. « Molecular Characterization of Propolis-Induced Cell Death in Saccharomyces cerevisiae ». Eukaryotic Cell 10, no 3 (30 décembre 2010) : 398–411. http://dx.doi.org/10.1128/ec.00256-10.
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ABSTRACTPropolis, a natural product of plant resins, is used by the bees to seal holes in their honeycombs and protect the hive entrance. However, propolis has also been used in folk medicine for centuries. Here, we apply the power ofSaccharomyces cerevisiaeas a model organism for studies of genetics, cell biology, and genomics to determine how propolis affects fungi at the cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochromecbut not endonuclease G (Nuc1p) is involved in propolis-mediated cell death inS. cerevisiae. We also observed that the metacaspaseYCA1gene is important for propolis-mediated cell death. To elucidate the gene functions that may be required for propolis sensitivity in eukaryotes, the full collection of about 4,800 haploidS. cerevisiaedeletion strains was screened for propolis sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis sensitivity compared to the corresponding wild-type strains. Systems biology revealed enrichment for genes involved in the mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein targeting to vacuoles, and cellular response to starvation. Validation studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis.
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Lang, Thomas, Steffen Reiche, Michael Straub, Monika Bredschneider et Michael Thumm. « Autophagy and the cvt Pathway Both Depend onAUT9 ». Journal of Bacteriology 182, no 8 (15 avril 2000) : 2125–33. http://dx.doi.org/10.1128/jb.182.8.2125-2133.2000.
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ABSTRACT In growing cells of the yeast Saccharomyces cerevisiae, proaminopeptidase I reaches the vacuole via the selective cytoplasm-to-vacuole targeting (cvt) pathway. During nutrient limitation, autophagy is also responsible for the transport of proaminopeptidase I. These two nonclassical protein transport pathways to the vacuole are distinct in their characteristics but in large part use identical components. We expanded our initial screen foraut − mutants and isolated aut9-1cells, which show a defect in both pathways, the vacuolar targeting of proaminopeptidase I and autophagy. By complementation of the sporulation defect of homocygous diploid aut9-1 mutant cells with a genomic library, in this study we identified and characterized the AUT9 gene, which is allelic withCVT7. aut9-deficient cells have no obvious defects in growth on rich media, vacuolar biogenesis, and acidification, but like other mutant cells with a defect in autophagy, they exhibit a reduced survival rate and reduced total protein turnover during starvation. Aut9p is the first putative integral membrane protein essential for autophagy. A biologically active green fluorescent protein-Aut9 fusion protein was visualized at punctate structures in the cytosol of growing cells.