Kei1: A Novel Subunit of Inositolphosphorylceramide Synthase,Essential for Its Enzyme Activity and Golgi Localization

Fungal sphingolipids have inositol-phosphate head groups, which are essential for the viability of cells. These head groups are added by inositol phosphorylceramide (IPC) synthase, and AUR1 has been thought to encode this enzyme. Here, we show that an essential protein encoded by KEI1 is a novel subunit of IPC synthase of Saccharomyces cerevisiae. We find that Kei1 is localized in the medial-Golgi and that Kei1 is cleaved by Kex2, a late Golgi processing endopeptidase; therefore, it recycles between the medial- and late Golgi compartments. The growth defect of kei1-1, a temperature-sensitive mutant, is effectively suppressed by the overexpression of AUR1, and Aur1 and Kei1 proteins form a complex in vivo. The kei1-1 mutant is hypersensitive to aureobasidin A, a specific inhibitor of IPC synthesis, and the IPC synthase activity in the mutant membranes is thermolabile. A part of Aur1 is missorted to the vacuole in kei1-1 cells. We show that the amino acid substitution in kei1-1 causes release of Kei1 during immunoprecipitation of Aur1 and that Aur1 without Kei1 has hardly detectable IPC synthase activity. From these results, we conclude that Kei1 is essential for both the activity and the Golgi localization of IPC synthase.     

AUR1, a novel gene conferring aureobasidin resistance onSaccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells

Aureobasidin A (AbA), a cyclic depsipeptide produced byAureobasidium pullulans R106, is highly toxic to fungi includingSaccharomyces cerevisiae. We isolated several dominant mutants ofS. cerevisiae which are resistant to more than 25 µg/ml of AbA. From a genomic library of one suchAUR1 mutant, theAUR1 ^R (foraureobasidinresistant) mutant gene was isolated as a gene that confers resistance to AbA on wild-type cells. Its nucleotide sequence showed that the predicted polypeptide is a hydrophobic protein composed of 401 amino acids, which contains several possible transmembrane domains and at least one predicted N-linked glycosylation site. Comparison of the mutant gene with the wild-typeaur1 ⁺ gene revealed that the substitution of Phe at position 158 by Tyr is responsible for acquisition of AbA resistance. We suggest that the gene product of the wild-typeaur1 ⁺ is a target for AbA on the basis of following results. Firstly, cells that overexpress the wild-typeaur1 ⁺ gene become resistant to AbA, just as cells with anAUR1 ^R mutation do. Secondly, disruption of theaur1 ⁺ gene demonstrated that it is essential for growth. Thirdly, in the cells with a disruptedaur1 locus, pleiotropic morphological changes including disappearance of microtubules, degradation of tubulin and abnormal deposition of chitin were observed. Some of these abnormalities are also observed when wild-type cells are treated with AbA. The abnormality in microtubules suggests that the Aur1 protein is involved in microtubule organization and stabilization.     

AUR1, a novel gene conferring aureobasidin resistance onSaccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells

Aureobasidin A (AbA), a cyclic depsipeptide produced byAureobasidium pullulans R106, is highly toxic to fungi includingSaccharomyces cerevisiae. We isolated several dominant mutants ofS. cerevisiae which are resistant to more than 25 µg/ml of AbA. From a genomic library of one suchAUR1 mutant, theAUR1 ^R (foraureobasidinresistant) mutant gene was isolated as a gene that confers resistance to AbA on wild-type cells. Its nucleotide sequence showed that the predicted polypeptide is a hydrophobic protein composed of 401 amino acids, which contains several possible transmembrane domains and at least one predicted N-linked glycosylation site. Comparison of the mutant gene with the wild-typeaur1 ⁺ gene revealed that the substitution of Phe at position 158 by Tyr is responsible for acquisition of AbA resistance. We suggest that the gene product of the wild-typeaur1 ⁺ is a target for AbA on the basis of following results. Firstly, cells that overexpress the wild-typeaur1 ⁺ gene become resistant to AbA, just as cells with anAUR1 ^R mutation do. Secondly, disruption of theaur1 ⁺ gene demonstrated that it is essential for growth. Thirdly, in the cells with a disruptedaur1 locus, pleiotropic morphological changes including disappearance of microtubules, degradation of tubulin and abnormal deposition of chitin were observed. Some of these abnormalities are also observed when wild-type cells are treated with AbA. The abnormality in microtubules suggests that the Aur1 protein is involved in microtubule organization and stabilization.     

Myrosin Cell Development Is Regulated by Endocytosis Machinery and PIN1 Polarity in Leaf Primordia of Arabidopsis thaliana

Myrosin cells, which accumulate myrosinase to produce toxic compounds when they are ruptured by herbivores, form specifically along leaf veins in Arabidopsis thaliana. However, the mechanism underlying this pattern formation is unknown. Here, we show that myrosin cell development requires the endocytosis-mediated polar localization of the auxin-efflux carrier PIN1 in leaf primordia. Defects in the endocytic/vacuolar SNAREs (syp22 and syp22 vti11) enhanced myrosin cell development. The syp22 phenotype was rescued by expressing SYP22 under the control of the PIN1 promoter. Additionally, myrosin cell development was enhanced either by lacking the activator of endocytic/vacuolar RAB5 GTPase (VPS9A) or by PIN1 promoter-driven expression of a dominant-negative form of RAB5 GTPase (ARA7). By contrast, myrosin cell development was not affected by deficiencies of vacuolar trafficking factors, including the vacuolar sorting receptor VSR1 and the retromer components VPS29 and VPS35, suggesting that endocytic pathway rather than vacuolar trafficking pathway is important for myrosin cell development. The phosphomimic PIN1 variant (PIN1-Asp), which is unable to be polarized, caused myrosin cells to form not only along leaf vein but also in the intervein leaf area. We propose that Brassicales plants might arrange myrosin cells near vascular cells in order to protect the flux of nutrients and water via polar PIN1 localization.     

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P₂ may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P₂. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P₂-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P₂ depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P₂ does not govern the steady-state pH of vacuoles or lysosomes.     

Regulation of Vacuolar Proton-translocating ATPase Activity and Assembly by Extracellular pH

Vacuolar proton-translocating ATPases (V-ATPases) are responsible for organelle acidification in all eukaryotic cells. The yeast V-ATPase, known to be regulated by reversible disassembly in response to glucose deprivation, was recently reported to be regulated by extracellular pH as well (Padilla-López, S., and Pearce, D. A. (2006) J. Biol. Chem. 281, 10273–10280). Consistent with those results, we find 57% higher V-ATPase activity in vacuoles isolated after cell growth at extracellular pH of 7 than after growth at pH 5 in minimal medium. Remarkably, under these conditions, the V-ATPase also becomes largely insensitive to reversible disassembly, maintaining a low vacuolar pH and high levels of V₁ subunit assembly, ATPase activity, and proton pumping during glucose deprivation. Cytosolic pH is constant under these conditions, indicating that the lack of reversible disassembly is not a response to altered cytosolic pH. We propose that when alternative mechanisms of vacuolar acidification are not available, maintaining V-ATPase activity becomes a priority, and the pump is not down-regulated in response to energy limitation. These results also suggest that integrated pH and metabolic inputs determine the final assembly state and activity of the V-ATPase.     

Abscisic Acid and photosynthesis in isolated leaf mesophyll cell

Abscisic acid (AbA) treatments of concentrations of up to 135 micromolar did not inhibit photosynthesis in enzymatically isolated leaf mesophyll cells of Phaseolus vulgaris, Nicotiana tabacum, and Lycopersicum esculentum over periods of up to 5 hours. Thin slices of leaves preincubated in hypertonic solutions identical to those used to isolate cells were shown to synthesize AbA rapidly, although accumulation of AbA in the cells was low due to extensive release of the newly synthesized AbA into the medium. The levels of endogenously made AbA in leaf cells of Phaseolus vulgaris rose from a low of 0.27 micromolar to a high of 6.74 micromolar during 2 hours preincubation. Exogenously applied AbA can be taken up by the cells as was demonstrated using ¹⁴[C]AbA. Thus, AbA applied at concentrations 19 times higher than endogenous levels does not change the rate of photosynthesis.     

Improving the yellow pigment content of bread wheat flour by selecting the three homoeologous copies of Psy1

The yellow pigment content (YPC) of endosperm affects the quality and nutritional value of wheat grain products. Major quantitative trait loci (QTL) for endosperm YPC have been repeatedly mapped on chromosomes 7A and 7B in durum and bread wheats. The genes coding for phytoene synthase (PSY1), which is involved in the biosynthesis of carotenoids, generally co-segregate with these QTL, indicating their role in determining YPC. Here, to study the genetic factors underlying endosperm YPC in bread wheat, the sequence polymorphism of the homoeologous A, B and D copies of genes coding for PSY1, Psy-A1, Psy-B1, and Psy-D1, was studied in a worldwide core collection, which was also phenotyped for flour YPC. Seven novel alleles of Psy-A1 and two novel alleles of Psy-B1 were detected, which confirms the high level of polymorphism of these genes. Two major QTL with respective candidate genes Psy-A1 and Psy-B1 were identified in the distal region of chromosomes 7A and 7B using progeny of a cross between Apache and Ornicar, high and low YPC cultivars, respectively. Association mapping confirms the role of these genes in YPC and shows that the D copy also significantly influences this trait. These results indicate that breeders need to consider all three Psy1 copies when seeking to improve the YPC of wheat endosperm.     

Structure and Properties of the Clathrin-Coated Vesicle and Yeast Vacuolar V-ATPases

The V-ATPases are a family of ATP-dependent proton pumps responsible foracidification of intracellular compartments in eukaryotic cells. This reviewfocuses on the the V-ATPases from clathrin-coated vesicles and yeastvacuoles. The V-ATPase of clathrin-coated vesicles is a precursor to thatfound in endosomes and synaptic vesicles, which function in receptorrecycling, intracellular membrane traffic, and neurotransmitter uptake. Theyeast vacuolar ATPase functions to acidify the central vacuole and to drivevarious coupled transport processes across the vacuolar membrane. TheV-ATPases are composed of two functional domains. The V₁ domain isa 570-kDa peripheral complex composed of eight subunits of molecular weight70—14 kDa (subunits A—H) that is responsible for ATP hydrolysis.The V₀ domain is a 260-kDa integral complex composed of fivesubunits of molecular weight 100—17 kDa (subunits a, d, c, c8 and c9)that is responsible for proton translocation. Using chemical modification andsite-directed mutagenesis, we have begun to identify residues that play arole in ATP hydrolysis and proton transport by the V-ATPases. A centralquestion in the V-ATPase field is the mechanism by which cells regulatevacuolar acidification. Several mechanisms are described that may play a rolein controlling vacuolar acidification in vivo. One mechanisminvolves disulfide bond formation between cysteine residues located at thecatalytic nucleotide binding site on the 70-kDa A subunit, leading toreversible inhibition of V-ATPase activity. Other mechanisms includereversible assembly and dissociation of V₁ and V₀domains, changes in coupling efficiency of proton transport and ATPhydrolysis, and regulation of the activity of intracellular chloride channelsrequired for vacuolar acidification.     

Sphingolipid biosynthesis is required for polar growth in the dimorphic phytopathogen Ustilago maydis

Sphingolipids participate in different biological processes such as cell growth, differentiation, virulence, etc. In recent years these lipids are becoming prime targets for antifungal research. In the dimorphic phytopathogen Ustilago maydis inhibition of sphingolipid biosynthesis with the antifungal drug aureobasidin A (AbA) did not alter cell cycle but caused loss of cell polarity and growth inhibition. The addition of AbA had effects on the assembly of sterol-rich domains (SRDs), which concentrate at the sites of active growth in U. maydis, the actin cytoskeleton, FM4-64 trafficking, and the localization of the motor protein Myo5 and the septin Sep1. However, AbA did not affect the localization of the scaffold proteins Bem1 and Spa2. The septin Sep1 and SRDs showed interdependent partial colocalization suggesting that both act in conjunction to maintain the stability of the polar axis. Time course experiments suggest that AbA provokes cell depolarization in three steps: disassembly of SRDs from the tips, delocalization of some polarity proteins, and lastly appearance of medial division septa.     

Proton-transporting and calcium ion-transporting ATPases of Entamoeba histolytica

Entamoeba histolytica is unique among human protozoan parasites in its ability to phagocytose bacteria and red blood cells and to destroy host epithelial cells via a contact-mediated cytolysis. Antagonists of vacuolar acidification and calcium ion-transport inhibit amebic lysis of epithelial cells in vitro. In this review, John Samuelson, Nnecka Azikiwe and Pei-Shen Shen describe the primary structures of E. histolytica V-type proton-transporting ATPase (V-ATPase) and P-type calcium-transporting ATPase, which probably mediate amebic vacuolar acidification and calcium ion sequestration, respectively. The function of the amebic V-ATPase is discussed with regard to pinocytosis, bacterial killing and host cell lysis. Phylogenetic trees incorporating the sequences of the proteolipid and catalytic peptides of the amebic V-ATPase are described. The amebic P-type calcium-transporting ATPase is compared to those of the red blood cell plasma membrane and yeast vacuolar membrane. Finally, the potential of the V-ATPase proteolipid and P-type calcium ion-transporting ATPase as targets for anti-amebic antibodies or for bacteria loaded with recombinant toxins is explored.     

Distinct roles for two CYP226 family cytochromes P450 in abietane diterpenoid catabolism by Burkholderia xenovorans LB400

The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 ± 0.01 μM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.     

Inhibition of Host Vacuolar H+-ATPase Activity by a Legionella pneumophila Effector

Legionella pneumophila is an intracellular pathogen responsible for Legionnaires'' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae.     

Organelle acidification is important for localisation of vacuolar proteins in Saccharomyces cerevisiae

The acidic environments in the vacuole and other acidic organelles are important for many cellular processes in eukaryotic cells. In this study, we comprehensively investigated the roles of organelle acidification in vacuolar protein localisation in Saccharomyces cerevisiae. After repressing the acidification of acidic compartments by treatment with concanamycin A, a specific inhibitor of vacuolar H⁺-ATPase (V-ATPase), we examined the localisation of GFP-fused proteins that were predicted to localise in the vacuolar lumen or on the vacuolar membrane. Of the 73 proteins examined, 19 changed their localisation to the cytoplasmic region. Localisation changes were evaluated quantitatively using the image processing programme CalMorph. The delocalised proteins included vacuolar hydrolases, V-ATPase subunits, transporters and enzymes for membrane biogenesis, as well as proteins required for protein transport. These results suggest that many alterations in the localisation of vacuolar proteins occur after loss of the acidification of acidic compartments.     

Essential Role for Vacuolar Acidification in Candida albicans Virulence

Fungal infections are on the rise, with mortality above 30% in patients with septic Candida infections. Mutants lacking V-ATPase activity are avirulent and fail to acidify endomembrane compartments, exhibiting pleiotropic defects in secretory, endosomal, and vacuolar pathways. However, the individual contribution of organellar acidification to virulence and its associated traits is not known. To dissect their separate roles in Candida albicans pathogenicity we generated knock-out strains for the V₀ subunit a genes VPH1 and STV1, which target the vacuole and secretory pathway, respectively. While the two subunits were redundant in many vma phenotypes, such as alkaline pH sensitivity, calcium homeostasis, respiratory defects, and cell wall integrity, we observed a unique contribution of VPH1. Specifically, vph1Δ was defective in acidification of the vacuole and its dependent functions, such as metal ion sequestration as evidenced by hypersensitivity to Zn²⁺ toxicity, whereas stv1Δ resembled wild type. In growth conditions that elicit morphogenic switching, vph1Δ was defective in forming hyphae whereas stv1Δ was normal or only modestly impaired. Host cell interactions were evaluated in vitro using the Caco-2 model of intestinal epithelial cells, and murine macrophages. Like wild type, stv1Δ was able to inflict cellular damage in Caco-2 and macrophage cells, as assayed by LDH release, and escape by filamentation. In contrast, vph1Δ resembled a vma7Δ mutant, with significant attenuation in host cell damage. Finally, we show that VPH1 is required for fungal virulence in a murine model of systemic infection. Our results suggest that vacuolar acidification has an essential function in the ability of C. albicans to form hyphae and establish infection.     

Mutations in the Yeast KEX2 Gene Cause a Vma−-Like Phenotype: a Possible Role for the Kex2 Endoprotease in Vacuolar Acidification

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 of vma mutants to accumulate the lysosomotropic dye quinacrine in their vacuoles, five new vma complementation groups (vma41 to vma45) were identified. The VMA45 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 KEX2 gene, 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 a kex2 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.     

Yeast Phosphofructokinase-1 Subunit Pfk2p Is Necessary for pH Homeostasis and Glucose-dependent Vacuolar ATPase Reassembly

V-ATPases are conserved ATP-driven proton pumps that acidify organelles. Yeast V-ATPase assembly and activity are glucose-dependent. Glucose depletion causes V-ATPase disassembly and its inactivation. Glucose readdition triggers reassembly and resumes proton transport and organelle acidification. We investigated the roles of the yeast phosphofructokinase-1 subunits Pfk1p and Pfk2p for V-ATPase function. The pfk1Δ and pfk2Δ mutants grew on glucose and assembled wild-type levels of V-ATPase pumps at the membrane. Both phosphofructokinase-1 subunits co-immunoprecipitated with V-ATPase in wild-type cells; upon deletion of one subunit, the other subunit retained binding to V-ATPase. The pfk2Δ cells exhibited a partial vma growth phenotype. In vitro ATP hydrolysis and proton transport were reduced by 35% in pfk2Δ membrane fractions; they were normal in pfk1Δ. In vivo, the pfk1Δ and pfk2Δ vacuoles were alkalinized and the cytosol acidified, suggestive of impaired V-ATPase proton transport. Overall the pH alterations were more dramatic in pfk2Δ than pfk1Δ at steady state and after readdition of glucose to glucose-deprived cells. Glucose-dependent reassembly was 50% reduced in pfk2Δ, and the vacuolar lumen was not acidified after reassembly. RAVE-assisted glucose-dependent reassembly and/or glucose signals were disturbed in pfk2Δ. Binding of disassembled V-ATPase (V₁ domain) to its assembly factor RAVE (subunit Rav1p) was 5-fold enhanced, indicating that Pfk2p is necessary for V-ATPase regulation by glucose. Because Pfk1p and Pfk2p are necessary for V-ATPase proton transport at the vacuole in vivo, a role for glycolysis at regulating V-ATPase proton transport is discussed.     

Rapid Structural Changes and Acidification of Guard Cell Vacuoles during Stomatal Closure Require Phosphatidylinositol 3,5-Bisphosphate

Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)–induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H⁺-ATPase vha-a2 vha-a3 and vacuolar H⁺-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P₂, which is essential for vacuolar acidification and convolution.     

Ceramide signaling targets the PP2A-like protein phosphatase Sit4p to impair vacuolar function,vesicular trafficking and autophagy in Isc1p deficient cells

The vacuoles play important roles in cellular homeostasis and their functions include the digestion of cytoplasmic material and organelles derived from autophagy. Conserved nutrient signaling pathways regulate vacuolar function and autophagy, ensuring normal cell and organismal development and aging. Recent evidence implicates sphingolipids in the modulation of these processes, but the impact of ceramide signaling on vacuolar dynamics and autophagy remains largely unknown. Here, we show that yeast cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase type 2, exhibit vacuolar fragmentation and dysfunctions, namely decreased Pep4p-mediated proteolysis and V-ATPase activity, which impairs vacuolar acidification. Moreover, these phenotypes are suppressed by downregulation of the ceramide-activated protein phosphatase Sit4p. The isc1Δ cells also exhibit defective Cvt and vesicular trafficking in a Sit4p-dependent manner, ultimately contributing to a reduced autophagic flux. Importantly, these phenotypes are also suppressed by downregulation of the nutrient signaling kinase TORC1, which is known to inhibit Sit4p and autophagy, or Sch9p. These results support a model in which Sit4p functions downstream of Isc1p in a TORC1-independent, ceramide-dependent signaling branch that impairs vacuolar function and vesicular trafficking, leading to autophagic defects in yeast.     

Lifespan Extension by Methionine Restriction Requires Autophagy-Dependent Vacuolar Acidification

Reduced supply of the amino acid methionine increases longevity across species through an as yet elusive mechanism. Here, we report that methionine restriction (MetR) extends yeast chronological lifespan in an autophagy-dependent manner. Single deletion of several genes essential for autophagy (ATG5, ATG7 or ATG8) fully abolished the longevity-enhancing capacity of MetR. While pharmacological or genetic inhibition of TOR1 increased lifespan in methionine-prototroph yeast, TOR1 suppression failed to extend the longevity of methionine-restricted yeast cells. Notably, vacuole-acidity was specifically enhanced by MetR, a phenotype that essentially required autophagy. Overexpression of vacuolar ATPase components (Vma1p or Vph2p) suffices to increase chronological lifespan of methionine-prototrophic yeast. In contrast, lifespan extension upon MetR was prevented by inhibition of vacuolar acidity upon disruption of the vacuolar ATPase. In conclusion, autophagy promotes lifespan extension upon MetR and requires the subsequent stimulation of vacuolar acidification, while it is epistatic to the equally autophagy-dependent anti-aging pathway triggered by TOR1 inhibition or deletion.