Sake brewing characteristics of a new type of cerulenin-resistant sake yeast mutant

Several new types of cerulenin-resistant mutants of sake yeast were isolated. These mutants showed respiratory deficiency and could grow on media containing a higher concentration of antibiotics than could the parent. Sakes brewed by the mutants produced less succinate than by both the parent yeast and the mutants with respiratory deficiency induced by ethidium bromide. In addition, the acidity of these mutants was decreased. Since low acidity is favourable in both sake and wine, these mutants might be applicable for both sake and wine brewing.     

Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake

Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)⁺ mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)⁺ mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.     

Mutational analysis of Vam4/Ypt7p function in the vacuolar biogenesis and morphogenesis in the yeast,Saccharomyces cerevisiae

Summary The vacuole is one of the most prominent compartments in yeast cells. The wild-type yeast cells have a large vacuolar compartment which occupies approximately a quarter of the cell volume, while thevam4 mutant cells exhibit highly fragmented vacuolar morphology. We isolated theVAM4 gene and found that theVAM4 is identical to theYPT7 which encodes a member of small GTP-binding protein superfamily. We introduced mutations to theVAM4/YPT7 which alter nucleotide binding characteristics of the gene product specifically, and their activities for the vacuolar morphogenesis were examined by transforming the mutant genes into yeast cells. The Thr22Asn mutation, which was expected to fix the protein in the GDP-bound state, resulted in loss of function in the vacuolar morphogenesis. Subcellular fractionation analysis indicated that the mutant molecule did not associate with intracellular membranes efficiently. In contrast, Vam4/Ypt7p with the Gln68Leu mutation, which was expected to be the GTP-bound form, complemented the fragmented vacuolar morphology of vam4 mutant cells. Vam4/Ypt7p with the Gln68Leu mutation also complemented the defects in the biogenesis of vacuolar alkaline phosphatase whose maturation requires the proper function of Vam4/Ypt7p. Overexpression of the mutant proteins in wild-type cells did not develop dominant-negative effects on the vacuolar assembly. These results indicated that the GTP-bound form of Vam4/Ypt7p promotes the biogenesis and morphogenesis of the yeast vacuolar compartment.Abbreviations ALP alkaline phosphatase - CDE centromeric - DNA element - CPY carboxypeptidase Y - GST glutathione S-transferase     

Cell Structure of Yeasts Grown Anaerobically in Aspergillus orpzae-Proteolipid-supplemented Media

Effects of Aspergillus oryzae-proteolipid (PL) on the formation of high concentrations of alcohol, more than 20% as in sake brewing, have been studied. Electron microscopy revealed that there were no vacuoles but many lipid deposits in cytoplasm of the alcoholtolerant cells of Saccharomyces sake Kyokai No. 7, which were obtained by the anaerobic culture supplemented with PL. Sphaeroplasts from the alcohol-tolerant cells were stable in 20% alcohol, whereas those from the untolerant cells grown anaerobically in the PL-unsupplemented media were ruptured. The cell membrane became alcohol-endurable in the anaerobic cultures with PL. Synthetic phospholipid promoted yeast growth and the fermentative activity, whereas a small amount of sterol ester enhanced the alcohoi-endurability. The supplementation of both lipids anaerobically induced physiological properties characteristic of the alcohol-tolerant cells.     

Ultracytochemical localization of the vacuolar marker enzymes alkaline phosphatase,adenosine triphosphatase,carboxypeptidase Y and aminopeptidase reveal new concept of vacuole biogenesis in Saccharomyces cerevisiae

Summary Logarithmic cultures of Saccharomyces cerevisiae strains LBG H 1022, FL-100, X 2180 1A and 1B were studied together with the mutants pep4-3, sec18-1 and sec7-1. The necessary ultrastructural observations showed that, as a rule, juvenile vacuoles were formed de novo from perinuclear endoplasmic reticulum cisternae (ER) packed and inflated with electron-dense (polyanionic) matrix material. This process was disturbed solely in the sec18-1 mutant ander non-permissive conditions. The vacuolar marker enzymes adenosine triphosphatase (ATPase) and alkaline phosphohydrolase (ALPase) were assayed by the ultracytochemical cerium precipitation technique. The neutral ATPase was active in vacuolar membranes and in the previously shown (coated) microglobules nearby. ALPase activity was detected in microglobules inside juvenile vacuoles, inside nucleus and in the cytoplasm as well as in the membrane vesicles and in the periplasm. The sites of vacuolar protease carboxypeptidase Y (CPY) activity were assayed using N-CBZ-l-tyrosine-4-methoxy-2-naphthylamide (CBZ-Tyr-MNA) as substrate and sites of the aminopeptidase M activity using Leu-MNA as substrate. Hexazotized p-rosaniline served as a coupler for the primary reaction product of both the above proteases (MNA) and the resulting azo-dye was osmicated during postfixation. The CPY reaction product was found in both polar layers of vacuolar membranes (homologous to ER) and in ER membranes enclosing condensed lipoprotein bodies which were taken up by the vacuoles of late logarithmic yeast. Both before and after the uptake into the vacuoles the bodies contained the CPY reaction product in concentric layers or in cavities. Microglobules with CPY activity were also observed. Aminopeptidase was localized in microglobules inside the juvenile vacuoles. These findings combined with the previous cytochemical localizations of polyphosphates and X-prolyl-dipeptidyl (amino)peptidase in S. cerevisiae suggest the following cytologic mechanism for the biosynthetic protein transport: coated microglobules convey metabolites and enzymes either to the cell surface for secretion or enter the vacuoles in all phases of the cell cycle. The membrane vesicles represent an alternative secretory mechanism present in yeast cells only during budding. The homology of the ER with the vacuolar membranes and with the surface membranes of the lipoprotein condensates (bodies) indicates a cotranslational entry of the CPY into these membranes: The secondary transfer of a portion of CPY into vacuoles is probably mediated by the lipoprotein uptake process.     

Formation of cytoplasmic P-bodies in sake yeast during Japanese sake brewing and wine making

Recent studies have revealed that cytoplasmic processing bodies (P-bodies) play important roles in the control of eukaryotic gene expression in response to stress. Since the formation of P-bodies is in dynamic competition with translation, the status of translation is reflected in the assembly and disassembly of P-bodies in eukaryotic cells. During the brewing of Japanese sake and the making of wine, yeast cells are exposed to stress caused by increases in the concentration of ethanol. Here we found that ethanol stress enhances the formation of P-bodies in yeast cells in SD medium. In the wine-making process, P-body formation was also enhanced as alcoholic fermentation proceeded, but the formation of P-bodies was not simply affected by the ethanol concentration in the sake mash. These findings suggest differences in the rate of translation and the cytoplasmic mRNA flux during the sake brewing and wine making processes.     

PXA Domain-Containing Protein Pxa1 Is Required for Normal Vacuole Function and Morphology in Schizosaccharomyces pombe

PhoX homology (PX) domain-containing proteins play critical roles in vesicular trafficking, protein sorting, and lipid modification in eukaryotic cells. Several proteins with PX domains contain an associated domain termed PXA (PX-associated). Although PXA domain-containing proteins are required for some important cellular processes, the function of the PXA domain is unknown. We identified three PXA domain-containing proteins in Schizosaccharomyces pombe. S. pombe Pxa1p (SPAC5D6.07c) contained only the PXA domain, not the PX domain. To elucidate the role of the PXA domain in eukaryotic cells, we constructed and characterized a disruption mutant, pxa1. The pxa1 disruptant contained enlarged vacuoles and exhibited mislocalization of vacuolar carboxypeptidase Y (CPY). The conversion rate from pro- to mature-CPY was greatly impaired in pxa1 cells, and fluorescence microscopy indicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. The mutants were also deficient in vacuolar sorting of a multivesicular body (MVB) marker, a ubiquitin–GFP–carboxypeptidase S (Ub–GFP–CPS) fusion protein. Taken together, these results indicate that Pxa1 protein is required for normal vacuole function and morphology in S. pombe.     

Loss of Rim15p in shochu yeast alters carbon utilization during barley shochu fermentation

ABSTRACT

Rim15p of the yeast Saccharomyces cerevisiae is a Greatwall-family protein kinase that inhibits alcoholic fermentation during sake brewing. To elucidate the roles of Rim15p in barley shochu fermentation, RIM15 was deleted in shochu yeast. The disruptant did not improve ethanol yield, but altered sugar and glycerol contents in the mash, suggesting that Rim15p has a novel function in carbon utilization.     

Proteomic characterization of extracellular vesicles produced by several wine yeast species

In winemaking, the use of alternative yeast starters is becoming increasingly popular. They contribute to the diversity and complexity of wine sensory features and are typically used in combination with Saccharomyces cerevisiae, to ensure complete fermentation. This practice has drawn the interest on interactions between different oenological yeasts, which are also relevant in spontaneous and conventional fermentations, or in the vineyard. Although several interactions have been described and some mechanisms have been suggested, the possible involvement of extracellular vesicles (EVs) has not yet been considered. This work describes the production of EVs by six wine yeast species (S. cerevisiae, Torulaspora delbrueckii, Lachancea thermotolerans, Hanseniaspora uvarum, Candida sake and Metschnikowia pulcherrima) in synthetic grape must. Proteomic analysis of EV-enriched fractions from S. cerevisiae and T. delbrueckii showed enrichment in glycolytic enzymes and cell-wall-related proteins. The most abundant protein found in S. cerevisiae, T. delbrueckii and L. thermotolerans EV-enriched fractions was the enzyme exo-1,3-β-glucanase. However, this protein was not involved in the here-observed negative impact of T. delbrueckii extracellular fractions on the growth of other yeast species. These findings suggest that EVs may play a role in fungal interactions during wine fermentation and other aspects of wine yeast biology.     

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.     

Critical importance of phytase for yeast growth and alcohol fermentation in Japanese sake brewing

The high phytase producing mutant of Aspergillus oryzae (KL-38) previously isolated was employed for koji making, and the produced koji rice then supplied for sake brewing. The alcohol fermentation was improved compared to that with the parent strain (A. oryzae BP-1). The effects of two phytase isozymes (Phy I and Phy II) produced by A. oryzae on yeast growth and inorganic phosphate liberation were investigated using a synthetic medium containing phytic acid as a sole phosphate source. Yeast growth and the liberation of inorganic phosphate were both enhanced by the combination of Phy I and Phy II at a ratio of 1 to 3, which was compatible with the production ratio in KL-38. Based on these results, phytase plays important role in sake brewing, and that the maximum inorganic phosphate liberation from phytic acid can be obtained by a suitable combination of Phy I and Phy II.     

Vba4p,a vacuolar membrane protein,is involved in the drug resistance and vacuolar morphology of Saccharomyces cerevisiae

In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4Δ cells, as well as vba1Δ and vba2Δ cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4Δ cells than in wild type, vba1Δ, and vba2Δ cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.     

Fine Structure of Silica Deposition and the Origin of Shell Components in a Testate Amoeba Netzelia tuberculata1

ABSTRACT Netzelia tuberculata secretes a test composed of siliceous particles cemented together by organic plaques forming a single-layered spheroidal shell. The siliceous particles are produced within cytoplasmic vacuoles by three mechanisms: 1) synthesis de novo by deposition of the silica on a matrix; 2) deposition of silica on particles remaining in digestive vacuoles, including starch grains and undigested walls of yeast cells; and 3) secretion of silica as a hollow sphere at the periphery of vacuoles enclosed by the silicasecreting membrane. The silicalemma (silica-secreting membrane) originates as fibril-containing vesicles (GFV) secreted by the Golgi body. Fusion of these vesicles with membranes surrounding digestive vacuoles or with membranes surrounding specialized vacuoles containing a silica-binding matrix apparently converts the vacuole into a silica-depositing organelle. Small spherules of silica occur on the vacuolar side of the membrane surrounding the developing test granules, marking the presence of silicalemma activity. These colloidal spherules become aggregated into larger spherules that condense to form the siliceous surface of the developing test particle. Other Golgi vesicles, designated Golgi plaque vesicles (GPV), produce the organic plaques that are deposited among the siliceous particles at the periphery of the cell during new test construction during cell division. The fine structure of the GFV and GPV and their role in test wall deposition are discussed in relation to other silica-biomineralizing protozoa, including radiolaria.     

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin‐related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin‐D‐induced depolymerization or phalloidin‐induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild‐type (WT) Arabidopsis plants. Light‐induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.     

Expression and activation of the vacuolar processing enzyme in Saccharomyces cerevisiae

Vacuolar processing enzymes (VPEs) are cysteine proteinases responsible for maturation of various vacuolar proteins in plants. A larger precursor to VPE synthesized on rough endoplasmic reticulum is converted to an active enzyme in the vacuoles. In this study, a precursor to castor bean VPE was expressed in a pep4 strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE. Two VPE proteins of 59 and 46 kDa were detected in the vacuoles of the transformant. They were glycosylated in the yeast cells, although VPE is not glycosylated in plant cells in spite of the presence of two N-linked glycosylation sites. During the growth of the transformant, the level of the 59 kDa VPE increased slightly until a rapid decrease occurred after 9 h. By contrast, the 46 kDa VPE appeared simultaneously with the disappearance of the 59 kDa VPE. Vacuolar processing activity increased with the accumulation of the 46 kDa VPE, but not of the 59 kDa VPE. The specific activity of the 46 kDa VPE was at a similar level to that of VPE in plant cells. The 46 kDa VPE instead of proteinase A mediated the conversion of procarboxypeptidase Y to the mature form. This indicates that proteinase A responsible for maturation of yeast vacuolar proteins can be replaced functionally by plant VPE. These findings suggest that an inactive VPE precursor synthesized on the endoplasmic reticulum is transported to the vacuoles in the yeast cells and then processed to make an active VPE by self-catalytic proteolysis within the vacuoles.     

Two Fluorescent Markers Identify the Vacuolar System of Schizophyllum commune

Vacuole-mediated proteolysis is important to sustained growth of filamentous wood-decaying fungi such as Schizophyllum commune. Demonstrating that specific proteases are vacuole associated has been difficult in these organisms due to the lack of specific markers for vacuolar compartments. We used 5-(and 6-)-carboxy-2′, 7′-dichlorofluorescein diacetate (carboxy-DCFDA) and a proprietary vacuolar membrane marker for yeast (MDY-64; Molecular Probes) for in situ fluorescent labeling of the vacuoles of S. commune mycelia grown on microscope slides. MDY-64 labels numerous small vesicles in S. commune mycelia in addition to larger vacuolar structures. In contrast, carboxy-DCFDA apparently is taken up by a subset of the MDY-64-labeled vesicles, accumulating primarily in larger vacuoles. Staining of mycelia with carboxy-DCFDA shows a transition from mostly cytoplasmic fluorescence in apical cells with little vacuolar fluorescence to nearly complete sequestration of the stain in vacuoles of older cells. In penultimate cells, both cytoplasm and vacuolar structures fluoresce. Vacuoles stained with carboxy-DCFDA typically were spherical and ranged in size from 0.4 μm to 3.2 μm in diameter with a mean of 1.8 um. Occasionally, in penultimate cells, tubular structures which stained with carboxy-DCFDA were found. ScPrB, a principal enzyme of nitrogen-limitation induced autolysis in S. commune, copurified in sucrose density gradients with carboxy-DCFDA and acid phosphatase, demonstrating its vacuolar localization. Received: 23 December 1998 / Accepted: 11 January 1999     

Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles ofSaccharomyces cerevisiae

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures ofSaccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0°C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits featured envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.     

A functional connection of Dictyostelium paracaspase with the contractile vacuole and a possible partner of the vacuolar proton ATPase

Dictyostelium discoideum possesses only one caspase family member, paracaspase (pcp). Two separate mutant cell lines were first analysed: one cell line was an over-expressed GFP-tagged Pcp (GFP-Pcp), while the other cell line was a pcp-null (pcp-). Microscopic analysis of cells expressing GFP-Pcp revealed that Pcp was associated with the contractile vacuole membrane consisting of bladder-like vacuoles. This association was disrupted when cells were exposed to osmotic stress conditions. Compared with wild-type cells, the GFP-Pcp-over-expressing cells were susceptible to osmotic stress and were seen to be very rounded in hypo-osmotic conditions and contained more abnormally swollen contractile vacuole. Cells with pcp- were also rounded but had few, if any, contractile vacuoles. These observations suggest that Pcp is essential for Dictyostelium osmotic regulation via its functioning in the contractile vacuole system. Subjecting these cells to selected contractile vacuole inhibitor provided additional support for these findings. Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the normal morphology of the cell.     

Interorganelle interactions and inheritance patterns of nuclei and vacuoles in budding yeast meiosis

Many of the mechanisms by which organelles are inherited by spores during meiosis are not well understood. Dramatic chromosome motion and bouquet formation are evolutionarily conserved characteristics of meiotic chromosomes. The budding yeast bouquet genes (NDJ1, MPS3, CSM4) mediate these movements via telomere attachment to the nuclear envelope (NE). Here, we report that during meiosis the NE is in direct contact with vacuoles via nucleus-vacuole junctions (NVJs). We show that in meiosis NVJs are assembled through the interaction of the outer NE-protein Nvj1 and the vacuolar membrane protein Vac8. Notably, NVJs function as diffusion barriers that exclude the nuclear pore complexes, the bouquet protein Mps3 and NE-tethered telomeres from the outer nuclear membrane and nuclear ER, resulting in distorted NEs during early meiosis. An increase in NVJ area resulting from Nvj1-GFP overexpression produced a moderate bouquet mutant-like phenotype in wild-type cells. NVJs, as the vacuolar contact sites of the nucleus, were found to undergo scission alongside the NE during meiotic nuclear division. The zygotic NE and NVJs were partly segregated into 4 spores. Lastly, new NVJs were also revealed to be synthesized de novo to rejoin the zygotic NE with the newly synthesized vacuoles in the mature spores. In conclusion, our results revealed that budding yeast nuclei and vacuoles exhibit dynamic interorganelle interactions and different inheritance patterns in meiosis, and also suggested that nvj1Δ mutant cells may be useful to resolve the technical challenges pertaining to the isolation of intact nuclei for the biochemical study of meiotic nuclear proteins.