Cytosol Of Yeast Cells Research Articles

Effect of selenium and methods of protein extraction on the proteomic profile of Saccharomyces yeast.

Selenium may influence the biosynthesis of individual proteins in the yeast cell cytosol. In this study, we used two-dimensional (2D) electrophoresis to identify proteins that are differentially expressed by the enrichment of selenium in Saccharomyces cerevisiae yeast cells. We chose eight protein fractions for further proteomic analysis. A detailed analysis was performed using the Ultraflextreme matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometer, which enables fast and accurate measurement of the molecular weight of the analysed proteins. This study, for the first time, provides evidence that selenium-enriched yeast contains higher levels of mitochondria malate dehydrogenase, adenosine-5′-triphosphate (ATP)-dependent RNA helicase dbp3, and tryptophan dimethylallyltransferase, and alanyl-tRNA editing protein AlaX than yeast without the addition of selenium. It should be emphasised that the proteomic variability obtained reflects the high biological and complexity of yeast metabolism under control and selenium-enriched conditions and can be properly used in the future as a model for further research aimed at determining the expression of appropriate metabolic genes.

A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast.

Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here, we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells, we observe drastic changes in organellar volume, leading to crowding on the micrometer scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.

GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria

GABA transaminase (GABA-T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA-T from Saccharomyces cerevisiae (ScGABA-TKG) is an α-ketoglutarate-dependent enzyme encoded by the UGA1 gene, while higher plant GABA-T is a pyruvate/glyoxylate-dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA-T). The GABA-T from A. thaliana is localized in mitochondria and mediated by an 18-amino acid N-terminal mitochondrial targeting peptide predicated by both web-based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA-TKG and AtGABA-T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA-TKG and AtGABA-T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA-TKG deletion mutant Δuga1 with AtGABA-T or ScGABA-TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat-induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA-T is functionally interchangeable with ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA-T is about half as efficient in doing so as ScGABA-TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate-limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces.

Characterization of a putative thioredoxin peroxidase prx1 of Candida albicans.

In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H(2)O(2)) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H(2)O(2) scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H(2)O(2), menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H(2)O(2). These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H(2)O(2), but its cellular function is specified to t-BOOH.

Free Oligosaccharides to Monitor Glycoprotein Endoplasmic Reticulum-associated Degradation in Saccharomyces cerevisiae

In eukaryotic cells, N-glycosylation has been recognized as one of the most common and functionally important co- or post-translational modifications of proteins. "Free" forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-beta-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide:N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole alpha-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae.

New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

pHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pH(in)) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH(in) for three prototrophic wild-types. The new construct was also used to determine the pH(in) in strains differing in the activity of the plasma membrane Pma1 H(+)-ATPase and the influence of glucose on pH(in). We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands-on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.

Lot6p from Saccharomyces cerevisiae is a FMN‐dependent reductase with a potential role in quinone detoxification

NAD(P)H:quinone acceptor oxidoreductases are flavoenzymes expressed in the cytoplasm of many tissues and afford protection against the cytotoxic effects of electrophilic quinones by catalyzing a strict two-electron reduction. Such enzymes have been reported from several mammalian sources, e.g. human, mouse and rat, and from plant species. Here, we report identification of Lot6p (YLR011wp), the first soluble quinone reductase from the unicellular model organism Saccharomyces cerevisiae. Localization studies using an antibody raised against Lot6p as well as microscopic inspection of Lot6p-GFP demonstrated accumulation of the enzyme in the cytosol of yeast cells. Despite sharing only 23% similarity to type 1 human quinone reductase, Lot6p possesses biochemical properties that are similar to its human counterpart. The enzyme catalyzes a two-electron reduction of a series of natural and artificial quinone substrates at the expense of either NADH or NADPH. The kinetic mechanism follows a ping-pong bi-bi reaction scheme, with K(M) values of 1.6-11 microm for various quinones. Dicoumarol and Cibacron Marine, two well-known inhibitors of the quinone reductase family, bind to Lot6p and inhibit its activity. In vivo experiments demonstrate that the enzymatic activity of Lot6p is consistent with the phenotype of both Deltalot6 and Lot6p overexpressing strains, suggesting that Lot6p may play a role in managing oxidative stress in yeast.

Application of photobleaching for measuring diffusion of prion proteins in cytosol of yeast cells

Measurement of fluorescence recovery after photobleaching (FRAP) is a non-invasive technique for studying protein dynamics in real time in living cells. FRAP studies are carried out on proteins tagged with green fluorescent protein (GFP) or one of its spectral variants. Illumination with high intensity laser light irreversibly bleaches the GFP fluorescence but has no effect on protein function. By photobleaching a limited region of the cytoplasm, the rate of fluorescence recovery provides a measure of the rate of protein diffusion. A detailed description of the FRAP technique is given, including its application to measuring the mobility of GFP-tagged Sup35p in [ psi −] and [ PSI +] cells.

The Zygosaccharomyces bailii antifungal virus toxin zygocin: cloning and expression in a heterologous fungal host.

Zygocin, a monomeric protein toxin secreted by a virus-infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double-stranded (ds)RNA killer virus (ZbV-M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N-terminal amino acid sequence was determined, and a full-length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238-amino-acid preprotoxin precursor (pptox) consisting of a hydrophobic N-terminal secretion signal, followed by a potentially N-glycosylated pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N-terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix-assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post-translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin-sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.

Mutational analysis of K28 preprotoxin processing in the yeast Saccharomyces cerevisiae.

K28 killer strains of Saccharomyces cerevisiae are permanently infected with a cytoplasmic persisting dsRNA virus encoding a secreted alpha/beta heterodimeric protein toxin that kills sensitive cells by cell-cycle arrest and inhibition of DNA synthesis. In vivo processing of the 345 aa toxin precursor (preprotoxin; pptox) involves multiple internal and carboxy-terminal cleavage events by the prohormone convertases Kex2p and Kex1p. By site-directed mutagenesis of the preprotoxin gene and phenotypic analysis of its in vivo effects it is now demonstrated that secretion of a biological active virus toxin requires signal peptidase cleavage after Gly(36) and Kex2p-mediated processing at the alpha subunit N terminus (after Glu-Arg(49)), the alpha subunit C terminus (after Ser-Arg(149)) and at the beta subunit N terminus (after Lys-Arg(245)). The mature C terminus of the beta subunit is trimmed by Kex1p, which removes the terminal Arg(345) residue, thus uncovering the toxin's endoplasmic reticulum targeting signal (HDEL) which--in a sensitive target cell--is essential for retrograde toxin transport. Interestingly, both toxin subunits are covalently linked by a single disulfide bond between alpha-Cys(56) and beta-Cys(340), and expression of a mutant toxin in which beta-Cys(340) had been replaced by Ser(340) resulted in the secretion of a non-toxic alpha/beta heterodimer that is blocked in retrograde transport and incapable of entering the yeast cell cytosol, indicating that one important in vivo function of beta-Cys(340) might be to ensure accessibility of the toxin's beta subunit C terminus to the HDEL receptor of the target cell.

Mft52, an Acid-bristle Protein in the Cytosol That Delivers Precursor Proteins to Yeast Mitochondria

We have identified a novel protein, Mft52, in the cytosol of yeast cells. Mft52 has a two-domain structure that includes a receptor-like carboxyl-terminal "acid-bristle" domain, which binds basic, amphipathic mitochondrial targeting sequences. Native Mft52, purified from the cytosol of yeast cells, is found as a large particle eluting in the void volume of a Superose 6 gel filtration column. Fusion proteins, consisting of mitochondrial targeting sequences fused to nonmitochondrial passenger proteins, are targeted to mitochondria in wild-type yeast cells, but defects in the gene encoding Mft52 drastically reduce the delivery of these proteins to the mitochondria. We propose that Mft52 is a subunit of a particle that is part of a system of targeting factors and molecular chaperones mediating the earliest stages of protein targeting to the mitochondria.

Enzymatic kinetic analyses that emplo high-performance liquid chromatography : Competition betwen orotate- and hypoxanthine/guanine-phosphoribosyltransferases for a common substrate

Enzymatic assay procedure that employ high-performance liquid chromatography (HPLC) have been proven to be sensitive and versatile methods for accomplishing kinetic analyses of enzyme-catalyzed reactions, with nucleotides as substrates or products. Both orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) have been purified from Baker's yeast and analyzed kineticaly using a modification of published HPLC procedures. Because these two enzymes exist in the cytosol of yeast and might compete for the limiting ( ≈15 μ M) concentration of phosphoribosyl α-1-pyrophosphate (PRibPP), we elected to examine both equilibrium and steady-state effects of one enzymatic reaction on the other with HPLC. First, under the condition of equivalent mass concentrations of OPRTase and HGPRTase, the initial rate of orotidine monophosphate synthesis and the equilibrium state were greatly affected by the presence of HGPRTase acitivity. In conrast, the presence of the OPRTase activity had no effect on the HGPRTase-catalyzed reaction under these conditions. Second, to examine a competition by these enzymes for PRibPP in vivo, we have established that the total activities (units/ml) of OPRTase and HGPRTase in yeast cell extracts were f740 units/ml and 450 units/ml, respectively )a 1.7:1 ratio). These relative activities were then employed in an in vitro reaction competition analysis. The results were similar to the those obtained from experiments where equivalent OPRTase and HGPRTase activities were employed and reveal profound initial velocity and equilibrium effecfs one one reaction on the other. Thus a real competition between these enzymes for PRibPP may occur in the yeast cell cytosol, as determined by this unique HPLC competition assay procedure.

Purification and properties of coproporphyrinogen oxidase from the yeast Saccharomyces cerevisiae.

Coproporphyrinogen oxidase has been located in the cytosol of yeast cells. The enzyme was purified to homogeneity from a heme mutant strain exhibiting a high specific activity (15-20 enzyme units/mg soluble protein compared to 1-2 enzyme units/mg soluble protein of by the wild-type strain). The final preparation was homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Mr = 35,000) and isoelectrofocusing (pI = 6.2). Gel filtration on AcA 44 gave a relative molecular mass of 70,000. N-terminal amino-acid sequence analysis revealed a single polypeptide chain. Thus the enzyme appears to be a dimer with identical subunits. Two iron atoms/molecule of native protein were detected; they could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. However the involvement of the iron atoms in the oxidative catalytic activity of the enzyme was not demonstrated. The Km value for coproporphyrinogen was 0.05 microM. The enzyme was active only when molecular oxygen was used as electron acceptor; no anaerobic activity could be detected. Thiol-directed reagents partially inhibited the enzyme, indicating that an SH group is required for activity. Yeast coproporphyrinogen oxidase was activated by phospholipids or neutral detergents as described for the bovine liver enzyme.

Exchange of phospholipids between mitochondria and microsomes in vitro stimulated by yeast cell cytosol

Yeast cell cytosol stimulated the exchange of phospholipids between yeast mitochondria and microsomes in vitro, and also between organelles isolated from rat liver. The major phospholipids exchanged in both cases were phosphatidylinositol and phosphatidylcholine, together with smaller amounts of phosphatidylethanolamine. Evidence was also obtained that interconversion of phospholipids occurred during the incubation, probably via base exchange mechanisms.