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PubMed1988

Infectious positive- and negative-strand transcript RNAs from bacteriophage Q beta cDNA clones.

Plasmids containing full-length cDNA copies of the Q beta RNA phage genome and flanking T7 promoters were constructed. Positive-strand Q beta RNA, generated by in vitro transcription of these plasmids with T7 RNA polymerase, was infectious to Escherichia coli spheroplasts. The Q beta replicase gene from the cloned DNA was subcloned and expressed in E. coli cells by means of a thermoinducible plasmid. Full-length, negative-strand Q beta transcripts were infectious when transfected into spheroplasts containing the induced replicase gene.

Cloning, Molecular↗
PubMed1988

Reconstitution of SEC gene product-dependent intercompartmental protein transport.

Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.

Adenosine Triphosphate↗
PubMed1990

Ligand autoradiographic receptor screening: receptor cDNA expression in replicas of transfected COS cells.

In this paper we validate a methodology, ligand autoradiographic receptor screening (LARS), for detecting expression of full length receptor cDNAs in COS cells. The method involves transfection of COS cells with receptor cDNAs by spheroplast fusion, production of filter replicas of the cell fragments, ligand binding to the receptors expressed in the membranes, and autoradiographic detection of bound ligand. A beta-adrenergic receptor cDNA cloned into a eukaryotic expression vector reliably induces high levels of beta-adrenergic receptor expression in 2-12% of COS cell colonies transfected with this plasmid after experimental conditions are optimized. Receptor expression is monitored by autoradiographic detection of 125iodocyanopindolol binding to COS cell fragments immobilized on polyester filter replicas. Binding displays appropriate pharmacological properties. The number of high-density binding spots per filter depends on the fraction of the spheroplasts in the fusion mixture that contain the beta-adrenergic receptor cDNA. A 100-plate LARS experiment can assess receptor expression in more than 10(4) transfected colonies. Thus detection of receptor-encoding sequences present in libraries in proportions as low as 1 in 10(4) should be possible. This technique may therefore be useful in detecting expression of other receptor cDNAs for which selective radioligands are available.

Animals↗
PubMed1993

Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution.

Mechanosensitive ion channels (MSCs) which could provide for fast osmoregulatory responses in bacteria, remain unidentified as molecular entities. MSCs from Escherichia coli (strain AW740) were examined using the patch-clamp technique, either (a) in giant spheroplasts, (b) after reconstitution by fusing native membrane vesicles with asolectin liposomes, or (c) by reassembly of octylglucoside-solubilized membrane extract into asolectin liposomes. MSC activities were similar in all three preparations, consisting of a large nonselective MSC of 3-nS conductance (in 200 mM KCl) that was activated by high negative pressures, and a small weakly anion-selective MSC of 1 nS activated by lower negative pressures. Both channels appeared more sensitive to suction in liposomes than in spheroplasts. After gel filtration of the solubilized membrane extract and reconstituting the fractions, both large MSC and small MSC activities were retrieved in liposomes. The positions of the peaks of channel activity in the column eluate, assayed by patch sampling of individual fractions reconstituted in liposomes, showed an apparent molecular mass under nondenaturing conditions of about 60-80 kDa for the large and 200-400 kDa for the small MSC. We conclude that (a) the large MSC and the small MSC are distinct molecular entities, (b) the fact that both MSCs were functional in liposomes following chromatography strongly suggests that these channels are gated by tension transduced via lipid bilayer, and (c) chromatographic fractionation of detergent-solubilized membrane proteins with subsequent patch sampling of reconstituted fractions can be used to identify and isolate these MS channel proteins.

Biomechanical Phenomena↗
PubMed2000

An efficient cell-free protein synthesis system using periplasmic phosphatase-removed S30 extract.

An efficient cell-free translation system was developed by removal of phosphatase localized in the periplasmic space, which hampers the translation reaction by hydrolyzing ATP. S30 extract was prepared from the spheroplast of Escherichia coli, and as much as 40% of ATP-hydrolysis activity of phosphatases could be easily removed by the spheroplast formation. The reaction period of translation using phosphatase-removed S30 extract could be prolonged and protein synthesis was enhanced by more than 30%.

Cell-Free System↗
PubMed2003

Cell volume changes during rapid temperature shifts.

The effect of a rapid temperature increase on the volume of different types of cells was investigated. Experiments were carried out using continuous microscopic image analysis. Volume variation of yeast cells, yeast spheroplasts and human leukaemia cells was measured during the transient phase after a thermal shift. The thermal shift was found to induce rapid increase in cell volume for cells lacking a cell wall (yeast spheroplasts and human leukaemia cells). This increase in cell volume is assumed to be a main cause of the heat shock-induced cell death. A theoretical mechanistic model that explains the behaviour of these cells is finally proposed.

Cell Size↗
PubMed2003

Underlying regularity in the shapes of nucleoids of Escherichia coli: implications for nucleoid organization and partition.

The genomic DNA of Escherichia coli is localized in one or a few compact nucleoids. Nucleoids in rapidly grown cells appear in complex shapes; the relationship of these shapes to underlying arrangements of the DNA is of structural interest and of potential importance in gene localization and nucleoid partition studies. To help assess this variation in shape, limited three-dimensional information on individual nucleoids was obtained by DNA fluorescence microscopy of cells as they reoriented in solution or by optical sectioning. These techniques were also applied to enlarged nucleoids within swollen cells or spheroplasts. The resulting images indicated that much of the apparent variation was due to imaging from different directions and at different focal planes of more regular underlying nucleoid shapes. Nucleoid images could be transformed into compact doublet shapes by exposure of cells to chloramphenicol or puromycin, consistent with a preexisting bipartite nucleoid structure. Isolated nucleoids and nucleoids in stationary-phase cells also assumed a doublet shape, supporting such a structure. The underlying structure is suggested to be two subunits joined by a linker. Both the subunits and the linker appear to deform to accommodate the space available within cells or spheroplasts ("flexible doublet" model).

Chloramphenicol↗
PubMed1989

Characterization of the bacterial cell associated calmodulin-sensitive adenylate cyclase from Bordetella pertussis.

Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase that is associated with the whole bacteria and released into its culture media. Preparations of this enzyme invade animal cells, causing elevations in intracellular cAMP levels. Cell-associated adenylate cyclase accounted for 28% of the total adenylate cyclase activity while 72% was released into the culture supernatant. Over 90% of the cell-associated adenylate cyclase activity was sensitive to trypsin treatment of whole cells, indicating that the catalytic domain of the enzyme is localized on the outer surface of the bacterial cells. Enzyme activity was released from whole cells by treatment with SDS. This activity was resolved as a large form (Mr 215,000) by SDS-polyacrylamide gel electrophoresis. In contrast, the culture supernatant contained only the 45,000-dalton catalytic subunit. Enzyme activity released from spheroplasts by sonication was resolved into a large form (Mr 215,000) and a small form (Mr 45,000). The appearance of the small form with spheroplast formation was probably the result of proteolytic degradation. Antibodies generated against the catalytic subunit purified from culture supernatants cross-reacted with and immunoprecipitated both the large and small forms of adenylate cyclase isolated from bacterial cells. Furthermore, incubation of the cell-associated enzyme with a crude bacterial extract resulted in a time-dependent disappearance of the 215,000-dalton form and a concomitant increase in the amount of the smaller 45,000-dalton form. There was also a parallel increase in the ability of the cell-associated preparation to elevate intracellular cAMP levels in N1E-115 mouse neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed2000

Insulin-like signaling in yeast: modulation of protein phosphatase 2A, protein kinase A, cAMP-specific phosphodiesterase, and glycosyl-phosphatidylinositol-specific phospholipase C activities.

Previously, we have described significant effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae under conditions of growth limitation. These regulations apparently rely on a transmembrane receptor capable of binding human insulin and responding by tyrosine/serine phosphorylation of a specific set of polypeptides [Müller, G., Rouveyre, N., Crecelius, A., and Bandlow, W. (1998) Biochemistry 37, 8683-8695; Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W. (1998) Biochemistry 37, 8696-8704; Müller, G., Rouveyre, N., Upshon, C., and Bandlow, W. (1998) Biochemistry 37, 8705-8713]. To characterize the molecular link between the initial steps in insulin-like signaling in yeast and the changes in the activities of glycogen synthase and glycogen phosphorylase, we examined here the effects of human insulin on a set of key regulatory enzymes of glycogen metabolism, protein phosphatase 2A (PP2A), cAMP-specific phosphodiesterase (cAMP-PDE), and protein kinase A (PKA). PP2A was activated about 2-fold by insulin in spheroplasts and in intact cells, whereas the fraction of active PKA was significantly reduced in a cAMP-independent manner as well as through a subsequent up to 3-fold increase in particulate cAMP-PDE activity accompanied by a 50% decrease in cytosolic cAMP levels. In addition, glycosyl-phosphatidylinositol-specific phospholipase C (GPI-PLC), which in isolated rat adipocytes is activated by insulin, was stimulated to up to 5-fold by glucose and 10-fold by glucose plus insulin in both yeast spheroplasts and intact cells leading to a concentration-dependent leftward shift of the glucose-response curve for activation of the GPI-PLC. GPI-PLC was most pronouncedly stimulated by authentic human insulin compared to various insulin analogues and insulin-like growth factor I. In addition to lipolytic cleavage by GPI-PLC, the GPI anchor of the cAMP-binding ectoprotein, Gce1p, was secondarily processed by a rapid proteolytic event. As the GPI-PLC reaction is rate limiting, the efficiency of the two-step anchor cleavage was significantly increased when insulin was present together with glucose as compared to glucose alone. The insulin concentrations effective in modulating PP2A, PKA, cAMP-PDE, and GPI-PLC activities correlate well with those required for half-saturation of the specific binding sites as well as for stimulation of protein phosphorylation and glycogen accumulation. The data suggest that mammalian insulin-sensitive cells and yeast share (part of) the key regulatory mechanism (consisting of PP2A, PKA, cAMP-PDE, and GPI-PLC) involved in the transduction of the insulin signal from the respective receptor systems to glycogen synthase and phosphorylase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Proton translocation and the respiratory nitrate reductase of Escherichia coli.

Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.

Anaerobiosis↗
PubMed1980

Photosynthetic electron transport in a cell-free preparation from the thermophilic blue-green alga Phormidium laminosum.

1. A cell-free preparation of membrane fragments was prepared from the thermophilic blue-green alga Phormidium laminosum by lysozyme treatment of the cells followed by osmotic shock to lyse the spheroplasts. The membrane fragments showed high rates of photosynthetic electron transport and O2 evolution (180-250 mumol of O2/h per mg of chlorophyll a with 2,6-dimethyl-1,4-benzoquinone as electron acceptor). O2-evolution activity was stable provided that cations (e.g. 10mM-Mg2+ or 100mM-Na+) or glycerol (25%, v/v) were present in the suspending medium. 2. The components of the electron-transport chain in P. laminosum were similar to those of other blue-green algae: the cells contained Pigment P700, plastocyanin, soluble high-potential cytochrome c-553, soluble low-potential cytochrome c-54 and membrane-bound cytochromes f, b-563 and b-559 (both low- and high-potential forms). The amounts and midpoint potentials of the membrane-bound cytochromes were similar to those in higher-plant chloroplasts. 3. Although O2 evolution in P. laminosum spheroplasts was resistant to high temperatures, thermal stability was not retained in the cell-free preparation. However, in contrast with higher plants, O2 evolution in P. laminosum membrane fragments was remarkably resistant to the non-ionic detergent Triton X-100.

Cell Membrane↗
PubMed1999

Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms.

The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Binding of [3H]diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2-1 pmol x mg-1 of membrane proteins). Inhibition of [3H]diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of heterotrimeric Gi,o-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists. Using individually purified Galpha-subunits re-associated with betagamma-dimers, we showed that alphao1, alphao2, alphai1, alphai2 and alphai3 reconstituted high-affinity agonist binding with equal efficiency. This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family. The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here.

Binding Sites↗
PubMed1987

Viruses in fungi: infection of yeast with the K1 and K2 killer viruses.

We demonstrate here that yeast killer viruses, previously thought to be transmitted only by cytoplasmic mixing during division, mating, or other induced forms of cell fusion, are capable of extracellular transmission. Viral particles from standard K1 and K2 killer strains were used to inoculate sensitive cells of Saccharomyces cerevisiae, rendered competent by spheroplasting, lithium acetate treatment, or by natural mating. Extracellular transmission of the killer viruses was judged by the following criteria and controls. Filter-sterilized virus inocula were shown to be free of viable yeast cells, and host cells treated in the absence of added virus did not yield killer progeny. Infected clones originating from spheroplasts or lithium acetate-treated cells were shown to possess the genotype of the host strain and the killer phenotype of the infecting virus. Infected clones derived from complementary mating pairs were found to be wild-type diploids, whose meiotic segregants exhibited 2:2 segregation for unlinked nutritional markers and 4:0 segregation for the killer phenotype. This technique is generally applicable to the study of interactions between yeast viruses and different hosts and suggests that extracellular transmission may be a natural route for the inheritance and dissemination of mycoviruses.

Crosses, Genetic↗
PubMed1996

Functional expression of the multidrug resistance-associated protein in the yeast Saccharomyces cerevisiae.

The multidrug resistance-associated protein (MRP) is a member of the ATP binding cassette superfamily of transporters which includes the mammalian P-glycoproteins (P-gp) family. In order to facilitate the biochemical and genetic analyses of MRP, we have expressed human MRP in the yeast Saccharomyces cerevisiae and have compared its functional properties to those of the mouse Mdr3 P-gp isoform. Expression of both MRP and Mdr3 in the anthracycline hypersensitive mutant VASY2563 restored cellular resistance to Adriamycin in this mutant. MRP and Mdr3 expression produced pleiotropic effects on drug resistance in this mutant, as corresponding VASY2563 transformants also acquired resistance to the anti-fungal agent FK506 and to the K+/H+ ionophore valinomycin. The appearance of increased cellular resistance to the toxic effect of Adriamycin (ADM) in MRP and Mdr3 transformants was concomitant with a reduced intracellular accumulation of [14C]ADM in spheroplasts prepared from these cells. Moreover, MRP and Mdr3, but not control spheroplasts, could mediate a time-dependent reduction in the overall cell-associated [14C]ADM from preloaded cells, suggesting the presence of an active ADM transport mechanism in MRP and Mdr3 transformants. Finally, human MRP was found to complement the biological activity of the yeast peptide pheromone transporter Ste6 and partially restored mating in a sterile ste6 null mutant. These findings suggest that despite their relatively low level of structural homology, MRP and P-gp share similar functional aspects, since both proteins can mediate transport of chemotherapeutic drugs and the a mating peptide pheromone in yeast.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1994

Introduction of YACs into intact yeast cells by a procedure which shows low levels of recombinagenicity and co-transformation.

Yeast artificial chromosomes (YACs) enable the cloning and analysis of large segments of genomic DNA and permit the isolation of sequences which are impossible to maintain in Escherichia coli. However, the construction of genome libraries in YAC vectors is beset by a number of technical problems, not least of which is the creation of cloned fragments which are not true representatives of the donor genome. These artefactual clones arise mainly due to intra-fragment rearrangements or inter-fragment chimaera formation, both phenomena resulting from the activity of the host yeast's mitotic recombination system. We demonstrate that this system is significantly stimulated by the spheroplasting step of the standard YAC transformation system. In contrast, the transformation of intact yeast cells by either the lithium method or a new lithium-free protocol is much less recombinagenic. It is not possible to introduce high molecular weight YACs into yeast using the lithium protocol, but we find that such molecules may be introduced into pde2-mutants using the lithium-free approach. Since intact cells are transformed by this method, automation of post-transformation steps in the construction of YAC libraries is facilitated. Moreover, the frequency of cotransformation (and, therefore, chimera formation) is significantly reduced. However, these advantages do incur a penalty. Yields of YAC transformants by this simplified intact cell approach are reduced some 25- to 30-fold compared to those obtained by the spheroplast transformation route. Nevertheless, the considerable advantages of the new system recommend it for a number of applications.

Chromosomes, Artificial, Yeast↗
PubMed1984

Redox properties of membrane-bound b-type cytochromes and a soluble c-type cytochrome of nitrate reductase in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans.

In order to identify the b-type cytochrome involved in the nitrate reduction in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, the b-type cytochromes in the spheroplast membranes were characterized. Difference spectra at 77K of spheroplast membranes indicated the presence of two b-type cytochromes with a bands at 556.5 and 562 nm. Three components considered to be of the b-type cytochrome were resolved by anaerobic potentiometric titration at 560-572 nm. Their midpoint potentials at pH 7, Em,7, were - 135 mV, +40 mV and +175 nm and their approximate reduced minus oxidized maxima were determined to be at 565 nm (562 nm at 77K), 560 nm (556.5 nm) and 560 nm (556.5 nm), respectively. These values are almost the same as those reported for R. sphaeroides. The Em,7 value of the cytochrome c involved in the nitrate reductase of this denitrifier was determined to be 250 mV. A b-type cytochrome reduced with NADH and FMN was oxidized by nitrate in chromatophore membranes. The possibility that cytochrome b (Em,7 = 175 mV) is involved in the nitrate reduction is discussed.

Cell Membrane↗
PubMed1986

Resistance of Providencia stuartii to chlorhexidine: a consideration of the role of the inner membrane.

Spheroplasts of three strains of Providencia stuartii (one sensitive, one moderately sensitive and one resistant to chlorhexidine) were induced by cefoxitin, glycine or a lysozyme-tris-EDTA combination, and their susceptibility to chlorhexidine-induced lysis investigated. Maximum lysis of spheroplasts occurred at a low (2.5-5 micrograms/ml) concentration of chlorhexidine and was greatest with the most sensitive strain, Pv2. The possible role of the inner membrane in chlorhexidine resistance is considered in the light of the findings obtained.

Bacteriolysis↗
PubMed1978

Studies on the biosynthesis of bacterio-opsin. Demonstration of the existence of protein species structurally related to bacterio-opsin.

1. The kinetics of processing newly synthesized bacterio-opsin from the non-crystalline state within the brown membrane to the crystalline state within the purple membrane was followed by pulse-chase experiments. 2. Biosynthesis of bacterio-opsin was found to be highly resistant to RNA-synthesis inhibitors like rifampicin and ethidium bromide. In the presence of ethidium bromide, only five protein species continue to be synthesized in halobacteria, one of them being bacterio-opsin. 3. In spheroplasts, synthesis of bacterio-opsin is found to be selectively disturbed. The purple membrane isolated from spheroplasts contains new, additional protein species with apparent molecular weights of 19 000, 23 000 and 29 000. These proteins share common amino acid sequences with bacterio-opsin. 4. In the halobacterial cell membrane, two membrane proteins with apparent molecular weights of 30 000 and 36 000 were detected which are structurally related to bacterio-opsin. 5. Bacterio-opsin as well as the 30 000 Mr and 36 000-Mr proteins contain covalently bound sulphate.

Amino Acid Sequence↗