0000000000041545
AUTHOR
Manfred J. Schmitt
Cloning and expression of a cDNA copy of the viral K28 killer toxin gene in yeast
The killer toxin K28, secreted by certain killer strains of the yeast Saccharomyces cerevisiae is genetically encoded by a 1.9 kb double-stranded RNA, M-dsRNA (M28), that is present within the cell as a cytoplasmically inherited virus-like particle (VLP). For stable maintenance and replication, M28-VLPs depend on a second dsRNA virus (LA), which has been shown to encode the major capsid protein (cap) and a capsid-polymerase fusion protein (cap-pol) that provides the toxin-coding M-satellites with their transcription and replicase functions. K28 toxin-coding M28-VLPs were isolated, purified and used in vitro for the synthesis of the single-stranded M28 transcript, which was shown to be of pl…
Yeast dsRNA viruses: replication and killer phenotypes
The cytoplasmic L-A dsRNA virus of Saccharomyces cerevisiae consists of a 4.5 kb dsRNA and the two gene products it encodes; the capsid (cap) and at least one copy of the capsid-polymerase (cap-pol) fusion protein. Virion cap-pol catalyses transcription of the plus (sense)-strand; this is extruded from the virus and serves as messenger for synthesis of cap and cap-pol. Nascent cap-pol binds to a specific domain in the plus strand to initiate encapsidation and then catalyses minus-strand synthesis to complete the replication cycle. Products of at least three host genes are required for replication, and virus copy number is kept at tolerable levels by the SKI antivirus system. S. cerevisiae k…
Complement Receptor Analogous Factors in Human Serum: I. Isolation of a Molecule Inhibitory for Complement Dependent Rosette Formation, its Identification as α1-Antitrypsin and its Functional Characterization
Abstract A glycoprotein was isolated from human plasma which partially inhibited C3 carrying erythrocytes from binding to complement receptor cells (CR + C). Based on its physicochemical characteristics and its antigenicity this glycoprotein was identified as aI-antitrypsin (α 1 -AT). The activity of α 1 -AT towards-C3 and its fragments was unaffected by heating but it was destroyed by periodic acid. The isolated carbohydrate moiety of α 1 -AT showed the same effect as the intact molecule. Using F(ab) 2 of IgG-anti-α 1 -AT, α 1 -AT could be demonstrated on Raji cells and human erythrocytes. Treatment of these CR + C with IgG-anti-α 1 -AT resulted in a blockade of their C3 receptor activity.…
Sequence of the M28 dsRNA: Preprotoxin Is Processed to an α/β Heterodimeric Protein Toxin
The killer and immunity phenotypes of K28 killer strains of Saccharomyces cerevisiae are determined by the 1.75-kb M28 dsRNA virus. In the plus strand, M28p, the K28 preprotoxin gene, comprises bases 13-1047 and is followed, after an additional 85 bases, by a 63-bp poly(A) sequence and a 553-base 3'-sequence. This 3'-sequence contains two potential stem-loop structures predicted to bind the L-A encoded cap-pol protein, initiating encapsidation; high-level expression results in curing of M1 dsRNA. Expression of M28p confers the complete K28 killer and immunity phenotype on a cell lacking M28 dsRNA. K28 toxin is a disulfide-bonded heterodimer of alpha (10.5 kDa) and beta (11 kDa) components w…
Killer toxin of Hanseniaspora uvarum
The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000. Additional investigations of the primary toxin binding sites within the cell wall of sensitiv…
Genetic analysis of maintenance and expression of L and M double-stranded RNAs from yeast killer virus K28
The killer phenotype expressed by Saccharomyces cerevisiae strain 28 differs from that of the more extensively studied K1 and K2 killers with respect to immunity, mode of toxin action and cell wall primary toxin receptor. We previously demonstrated that the M28 and L28 dsRNAs found in strain 28 are present in virus-like particles (VLPs) and that transfection with these VLPs is sufficient to confer the complete K28 phenotype on a dsRNA-free recipient cell. We also demonstrated that L28, like the L-A-H species in K1 killers, has [HOK] activity required for maintenance of M1-dsRNA, and predicted that M28 would share with M1 dependence on L-A for replication. We now confirm this prediction by g…
Killer-toxin-resistant kre12 mutants of Saccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor.
The Saccharomyces cerevisiae killer toxin K1 is a secreted alpha/beta-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to beta-1,6-D-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of the KRE genes whose products are involved in synthesis and/or assembly of cell wall beta-D-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane leve…
Killer Toxins of Yeasts: Inhibitors of Fermentation and Their Adsorption.
The killer toxin (KT 28), a glycoprotein of Saccharomyces cerevisiae strain 28, was almost completely adsorbed by bentonite, when applied at a concentration of 1 g per liter. No significant differences were found between several types of bentonite. Killer toxin KT 28 is similarly adsorbed by intact yeast cells or by a commercial preparation of yeast cell walls that has been recommended to prevent stuck fermentations. An investigation of the cell wall fractions revealed that the toxin KT 28 was mainly adsorbed by mannan, that removed the toxin completely. The alkali-soluble and the alkali-insoluble β-1,3- and β-1,6-D-glucans lowered the toxin concentration to one tenth of the original amount…
Immunochemical analysis of the carbohydrate moiety of yeast killer toxin K28
Killer toxin K28, a 16 kd protein secreted by the wine yeast Saccharomyces cerevisiae strain 28, was reversibly bound by a column of Concanavalin A-Sepharose, confirming its glycoprotein nature. HPLC analysis of acid hydrolyzates of K28 toxin as well as Western-blots of beta-eliminated and/or endo H-treated killer toxin preparations probed with polyclonal alpha-toxin antibodies revealed that the carbohydrate moiety of K28 consists of D-mannose only, which is O-glycosidically linked via Ser/Thr residues to the protein part. The change in gel mobility of K28 after beta-elimination was caused by a decrease in molecular mass of about 1,800, corresponding to a carbohydrate moiety of 10 mannose r…
Role of β1H for the binding of C3b-coated particles to human lymphoid and phagocytic cells
Coating of EAC14oxy23b with highly purified human serum beta 1H globulin (beta 1H) led to acceleration of rosette formation with human peripheral blood lymphocytes (PBL), tonsil lymphocytes, B lymphoblastoid (Raji) cells, granulocytes and monocytes. This reaction was discernible from C3bi-dependent rosette formation. Enhancement of rosette formation of C3b cells by beta 1H was most effective at limiting amounts of C3 per EAC14oxy23b. The beta 1H effect was not due to trace contamination with C3b inactivator. beta 1H-dependent rosette formation with the various lymphoid and phagocytic cells could be suppressed by the F(ab')2 fragment of anti-beta 1H suggesting beta 1H-mediated binding of bet…
Cell cycle studies on the mode of action of yeast K28 killer toxin.
The virally encoded K28 killer toxin of Saccharomyces cerevisiae kills sensitive cells by a receptor-mediated process. DNA synthesis is rapidly inhibited, cell viability is lost more slowly and cells eventually arrest, apparently in the S phase of the cell cycle with a medium-sized bud, a single nucleus in the mother cell and a pre-replicated (1n) DNA content. Cytoplasmic microtubules appear normal, and no spindle is detectable. Arrest of a sensitive haploid yeast strain by alpha-factor at START gave complete protection for at least 4 h against a toxin concentration that killed non-arrested cells at the rate of one log each 2.5 h. Cells released from alpha-factor arrest were killed by toxin…
Mannoprotein of the yeast cell wall as primary receptor for the killer toxin of Saccharomyces cerevisiae strain 28.
The killer toxin KT 28 of Saccharomyces cerevisiae strain 28 is primarily bound to the mannoprotein of the cell wall of sensitive yeasts. The mannoprotein of S. cerevisiae X 2180 was purified; gel filtration and SDS-PAGE indicated an estimated Mr of 185,000. The ability to bind killer toxin KT 28 increased during purification of the mannoprotein. Removing the protein part of the mannoprotein by enzymic digestion or removing the alkali-labile oligosaccharide chains by beta-elimination did not destroy the ability to bind killer toxin KT 28. However, binding activity was lost when the 1,6-alpha-linkages of the outer carbohydrate backbone were hydrolysed by acetolysis. The separated oligomannos…