Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae

By the kar1-mediated cytoduction, linear double-stranded DNA plasmids pGKL1 and pGKL2, encoding killer toxin complex, have been successfully transferred to the recipient strains with about 30% frequency. The killer toxin was found to be secreted through the normal yeast secretory pathway by introducing pGKL plasmids into the several Saccharomyces cerevisiae sec mutants and examining the secretion of killer toxin. S. cerevisiae cells, harboring newly isolated deletion plasmid pGKL1D, expressed only the 28K protein among three killer subunits, and secreted the 28K subunit at a level of zero to 20% efficiency of the cells containing intact pGKL1 plasmid. These data indicated that subunit interaction (cosecretion) of killer proteins is required for the efficient secretion of 28K subunit. The 28K precursor protein was found to translocate across the canine pancreatic endoplasmic reticulum membrane under the direction of its own signal peptide in vitro without any other subunits. From kex2 mutant cells harboring pGKL1 plasmid, the 97K subunit, and its precursor 128K protein were not secreted, however, the 28K subunit was secreted in the same amount as that secreted from KEX2 cells. These lines of evidence suggest that the final assembly of killer toxin complex after KEX2 site of Golgi apparatus is not essential for the secretion of 28K subunit, and therefore, that putative interaction between 128K protein and 28K subunit for the transport between endoplasmic reticulum and Golgi apparatus may be required for the efficient secretion of 28K subunit.     

DNA replication in vivo of linear DNA killer plasmids pGKL1 and pGKL2 in Saccharomyces cerevisiae

Abstract Electron microscopic observation demonstrated that linear DNA plasmids, pGKL1 and pGKL2, were replicated by a strand displacement mechanism similar to adenovirus and Bacillus subtilis ø 29 phage. Moreover, their DNA replication was prevented by α-factor, a mating hormone which prevents the replication of chromosomal DNA and 2 μm plasmid in Saccharomyces cerevisiae mating type a cells. This result suggests that the replication of pGKL plasmids is controlled by the same genes that control the initiation or maintenance of chromosomal DNA and 2 μm plasmid replications.     

The yeast linear DNA killer plasmids, pGKL1 and pGKL2, possess terminally attached proteins.

The terminal structures of linear DNA killer plasmids from yeast, pGKL1 and pGKL2, were analyzed. Results obtained by exonuclease treatments of these plasmids show that both pGKL plasmids have free hydroxyl 3'-ends and blocked 5'-ends. Electrophoretic analysis of the terminal restriction fragments treated with proteases revealed that pGKL1 and pGKL2 have proteins bound at 5'termini and that the terminal protein of pGKL1 is distinct from that of pGKL2. This is the first linear DNA-terminal protein association found in yeast.     

Cloning and nucleotide sequences of the linear DNA killer plasmids from yeast.

The linear DNA killer plasmids (pGKL1 and pGKL2) isolated from a Kluyveromyces lactis killer strain are also maintained and expressed its killer character in Saccharomyces cerevisiae. After these killer plasmid DNAs isolated from S. cerevisiae were treated with alkali, four terminal fragments from each plasmid DNAs were cloned separately. Using these and other cloned DNA fragments, the terminal nucleotide sequences of pGKL2 and the complete nucleotide sequence of pGKL1 were determined. The inverted terminal repetitions of 202 bp and 182 bp were found in pGKL1 and pGKL2, respectively. The pGKL1 sequence showed an extremely high A + T content of 73.2% and it contained five large open reading frames. The largest of these open reading frame was suggested to code for a membrane-bound precursor of glycoprotein subunit of the killer toxin.     

Expression and identification of immunity determinants on linear DNA killer plasmids pGKL1 and pGKL2 in Kluyveromyces lactis.

The linear dsDNA plasmids, pGKL1 (8.9 kb) and pGKL2 (13.4 kb) discovered in Kluyveromyces lactis, confer killer and immunity characteristics upon various yeast strains. We have devised an immunity assay and have been able to show the expression of an immunity phenotype in the K. lactis transformants harbouring conventional circular plasmids which contain DNA fragments of pGKL1. Using this expression system, the immunity determinant on pGKL1 was identified as ORF5. In addition, the presence of pGKL2 was proved to be essential for the expression of the immunity phenotype. This is the first demonstration of this new pGKL2 function, as distinct from its known functions for the replication and maintenance of pGKL1 in yeast cells.     

Palindrome-hairpin linear plasmids possessing only a part of the ORF1 gene of the yeast killer plasmid pGKL1

Summary The yeast Kluyveromyces lactis haboring linear DNA plasmids pGKL1 and pGKL2 exhibits killer and killer-resistant phenotypes. Two new linear plasmids pK192L and pK192S were found in the weak killer mutant KUV192 induced by UV irradiation. pK192S was always accompanied by pK192L in subclones of KUV192. Both plasmids were derived from pGKL1 by deletion of the large right part of it. pK192L was 4.9 kb in size and had a palindromic structure consisting of 2.35 kb inverted terminal repetitions and a 215 base unique sequence. Analysis of denatured and renatured DNA strands suggested that pK192S was a hairpin-like form of pK192L. The pK192 plasmids were maintained only in cells haboring either pGKL1 or pGKL1S in addition to pGKL2 and competed with pGKL1 or pGKL1S for their maintenance. Since no complete ORF1 was conserved in pK192 plasmids, these results lead to the conclusion that the ORF1 gene is necessary for the replication and/or maintenance of pGKL1.     

A novel yeast secretion signal isolated from 28K killer precursor protein encoded on the linear DNA plasmid pGKL1.

Saccharomyces cerevisiae harboring linear dsDNA plasmids, pGKL1 and pGKL2, secretes a killer toxin consisting of 97, 31 and 28 kilodalton subunits (Nucleic Acids Res., 15, 1031-1046, 1987). We isolated the DNA encoding the N-terminal pre-sequence of the 28K precursor protein and constructed a new secretion vector in S. cerevisiae. Mouse alpha-amylase fused to the 28K signal sequence was secreted into the culture medium with a high efficiency similar to those fused to the mating factor alpha and 97K-31K killer signal sequences. This data clearly indicates that 28K presequence functions as a secretion signal. Glycosylated and nonglycosylated alpha-amylase molecules were detected in the culture medium. The secretion of alpha-amylase was blocked by sec18-1 mutation. The secreted alpha-amylase recovered from the medium was found to migrate faster in SDS-polyacrylamide gel than the precursor form of alpha-amylase synthesized in vitro. These lines of evidence suggest that mouse alpha-amylase fused to 28K killer signal sequence was processed, glycosylated and secreted through the normal secretion pathway of the yeast.     

Expression of pGKL killer 28K subunit in Saccharomyces cerevisiae: identification of 28K subunit as a killer protein.

Saccharomyces cerevisiae and other yeast cells harboring the linear double stranded (ds) DNA plasmids pGKL1 and pGKL2 secrete a killer toxin consisting of 97K, 31K and 28K subunits into the culture medium (EMBO J. 5, 1995-2002 (1986), Nucleic Acids Res., 15, 1031-1046 (1987]. The 28K subunit of the killer toxin was successfully expressed in S. cerevisiae when it was cloned on a circular plasmid with its putative promoter region replaced with that of S. cerevisiae chromosomal genes. The expression of the 28K subunit of the killer toxin in killer-sensitive cells resulted in the death of the host cells. This killing activity by the 28K subunit was prevented by the expression of the killer immunity, indicating that the killing activity of the killer toxin complex was carried out by the 28K subunit. Although the 28K subunit was synthesized as a intact precursor protein with its own signal sequence, it was not secreted into the culture medium but remained in the host cells. This indicated that 28K subunit killed host cells from inside of the cells rather than from outside. We further suggested that 28K killer subunit without 97K and 31K subunits did not kill the killer-sensitive cells from outside.     

Transformation of Saccharomyces cerevisiae with linear DNA killer plasmids from Kluyveromyces lactis.

Protoplasts of Saccharomyces cerevisiae were mixed with linear DNA plasmids, pGKl1 and pGKl2, isolated from a Kluyveromyces lactis killer strain and treated with polyethylene glycol. Out of 2,000 colonies regenerated on a nonselective medium, two killer transformants were obtained. The pGKl plasmids and the killer character were stably maintained in one (Pdh-1) of them. Another transformant, Pdl-1, was a weak killer, and the subclones consisted of a mixture of weak and nonkiller cells. The weak killers were characterized by the presence of pGKl1 in a decreased amount, and nonkillers were characterized by the absence of pGKl1. The occurrence of two new plasmids which migrated faster than pGKl1 in an agarose gel was observed in Pdl-1 and its subclones, whether weak or nonkillers. Staining with 4',6-diamidino-2-phenylindole revealed that the pGKl plasmids exist in the cytosol of transformant cells with numerous copy numbers.     

Hairpin plasmid--a novel linear DNA of perfect hairpin structure.

The terminal structures of deletion derivatives of linear DNA killer plasmid from yeast were analyzed. The yeast Kluyveromyces lactis harbors two unique double-stranded linear DNA killer plasmids, pGKL1 of 8.9 kb and pGKL2 of 13.4 kb. The killer toxin and the resistance to the killer are coded by pGKL1, while pGKL2 is required for the maintenance of pGKL1 in the cell. When the pGKL plasmids from K. lactis were transferred into Saccharomyces cerevisiae by transformation, non-killer transformants harboring pGKL2 and new plasmids, F1 of 7.8 kb and F2 of 3.9 kb, were obtained. F2 was shown to be a linear DNA arising from a 5-kb deletion of the right part of pGKL1. F1 was an inverted dimer of F2. Here we show that F2 has two different terminal structures: one end has a protein attached at the 5' terminus whereas the two strands of duplex are linked together at the other end, thus forming a hairpin structure. This is a novel type of autonomously replicating DNA molecule.     

Mice with only rat mtDNA are required as models of mitochondrial diseases

We examined the possibility of generation of mice expressing mitochondrial dysfunction by introduction of exogenous mtDNA from different species using mouse mtDNA-less (rho(0)) cells as mtDNA recipients. For determination of how genetically distant species of mtDNA could replicate in cells with only the mouse nuclear genome, we introduced mtDNA of the Syrian hamster (Mesocricetus auratus) into mouse rho(0) cells, and found that its replication was not sufficient to propagate to following generations, probably due to significant incompatibility between mouse-nuclear and Syrian hamster-mitochondrial genomes. On the other hand, rat mtDNA, which propagated stably and expressed mitochondrial dysfunction in mouse cells, also disappeared rapidly by exogenous introduction of mouse mtDNA, suggesting that mouse mtDNA in mouse cells must be excluded completely before introduction of rat mtDNA for generation of mice with rat mtDNA as mitochondrial disease models.     

DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.     

A novel yeast secretion vector utilizing secretion signal of killer toxin encoded on the yeast linear DNA plasmid pGKL1

The NH2-terminal signal region comprising of approximately 70% length of the prepro-sequence of the pGKL killer precursor protein was found to direct an efficient secretion of the mouse alpha-amylase into the culture medium of Saccharomyces cerevisiae. The alpha-amylase molecule secreted into the culture medium was identified by both immuno-blotting and assay of the enzyme activity. The amount of alpha-amylase secreted via the killer toxin signal was comparable to that directed by the leader sequence of mating factor alpha. The secretion of alpha-amylase using the killer toxin signal was blocked at 37C but not at 25C in sec18-1 host, indicating that alpha-amylase is exported through the normal secretion pathway of S. cerevisiae.     

Oltipraz-induced phase 2 enzyme response conserved in cells lacking mitochondrial DNA

Oltipraz, a member of a class of 1,2-dithiolethiones, is a potent phase 2 enzyme inducing agent used as a cancer chemopreventive. In this study, we investigated regulation of the phase 2 enzyme response and protection against endogenous oxidative stress in lymphoblastic leukemic parental CEM cells and cells lacking mitochondrial DNA (mtDNA) (rho0) by oltipraz. Glutathione (GSH) levels (total and mitochondrial) and glutathione S-transferase (GST) activity were significantly increased after pretreatment with oltipraz in both parental (rho+) and rho0 cells, and both cell lines were resistant to mitochondrial oxidation, loss of mitochondrial membrane potential, and cell death in response to the GSH depleting agent diethylmaleate. These results show that the phase 2 enzyme response, by enhancing GSH-dependent systems involved in xenobiotic metabolism, blocks endogenous oxidative stress and cell death, and that this response is intact in cells lacking mtDNA.     

The bivious suppressiveness of cytoplasmic petites of S. cerevisiae lacking in mitochondrial DNA

Summary When crossed to strain GR25 [rho+], petites lacking in mtDNA were neutral, but when crossed to a related [rho+] strain (GR25a) they were found to be suppressive (Table 1). Likewise, crosses of GR25 [rho+] and GR25a [rho+] to a common [rho+] parent were, respectively, neutral and suppressive (Table 1). The suppressive phenotype observed in these crosses was attributed to a factor in the [rho+] strain GR25a. Strain GR25a also differed from strain GR25 in having a decreased [rho+] stability (Table 2) and a decreased transmission of its cytoplasmically-inherited erythromycin-resistance marker to zygote progeny (Table 4). These three phenotypes of GR25a are, discussed in terms of a nuclear mutation in a gene responsible for the maintenance of the [rho+] state.     

Preliminary characterisation of DML1, an essential Saccharomyces cerevisiae gene related to misato of Drosophila melanogaster

A genetic and cell-biological analysis is provided for Saccharomyces cerevisiae DML1 (YMR211w) encoding a Drosophila melanogaster Misato-like protein. Misato and Dml1p are descendants of an ancestral tubulin-like protein, and exhibit regions with similarity to members of a GTPase family that include eukaryotic tubulin and prokaryotic FtsZ. Deletion of DML1 was lethal to haploid cells; sporulated DML1/dml1Delta heterozygotes from different genetic backgrounds gave rise to no more than two viable spores per tetrad. DAPI staining for DNA in combination with Southern analysis using the mitochondrial genes COX3, 15S_rRNA_2, and COB revealed that a significant portion of the surviving meiotic progeny were [rho(0)] lacking mtDNA. In addition, meiotic transmission of centromeric plasmids also appeared to be impaired. Self-complementation using extra-chromosomal copies of DML1 efficiently restored meiotic inheritance of mtDNA, but improved spore viability ratios only in part. Inheritance of mtDNA could also be restored using misato cDNA. Unscheduled expression of DML1 tethered to the inducible ADH2 promoter altered both mitochondrial dispersion and general cell morphology. We propose that Dml1p and Misato have been co-opted into a role in mtDNA inheritance in yeast, and into a cell division-related mechanism in flies, respectively. Dml1p might additionally function in the partitioning of the mitochondrial organelle itself, or in the segregation of chromosomes, thereby explaining its essential requirement.     

Intergeneric transfer of deoxyribonucleic acid killer plasmids, pGKl1 and pGKl2, from Kluyveromyces lactis into Saccharomyces cerevisiae by cell fusion

Two novel linear deoxyribonucleic acid plasmids, pGKl1 and pGKl2, were isolated from the yeast Kluyveromyces lactis. K. lactis strains harboring the pGK1 plasmids killed a certain group of yeasts, including Saccharomyces cerevisiae, Saccharomyces italicus, Saccharomyces rouxii, K. lactis, Kluyveromyces thermotolerans, Kluyvermyces vanudenii, Torulopsis glabrata, Candida utilis, and Candida intermedia. In this experiment, the pGKl1 and pGKl2 plasmids were intergenerically transferred from a K. lactis killer strain into a non-killer (killer-sensitive) strain of S. cerevisiae by the use of a protoplast fusion technique. Both of the pGKl plasmids replicated autonomously and stably in the new host cells of S. cerevisiae and could coexist with the resident 2-micrometers deoxyribonucleic acid plasmid. The S. cerevisiae cells which accepted the pGKl plasmids expressed the same killer phenotype as that of the donor K. lactis killer and became resistant to the K. lactis killer. The pGKl plasmids existing in the S. cerevisiae cells were cured by treatment with ethidium bromide, and the killer and resistance characters were simultaneously lost. From there results, it was concluded that both the killer and the resistance genes are located on the pGKl plasmids.     

Nuclear-mitochondrial epistasis for fitness in Saccharomyces cerevisiae

In addition to the familiar possibility of epistasis between nuclear loci, interactions may evolve between the mitochondrial and nuclear genomes in eukaryotic cells. We looked for such interactions in Saccharomyces cerevisiae genotypes evolved independently and asexually in the laboratory for 2000 generations, and in an ecologically distinct pathogenic S. cerevisiae strain. From these strains we constructed derivatives entirely lacking mitochondrial DNA and then used crosses to construct matched and unmatched pairings of nuclear and mitochondrial genomes. We detected fitness effects of such interactions in an evolved laboratory strain and in crosses between the laboratory and pathogen strains. In both cases, there were significant contributions to progeny fitness of both nuclear and mitochondrial genomes and of their interaction. A second evolved genotype showed incompatibility with the first evolved genotype, but the nuclear and mitochondrial contributions to this incompatibility could not be resolved. These results indicate that cytonuclear interactions analogous to those already known from plants and animals can evolve rapidly on an evolutionary timescale.     

Nuclear complementation restores mtDNA levels in cultured cells from a patient with mtDNA depletion.

We have studied cultured skin fibroblasts from a patient with a fatal mitochondrial disease manifesting soon after birth. These fibroblasts were found to grow only in the presence of pyruvate and uridine, a characteristic of cells lacking mtDNA (rho0 cells). Southern blot and PCR analyses confirmed that the patient's fibroblasts contained less than 2% of control levels of mtDNA. Biochemical analyses indicated that the activities of all the respiratory-chain enzymes were severely decreased in mitochondria isolated from these fibroblasts. In order to elucidate the underlying molecular defect, cell fusions were performed between enucleated fibroblasts from this patient and a human-derived rho0 cell line (rho0 A549.B2). The resulting cybrids were plated in medium lacking pyruvate and uridine, to select for the restoration of respiratory-chain function. Complementation was observed between the nuclear genome of the rho0 A549.B2 cells and the mtDNA of the patient's cells, restoring mtDNA levels and respiratory-chain function in the cybrid cells. These results indicate that mtDNA depletion in our patient is under the control of the nuclear genome.