Biomedical subjects

D J Tipper

Publications and source records attributed to D J Tipper.

At least 19 recordsLinked to original sources

PubMed1999

A novel membrane protein influencing cell shape and multicellular swarming of Proteus mirabilis.

Swarming in Proteus mirabilis is characterized by the coordinated surface migration of multicellular rafts of highly elongated, hyperflagellated swarm cells. We describe a transposon mutant, MNS185, that was unable to swarm even though vegetative cells retained normal motility and the ability to differentiate into swarm cells. However, these elongated cells were irregularly curved and had variable diameters, suggesting that the migration defect results from the inability of these deformed swarm cells to align into multicellular rafts. The transposon was inserted at codon 196 of a 228-codon gene that lacks recognizable homologs. Multiple copies of the wild-type gene, called ccmA, for curved cell morphology, restored swarming to the mutant. The 25-kDa CcmA protein is predicted to span the inner membrane twice, with its C-terminal major domain being present in the cytoplasm. Membrane localization was confirmed both by immunoblotting and by electron microscopy of immunogold-labelled sections. Two forms of CcmA were identified for wild-type P. mirabilis; they were full-length integral membrane CcmA1 and N-terminally truncated peripheral membrane CcmA2, both present at approximately 20-fold higher concentrations in swarm cells. Differentiated MNS185 mutant cells contained wild-type levels of the C-terminally truncated versions of both proteins. Elongated cells of a ccmA null mutant were less misshapen than those of MNS185 and were able to swarm, albeit more slowly than wild-type cells. The truncated CcmA proteins may therefore interfere with normal morphogenesis, while the wild-type proteins, which are not essential for swarming, may enhance migration by maintaining the linearity of highly elongated cells. Consistent with this view, overexpression of the ccmA gene caused cells of both Escherichia coli and P. mirabilis to become enlarged and ellipsoidal.

Amino Acid Sequence↗

PubMed1998

Transmembrane protein insertion orientation in yeast depends on the charge difference across transmembrane segments, their total hydrophobicity, and its distribution.

The determinants of transmembrane protein insertion orientation at the endoplasmic reticulum have been investigated in Saccharomyces cerevisiae using variants of a Type III (naturally exofacial N terminus (Nexo)) transmembrane fusion protein derived from the N terminus of Ste2p, the alpha-factor receptor. Small positive and negative charges adjacent to the transmembrane segment had equal and opposite effects on orientation, and this effect was independent of N- or C-terminal location, consistent with a purely electrostatic interaction with response mechanisms. A 3:1 bias toward Nexo insertion, observed in the absence of a charge difference, was shown to reflect the Nexo bias conferred by longer transmembrane segments. Orientation correlated best with total hydrophobicity rather than length, but it was also strongly affected by the distribution of hydrophobicity within the transmembrane segment. The most hydrophobic terminus was preferentially translocated. Insertion orientation thus depends on integration of responses to at least three parameters: charge difference across a transmembrane segment, its total hydrophobicity, and its hydrophobicity gradient. Relative signal strengths were estimated, and consequences for topology prediction are discussed. Responses to transmembrane sequence may depend on protein-translocon interactions, but responses to charge difference may be mediated by the electrostatic field provided by anionic phospholipids.

Amino Acid Sequence↗

PubMed1997

A nonswarming mutant of Proteus mirabilis lacks the Lrp global transcriptional regulator.

Proteus swarming is the rapid cyclical population migration across surfaces by elongated cells that hyperexpress flagellar and virulence genes. The mini-Tn5 transposon mutant mns2 was isolated as a tight nonswarming mutant that did not elongate or upregulate flagellar and hemolysin genes. Individual cell motility was retained but was reduced. The transposon had inserted in the gene encoding the global transcriptional regulator Lrp (leucine-responsive regulatory protein), expression of which was upregulated in differentiating swarm cells. Swarming was restored to the lrp mutant by artificial overexpression of the flhDC flagellar regulatory master operon. Lrp may be a key component in generating or relaying signals that are required for flagellation and swarming, possibly acting through the flhDC operon.

Amino Acid Sequence↗

PubMed1996

The role of charged residues in determining transmembrane protein insertion orientation in yeast.

The first 79 residues of the yeast Ste2p G protein-coupled pheromone receptor, including the negatively charged N-terminal domain, the first transmembrane segment, and the following positively charged cytoplasmic loop, has been fused to a Kex2p-cleavable beta-lactamase reporter. Insertion orientation was determined by analysis of cell-associated and secreted beta-lactamase activities and independently corroborated by analysis of membrane association and glycosylation patterns. This fusion inserts with exclusively N terminus exofacial (Nexo) topology, serving as a model type III membrane protein. Orientation is unaffected by removal of all three positively charged residues in the cytoplasmic loop or by deletion of all but 12 residues from the N-terminal domain. The residual -2 N-terminal charge apparently provides a signal sufficient to determine Nexo topology. This is entirely consistent with the statistically derived rule in which the charge difference, Delta(C-N), counted for the 15 immediately flanking residues, is the primary topology determinant. Mutations altering Delta(C-N) to zero favors Nexo insertion by 3 to 1, whereas increasingly negative values cause increasing inversion of orientation. All results are consistent with the charge difference rule and indicate that whereas positive charges promote cytoplasmic retention, negative charges promote translocation.

Amino Acid Sequence↗

PubMed1996

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 at a similar rate; arrest occurred with medium-sized buds within the same cell cycle. Cells arrested by hydroxyurea, with unreplicated DNA, or by the spindle poison methylbenzimidazol-2yl-carbamate, with unseparated chromosomes, both arrest at the checkpoint at the G2/M boundary; these arrested cells were not protected against toxin, losing about one log of viability every 4 h. Following release from the cell cycle block, a majority of these toxin-exposed cells progressed through the cell cycle and arrested in the following S-phase, again with medium-sized buds. Killing by K28 toxin apparently requires entry into the nuclear division and bud cycles, but can result from inhibition of either early or late events in these cycles. Morphogenesis in moribund cells is uniformly blocked in early S-phase with an immature bud. Toxin action causes either independent blockage of both DNA synthesis and the budding cycle, or inhibits some unknown step required for both events.

4-Butyrolactone↗

PubMed1995

Sequence of the M28 dsRNA: preprotoxin is processed to an alpha/beta 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 whose N-termini correspond to M28p residues 50-61 and 246-257, respectively. alpha is preceded by a potentially redundant pair of secretion signal peptides; deletion of the first reduces toxin secretion by 75%. While M28p bears no sequence similarity to M1p, the K1 preprotoxin, the predicted patterns of processing by glycosylation and cleavage are remarkably similar. The beta N- and C-termini are probably processed by Kex2p and Kex1p, respectively; the mechanism of cleavage at the less typical sites bounding the alpha component is under investigation. While a kex2 delta mutation prevents toxin secretion, secreted toxin retains 20% activity in a kex1 delta mutant. Neither mutation affects immunity.

Amino Acid Sequence↗

PubMed1994

Use of beta-lactamase as a secreted reporter of promoter function in yeast.

K1 preprotoxin is the 316 residue precursor of the K1 killer toxin secreted by the yeast Saccharomyces cerevisiae. The SP beta la reporter consists of the mature, secreted form of beta-lactamase (beta la) fused to S and P, two fragments of preprotoxin. S is the N-terminal 34 residues, including the secretion signal. P, a 67 residue 'processing' segment with three sites for N-glycosylation, terminates in a Lys Arg site for cleavage by the Kex2 protease. Expression of SP beta 1a in yeast results in efficient secretion, processing by signal peptidase and glycosylation in the endoplasmic reticulum, producing pro beta la. Kex2 cleavage of pro beta la in the lumen of a late Golgi compartment releases beta la, which accumulates stably in culture media buffered at pH 5.8-7. The half-life of secretion is 11 min at 30 degrees C; 10-12% of the total activity in exponential-phase cells is intracellular, mostly in the form of pro beta la, indicating that transit from the endoplasmic reticulum to the Golgi is rate limiting. We have used SP beta la expression in single- and multi-copy vectors to compare the PGK, GAL1, GAL10, PHO5 and CUP1 promoters under varying nutritional conditions. In exponential-phase cells, secretion of beta la over a 40-fold range and up to several micrograms/ml was proportional to transcript level, demonstrating that SP beta la can be employed as a convenient secreted reporter of promoter function in yeast.

Amino Acid Sequence↗

PubMed1993

Role of the gamma component of preprotoxin in expression of the yeast K1 killer phenotype.

K1 killer strains of Saccharomyces cerevisiae secrete a polypeptide toxin to which they are themselves immune. The alpha and beta components of toxin comprise residues 45-147 and 234-316 of the 316-residue K1 preprotoxin. The intervening 86-residue segment is called gamma. A 26-residue signal peptide is removed on entry into the endoplasmic reticulum. The Kex2 protease excises the toxin components from the 290-residue glycosylated protoxin in a late Golgi compartment. Expression of a cDNA copy of the preprotoxin gene confers the complete K1 killer phenotype on sensitive cells. We now show that expression of immunity requires the alpha component and the N-terminal 31 residues of gamma. An additional C-terminal extension, either eight residues of gamma or three of four unrelated peptides, is also required. Expression of preprotoxin terminating at the alpha C-terminus, or lacking the gamma N-terminal half of gamma causes profound but reversible growth inhibition. Inhibition is suppressed in cis by the same 31 residues of gamma required for immunity to exocellular toxin in trans, but not by the presence of beta. Both immunity and growth inhibition are alleviated by insertions in alpha that inactivate toxin. Inhibition is not suppressed by kex2, chc1 or kre1 mutations, by growth at higher pH or temperature, or by normal K1 immunity. Inhibition, therefore, probably does not involve processing of the alpha toxin component at its N-terminus or release from the cell and binding to glucan receptors. Some insertion and substitution mutations in gamma severely reduce toxin secretion without affecting immunity. They are presumed to affect protoxin folding in the endoplasmic reticulum and translocation to the Golgi.

Amino Acid Sequence↗

PubMed1993

Suppression of a dominant G-protein beta-subunit mutation in yeast by G alpha protein expression.

SCG1/GPA1, STE4 and STE18 encode the alpha, beta and gamma components of the G protein involved in mating pheromone signal transduction in Saccharomyces cerevisiae. Responses, including G1 arrest and expression of genes such as FUS1, are activated by beta gamma, which is negatively controlled by alpha(GDP). We previously demonstrated that overexpression of Scg1 suppresses responses to alpha factor and that expression of certain hybrids between Scg1 and mammalian G alpha proteins has the same effect and also suppresses growth arrest in an scg1-null mutant. Effects were attributed to sequestration of beta gamma. We now show that effects on growth rate, morphology and FUS1 expression are consistent with this model. The STE4HPL allele causes dominant activation of the response pathway, and is presumed to encode a beta subunit insensitive to control by alpha(GDP). Scg1 overexpression suppresses the growth arrest due to STE4HPL; normal alpha-factor responses and fertility are restored. A model based on sequestration of beta gamma reconciles this result with the apparent paradox that the same level of Scg1 overexpression inhibits responses and mating in wild-type cells. A G alpha i hybrid also restores growth and allows inefficient mating in the STE4HPL strain.

Cell Cycle↗

PubMed1992

Efficient secretion in yeast based on fragments from K1 killer preprotoxin.

The alpha and beta components of the secreted K1 killer toxin of Saccharomyces cerevisiae are derived from residues 45-147 and 234-316, respectively, of the 316 residue preprotoxin (ppTox). The beta N-terminus is produced by Kex2 cleavage after Lys Arg233; when beta la (the mature sequence of beta-lactamase) is fused at this site and the fusion is expressed from the PGK promoter in pDT17, a multicopy plasmid, unexpectedly modest levels of beta la secretion resulted. Over-expression of Kex2 failed to increase beta la secretion while a kex2-null mutation reduced secretion by 98%. beta la secretion in a Kex+ strain was not enhanced by inactivation of the alpha toxin component or by deletion of most of its central hydrophobic segments. However SP-beta la, produced by deletion of ppTox residues 35-176, expressed 10-fold higher beta la activity and the precursor was now secreted with similar efficiency in a kex2-null strain. Fusions of beta la to ppTox at Ala34 or Ala46 also led to efficient secretion in both KEX2 and kex2-null strains. Since these beta la fusions differ only in segments well downstream of the signal peptide and all had similar transcript levels, the efficiency of beta la secretion is apparently determined by the efficiency with which these fusions are translocated to the Golgi compartment where Kex2 is active. Efficiency is high for the shorter fusions, but is 10% or less for the longer fusions; even this fraction is apparently diverted to the vacuole if not cleaved by Kex2. SP-beta la was the most efficient construct tested; secreted beta la reached 4% of total cell protein, modestly exceeding levels produced by fusion to the MF alpha 1-encoded prepro alpha-factor, suggesting potential for the production of foreign proteins in yeast.

Amino Acid Sequence↗

PubMed1992

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 genetic and biochemical analysis of the effects of representative mak, ski and mkt mutations on M28 maintenance, demonstrating that M28 replication resembles M1 in all respects. We also show that L28 is an L-A-H species lacking [B] activity, and that M28 excludes both M1 and M2 from the same cytoplasm. Stable coexpression of K28 phenotype from M28 and of K1 phenotype from an M1-cDNA clone was demonstrated. Exclusion, therefore, acts at the level of dsRNA replication, presumably reflecting competition for the L-A-H encoded capsid and cap-pol fusion protein, rather than reflecting incompatibility of toxin or immunity expression. Finally, we show that expression of active K28 toxin, but not of K28 immunity, requires the Kex2 endoprotease.

Fungal Proteins↗

PubMed1992

Kex2-dependent processing of yeast K1 killer preprotoxin includes cleavage at ProArg-44.

The K1 killer toxin of Saccharomyces cerevisiae consists of 103- and 83-residue alpha and beta components whose derivation, from a 316-residue precursor preprotoxin, requires processing at the alpha N-terminus (after ProArg-44), the alpha C-terminus (after ArgArg-149) and at the beta N-terminus (after LysArg-233). These processing events occur after translocation to the Golgi and have been investigated using beta-lactamase fusions. Signal peptidase cleavage of the precursor, predicted to occur after Ala-26, was confirmed by N-terminal sequence analysis of Ala-34 and Ile-52 fusions. Cleavage at all of the other predicted processing sites, including ProArg-44, is dependent on activity of the Kex2 protease. A fourth Kex2-dependent cleavage occurs at LysArg-188. Implications for the specificity of Kex2 cleavage and preprotoxin processing are discussed.

Amino Acid Sequence↗

PubMed1991

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 killer viruses are satellite dsRNAs that use a similar encapsidation domain to parasitize the L-A replication machinery. They encode precursors of secreted polypeptide toxins and immunity (specific resistance) determinants and are self-selecting. Three unique killer types, K1, K2 and K28, are currently recognized. They are distinguished by an absence of cross-immunity and by toxin properties and lethal mechanisms; while K1 and K2 toxins bind to cell-wall glucan and disrupt membrane functions, K28 toxin binds to mannoprotein and causes inhibition of DNA synthesis.

DNA Topoisomerases, Type I↗

PubMed1991

In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions.

Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.

Amino Acid Sequence↗

PubMed1990

Effects of expression of mammalian G alpha and hybrid mammalian-yeast G alpha proteins on the yeast pheromone response signal transduction pathway.

Scg1, the product of the Saccharomyces cerevisiae SCG1 (also called GPA1) gene, is homologous to the alpha subunits of G proteins involved in signal transduction in mammalian cells. Scg1 negatively controls the pheromone response pathway in haploid cells. Either pheromonal activation or an scg1 null mutation relieves the negative control and leads to an arrest of cell growth in the G1 phase of the cell cycle. Expression of rat G alpha s was previously shown to complement the growth defect of scg1 null mutants while not allowing mating. We have extended this analysis to examine the effects of the short form of G alpha s (which lacks 15 amino acids present in the long form), G alpha i2, G alpha o, and Scg1-mammalian G alpha hybrids. In addition, we have found that constructs able to complement scg1 are also able to inhibit the response to pheromone and mating when expressed in a wild-type SCG1 strain. Overexpression of Scg1 has a similar inhibitory effect. These results are consistent with a model proposed for the action of Scg1 as the alpha component of a heterotrimeric G protein in which the beta gamma component (Ste4/Ste18) activates the pheromone response after dissociation from Scg1. They suggest that the G alpha constructs able to complement scg1 can interact with beta gamma to prevent activation of the pathway but are unable to interact with pheromone receptors to activate the pathway.

Animals↗

PubMed1990

K28, a unique double-stranded RNA killer virus of Saccharomyces cerevisiae.

The double-stranded RNA (dsRNA) viruses of Saccharomyces cerevisiae consist of 4.5-kilobase-pair (kb) L species and 1.7- to 2.1-kb M species, both found in cytoplasmic viruslike particles (VLPs). The L species encode their own capsid protein, and one (LA) has been shown to encode a putative capsid-polymerase fusion protein (cap-pol) that presumably provides VLPs with their transcriptase and replicase functions. The M1 and M2 dsRNAs encode the K1 and K2 toxins and specific immunity mechanisms. Maintenance of M1 and M2 is dependent on the presence of LA, which provides capsid and cap-pol for M dsRNA maintenance. Although a number of different S. cerevisiae killers have been described, only K1 and K2 have been studied in any detail. Their secreted polypeptide toxins disrupt cytoplasmic membrane functions in sensitive yeast cells. K28, named for the wine S. cerevisiae strain 28, appears to be unique; its toxin is unusually stable and disrupts DNA synthesis in sensitive cells. We have now demonstrated that 4.5-kb L28 and 2.1-kb M28 dsRNAs can be isolated from strain 28 in typical VLPs, that these VLPs are sufficient to confer K28 toxin and immunity phenotypes on transfected spheroplasts, and that the immunity of the transfectants is distinct from that of either M1 or M2. In vitro transcripts from the M28 VLPs show no cross-hybridization to denatured M1 or M2 dsRNAs, while L28 is an LA species competent for maintenance of M1. K28, encoded by M28, is thus the third unique killer system in S. cerevisiae to be clearly defined. It is now amenable to genetic analysis in standard laboratory strains.

Blotting, Northern↗

PubMed1987

The major acid-soluble proteins of Bacillus subtilis spores: partial amino acid sequence and forespore location of their mRNAs.

In Bacillus subtilis the alpha, beta, gamma and delta components comprise 80-90% of the total acid-soluble spore proteins (ASSPs). Sequence analysis demonstrates that alpha and beta share 32 of their first 36 amino acids and are closely related to the A and C ASSPs of Bacillus megaterium spores, confirming the results of analysis of their cloned genes. Despite the difference in apparent size of gamma and delta, they have identical N-terminal sequences (37 residues). Unless gamma and delta derive from very recently duplicated genes, it appears that gamma is derived from delta, either in vivo or during isolation. Although the sequenced regions of gamma and delta have no homology to alpha and beta, outside of the previously recognized pentapeptide recognition sequence for the spore endopeptidase, they share 10 and 15 residue peptides flanking this sequence with ASSP B of B. megaterium, but in reverse order. At least two groups of ASSPs have, therefore, been conserved between B. subtilis and B. megaterium: the multigene AC alpha beta family and the B gamma (delta) group. Sequence conservation in each group implies selection for functions in addition to storage. Both the alpha and beta components of B. subtilis ASSPs and their mRNAs are located in the forespore compartment of cells at t5.5 of sporulation, the time of most rapid ASSP synthesis. The sizes of these transcripts (250-350 bp) and their ability to direct the in vitro synthesis of ASSPs of mature size, indicate that genes for these ASSPs are monocistronic, consistent with dispersed map location. Synthesis of ASSPs is, therefore, coordinately controlled by selective transcription in the forespore.

Amino Acid Sequence↗

PubMed1986

Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin.

Strains of Saccharomyces cerevisiae harboring M1-dsRNA, the determinant of type 1 killer and immunity phenotypes, secrete a dimeric 19-kd toxin that kills sensitive yeast cells by the production of cation-permeable pores in the cytoplasmic membrane. The preprotoxin, an intracellular precursor to toxin, has the domain sequence delta-alpha-gamma-beta where alpha and beta are the 9.5-and 9.0-kd subunits of secreted toxin. Plasmids containing a partial cDNA copy of M1, in which alpha, gamma, and beta are fused to the PH05 promoter and signal peptide, have previously been shown to express phosphate-repressible toxin production and immunity. Here the construction of a complete DNA copy of the preprotoxin gene and its mutagenesis are described. Analysis of the expression of these mutants from the PH05 promoter elucidates the functions of the preprotoxin domains. delta acts as a leader peptide and efficiently mediates the secretion, glycosylation and maturation of killer toxin. Mutations within the beta subunit indicate it to be essential for binding of toxin to and killing of whole cells but unnecessary for the killing of spheroplasts. Mutations within the putative active site of alpha prevent killing of both cells and spheroplasts. The probable role of beta is therefore recognition and binding to the cell wall receptor whereas alpha is the active ionophore. Mutations within alpha causing loss of toxicity also cause loss of immunity, while the mutants described within gamma and beta retain partial or complete immunity. Expression of gamma without alpha or beta confers no phenotype. The immunity determinant may minimally consist of the alpha domain and the N-terminal portion of gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗