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FEMS Microbiology Reviews 26 (2002) 257^276
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The viral killer system in yeast:
from molecular biology to application
Manfred J. Schmitt , Frank Breinig
Angewandte Molekularbiologie (FR 8.3 ^ Mikrobiologie), Universita«t des Saarlandes, Im Stadtwald, Geba«ude 2, D-66123 Saarbru«cken, Germany
Received 5 March 2002; received in revised form 26 March 2002; accepted 28 March 2002
First published online 8 May 2002
Abstract
Since the initial discovery of the yeast killer system almost 40 years ago, intensive studies have substantially strengthened our knowledge
in many areas of biology and provided deeper insights into basic aspects of eukaryotic cell biology as well as into virus^host cell
interactions and general yeast virology. Analysis of killer toxin structure, synthesis and secretion has fostered understanding of essential
cellular mechanisms such as post-translational prepro-protein processing in the secretory pathway. Furthermore, investigation of the
receptor-mediated mode of toxin action proved to be an effective means for dissecting the molecular structure and in vivo assembly of
yeast and fungal cell walls, providing important insights relevant to combating infections by human pathogenic yeasts. Besides their
general importance in understanding eukaryotic cell biology, killer yeasts, killer toxins and killer viruses are also becoming increasingly
interesting with respect to possible applications in biomedicine and gene technology. This review will try to address all these
aspects. , 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Yeast; Prepro-protein processing ; Toxin secretion; Receptor-mediated toxicity; Endocytosis ; Retrograde transport ; Antifungal ; Killer virus;
Heterologous protein secretion
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Molecular biology of killer yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Genetic basis of killer phenotype expression: cytoplasmic RNA viruses . . . . . . . . . . .
2.2. dsRNA viruses in S. cerevisiae: L-A and its toxin-coding M satellites . . . . . . . . . . . . .
2.3. Preprotoxin processing and toxin secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Toxicity of the killer proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Toxin immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6. Virus^host cell interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7. Killer viruses in the non-conventional yeasts Hanseniaspora uvarum and Zygosaccharomyces bailii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Killer yeasts in wine fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Killer toxins as potential antifungals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Killer toxin expression in transgenic plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Heterologous protein secretion via pptox secretion and processing signals . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3.
4.
* Corresponding author. Tel. : +49 (681) 302 4730 ; Fax: +49 (681) 302 4710.
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E-mail address: [email protected] (M.J. Schmitt).
0168-6445 / 02 / $22.00 , 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
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Fig. 1. Phenotype of a K28 killer and a non-killer strain of the yeast S. cerevisiae. A: A toxin-secreting K28 killer strain expressing functional toxin immunity (Kþ Iþ ) was streaked onto methylene blue agar (MBA; pH 4.7) that had been seeded with a lawn of a sensitive indicator non-killer strain.
After 3 days of incubation at 20‡C, clear zones of growth inhibition around the streak of the killer strain indicate K28 toxin secretion. B: A non-killer
strain lacking functional immunity (K3 I3 ) was streaked onto an MBA plate that had been seeded with a K28 toxin-secreting killer strain. Secreted
K28 toxin di¡uses into the agar and greatly impairs cell growth of the K3 I3 non-killer strain. The dark blue colony staining indicates that the cells
are being killed by the toxin.
1. Introduction
In contrast to antibacterial antibiotics, bacteriophages
and bacteriocins, that were described at the beginning of
the last century, a similar antibiotic phenomenon in yeast
was only demonstrated much later. In 1963, Bevan and
Makower discovered the killer phenomenon in a Saccharomyces cerevisiae strain which was isolated as a brewery
contaminant [1]. The killer phenotype they described is
based on the secretion of a low molecular mass protein
or glycoprotein toxin (the so-called killer toxin) which kills
sensitive cells of the same or related yeast genera without
direct cell^cell contact. The killer strains themselves are
immune to their own toxin, but remain susceptible to
the toxins secreted by other killer yeasts (Fig. 1). Besides
toxin-secreting killer strains, a signi¢cant number of nonkiller yeasts can be isolated that have lost their ability to
produce various killer toxins but nevertheless retain immunity. The killer phenotype, i.e. toxin production and
functional immunity, is very frequent among yeasts and
can be found both in natural yeast isolates and in laboratory yeast strain collections. In particular, for yeast strains
living in their natural habitat it has been shown that toxin
production can confer a marked advantage in the competition with sensitive yeast strains for limited available nutrients [2,3]. After the initial discovery of the killer phenomenon in S. cerevisiae, it soon became evident that
killer strains are not restricted to the genus Saccharomyces
but can also be found among many other yeast genera; up
to now, toxin-producing killer yeasts have been identi¢ed
in Candida, Cryptococcus, Debaryomyces, Hanseniaspora,
Hansenula, Kluyveromyces, Metschnikowia, Pichia, Ustilago, Torulopsis, Williopsis and Zygosaccharomyces, indicating that the killer phenomenon is indeed widespread
among yeasts [2,4,5]. Interestingly, the genetic basis for
killer phenotype expression can be quite variable; in the
few cases where killer determinants have clearly been identi¢ed, they are either cytoplasmically inherited encapsulated dsRNA viruses, linear dsDNA plasmids or nuclear
genes (see below and Table 1). Among the plasmid-encoded killer toxins, the killers of Kluyveromyces lactis
and Pichia acaciae are most intensively studied and have
been nicely described in recent reviews [6,7].
During the past three decades, intensive investigations
Table 1
Genetic basis for killer phenotype expression in yeast
Yeast
Genetic basis
Toxin gene
Ref.
S. cerevisiae
H. uvarum
Z. bailii
U. maydis
K. lactis
P. acaciae
Pichia inositovora
Pichia kluyveri
Pichia farinosa
P. anomala
Williopsis mrakii
dsRNA virus
dsRNA virus
dsRNA virus
dsRNA virus
linear dsDNA plasmid
linear dsDNA plasmid
linear dsDNA plasmid
chromosomal
chromosomal
chromosomal
chromosomal
M1-, M2-, M28-dsRNA
M-dsRNA
M-dsRNA
M-dsRNA
pGKl1
pPac1
pPin1
not identi¢ed
SMK1
not identi¢ed
HMK
[48,49,13,168]
[9]
[14]
[159]
[169]
[26]
[170]
[2]
[171]
[172]
[4]
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of the killer system particularly in S. cerevisiae resulted in
substantial progress in many di¡erent ¢elds of biology,
providing important insights into basic and more general
aspects of eukaryotic cell biology, virus^host cell interactions and yeast virology. Moreover, since killer toxins
resemble naturally secreted proteins or glycoproteins, detailed analysis of their structure and synthesis also substantially strengthened our knowledge of the mechanisms
of post-translational prepro-protein processing in the eukaryotic secretion pathway. In addition, analysis of killer
toxins and their receptor-mediated mode of action has
proved to be an e¡ective means for investigating the molecular structure and in vivo assembly of yeast and fungal
cell walls, and for providing information important to
combating yeast infections caused by certain human
pathogenic strains of the yeasts Candida albicans and/or
Sporothrix schenkii.
The main purpose of this review is to summarize the
most recent data within the fast-growing ¢eld of the viral
killer systems in S. cerevisiae and to focus on the molecular basis of the killer phenomenon and its possible applications, thereby stressing aspects that are equally relevant
to yeast molecular biology, biomedicine and biotechnology. These are aspects of this topic which have not been
thoroughly addressed in previous reviews.
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presence of cytoplasmically inherited double-stranded
RNA (dsRNA) viruses (reviewed in [7,8]). Persistent infection of yeast cells with these viruses is symptomless, and in
contrast to certain fungal viruses that are associated with
adverse phenotypic e¡ects (like La France disease in Agaricus bisporus or plaque formation in Penicillium), yeast
dsRNA viruses have no (so far recognized) disadvantage
for a single cell. In addition, yeast dsRNA viruses are
considered non-infectious since no naturally occurring
extracellular route of transmission has been identi¢ed.
They were therefore designated cryptic viruses or ‘viruslike particles’ (VLPs). In contrast to the horizontal transfer of most pathogenic plant and animal RNA viruses,
yeast dsRNA viruses only spread vertically during mating
and/or heterokaryon formation in vivo and it has been
suggested that this kind of virus transmission most likely
represents a special adaptation since the frequent occurrence of mating and hyphal fusion in yeast and higher
fungi makes an extracellular route of virus transmission
dispensable [9^11]. However, since it was shown that nonkiller strains of S. cerevisiae can be successfully transfected
with isolated and puri¢ed VLPs in vitro, it cannot be excluded that a natural route of VLP infection also exists in
natural yeast and fungal habitats [12^14].
2.2. dsRNA viruses in S. cerevisiae: L-A and its
toxin-coding M satellites
2. Molecular biology of killer yeast
2.1. Genetic basis of killer phenotype expression:
cytoplasmic RNA viruses
In S. cerevisiae, the killer phenomenon is based on the
Based on the lack of cross-immunity, their molecular
mode of action and their killing pro¢les, toxin-producing
S. cerevisiae killer strains have been classi¢ed into three
major groups (K1, K2 and K28), each of them secreting a
unique killer toxin as well as a speci¢c but as yet uniden-
Table 2
Viral dsRNA genomes involved in killer phenotype expression in di¡erent yeasts
Virus
Function of virus
dsRNA genome (kb)
Encoded protein(s)
Ref.
ScV-L-A
Helper virus
4.6
[24]
ScV-M1
Satellite virus (‘killer’ virus)
1.6
ScV-M2
Satellite virus (‘killer’ virus)
1.5
ScV-M28
Satellite virus (‘killer’ virus)
1.8
UmV-P1
‘Killer’ virus
1.4
UmV-P4
UmV-P6
‘Killer’ virus
‘Killer’ virus
1.0
1.2
HuV-L
Helper virus
4.6
HuV-M
ZbV-L
Satellite virus ‘killer’ virus
Helper virus
1.0
4.6
ZbV-M
Satellite virus ‘killer’ virus
2.1
Gag, major capsid protein; Pol, RNA-dependent RNA
polymerase (Pol is in vivo expressed as Gag-Pol fusion
protein)
K1 preprotoxin (unprocessed K1 toxin precursor and
immunity determinant)
K2 preprotoxin (unprocessed K2 toxin precursor and
immunity determinant)
K28 preprotoxin (unprocessed K28 toxin precursor and
immunity determinant)
KP1 preprotoxin (precursor of an K/L heterodimeric
protein toxin)
KP4 monomeric protein toxin
KP6 preprotoxin (precursor of an K/L heterodimeric
protein toxin)
Gag, major capsid protein; Gag-Pol, RNA-dependent
RNA polymerasea
Precursor of a monomeric protein toxin
Gag, major capsid protein; Gag-Pol, RNA-dependent
RNA polymerasea
Precursor of a monomeric protein toxin
a
[173]
[49]
[39]
[161]
[159]
[174]
[9]
[9]
[14]
[14]
The dsRNA sequence has not yet been identi¢ed and, therefore, the postulated viral gene products Gag and Gag-Pol are hypothetical and only predicted by analogy to Gag and Gag-Pol of ScV-L-A.
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ti¢ed immunity component that renders killer cells immune to their own toxin [15^17]. The previously described
killer type K3 in S. cerevisiae [18] is not clearly distinct
from K2. In a killer yeast, production of either killer toxin
K1, K2 and/or K28 is associated with the presence of a
cytoplasmically inherited M-dsRNA satellite virus (designated ScV-M1, ScV-M2 or ScV-M28 for S. cerevisiae virus), which depends on the coexistence of L-A helper virus
to be stably maintained and replicated within the cytoplasm of the infected host cell (Table 2). Cells containing
neither dsRNA or only L-A are toxin-sensitive non-killers,
while those also containing ScV-M1, ScV-M2 or ScV-M28
are immune killers. The killer viruses self-select by killing
any virus-free segregants. Sensitive yeast strains survive
mating with killers, and cytoplasmic mixing of the multiple ScV-M killer virion copies during mating accounts for
the non-Mendelian inheritance pattern during subsequent
meiosis [8]. The toxin-coding killer viruses are incompatible at the replicative level, i.e. they exclude one another in
mixed infection and are unstable as multiple killer viruscontaining strains. This exclusion means that the viruses
remain isolated in their host and might explain why ScVM killer virions remained distinct and have not been
found as recombinants or hybrids [19].
L-A itself is an autonomously replicating mycovirus neither conferring a detectable phenotype upon its host
(yeast) cell, nor requiring the presence of an M-dsRNA
satellite virus for stable maintenance or replication. L-A
belongs to the Totiviridae virus family. Viruses of this
family have a genome encapsidated in an icosahedral shell
that is a single segment of dsRNA which encodes a major
coat protein and a RNA-dependent RNA polymerase [20].
L-A particles contain one and M particles contain two
copies of the corresponding dsRNA genome. Each contains 120 copies of the 76-kDa major capsid protein
Gag, and an estimated two copies of a minor 171-kDa
Gag-Pol fusion protein. From cryoelectron microscopy
and image reconstruction on the S. cerevisiae virus L-A
as well as on the Ustilago maydis killer virus P4 it has been
P diameter
shown that both viruses have similar 430-A
structures, consisting of 120 subunits arranged as 60 asymmetric dimers in a T = 1 lattice. The capsid wall contains
P in diameter
about 60 small openings each of them 10^15 A
which extend throughout the virus surface and probably
serve as molecular sieves which are large enough to allow
in£ux of metabolites that are needed for RNA synthesis
and replication and for exit of new viral transcripts, but
small enough to ensure protection of the viral RNA genome from degradation [21^23].
Each L-A particle contains a single copy of a linear
4.6-kb L-A dsRNA, and the codogenic L-A plus strand
(L-A(+)ssRNA) has two open reading frames (ORFs)
that overlap by 130 nucleotides, with the 3P ORF in the
31 frame relative to the 5P ORF [24]. The latter encodes
the major capsid protein Gag which is necessary for encapsulation and responsible for viral particle structure,
while the 3P ORF shows the consensus amino acid sequence pattern typical of RNA-dependent RNA polymer-
Fig. 2. Replication cycle of L-A and its toxin-coding M satellite virus. Both dsRNA viruses compete for the L-A encoded viral proteins Gag and GagPol which are essential for viral particle structure, viral (+)ssRNA packaging, in vivo RNA replication (i.e. minus-strand RNA synthesis), and
(+)ssRNA synthesis on the double-stranded RNA template.
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Fig. 3. Comparison of the coding capacity and the potential VBS/IRE and 3P-TRE sequence elements on the killer virus transcripts M1(+)ssRNA (B)
and M28(+)ssRNA (C) with those on L-A(+)ssRNA (A). Two cis-active viral binding sites (VBS1, VBS2) on the M transcripts are necessary for ssRNA
binding and packaging. The 3P-TRE might be all that is required for in vivo ssRNA replication. Numbers are shown for distance from the 3P-terminus
of the unpaired A residues which interrupt each VBS stem. (The indicated 5P-(+)ssRNA sequence (GAAAAA) is characteristic for yeast dsRNA viruses.)
ases of plus-strand ssRNA and dsRNA viruses (Table 2
[25]). In analogy to the nomenclature of retroviral genes,
the Gag encoding 5P ORF of L-A has been designated gag
and the Pol coding 3P ORF pol. The gene product of the
3P ORF is expressed in vivo as a Gag-Pol fusion protein
by a 31 ribosomal frameshift event during translation
[4,24,26^30]. The Gag-Pol fusion itself is the key viral
component in the replication of both, L-A and M virions;
through its C-terminal Pol domain, it recognizes a com-
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plex encapsulation signal of stem loop structures and an
overlapping direct repeat within the 3P-terminal 400 bases
of the L-A(+)ssRNA [31]. It is presumed that the N-terminal Gag domain within the Gag-Pol/ssRNA complex
primes capsid (Gag) polymerization. In contrast to L-A,
each of the three M-dsRNA genomes contains a single
ORF encoding a preprotoxin protein (pptox) representing
the unprocessed precursor of the mature and secreted killer toxin as well as a functional but so far unidenti¢ed
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Fig. 4. Secretory pathway of killer toxin K28 in S. cerevisiae. After in vivo translation of the pptox coding viral M28 transcript, pptox is (post-translationally) imported into the lumen of the ER and signal peptidase (SP) cleavage removes the toxin’s N-terminal secretion signal (pre-region). In a late
Golgi compartment, the Kex2p endopeptidase cleaves the pro-region, removes the intramolecular Q sequence and ¢nally leads to the secretion of the mature K/L heterodimeric protein toxin (modi¢ed after [167]).
immunity component (Table 2). Interestingly, the toxincoding M genomes exclude each other at the replicative
level and, therefore, simultaneous coexistence of all three
ScV-M killer virions in a single yeast cell does not occur in
vivo, but can be by-passed by co-expressing cDNA copies
of di¡erent pptox genes [8,32].
Intensive work on L-A, mainly by the group of Reed
Wickner, provided deeper insight into important cellular
aspects such as RNA transcription, dsRNA replication,
ssRNA packaging, ribosomal frameshifting, proteolytic
precursor processing, and virus^host as well as virus^virus
interactions. These aspects, which are quite common
among RNA viruses showing striking similarities to certain mammalian viruses such as HIV, are nicely addressed
in recent reviews [7,8,22,33]. L-A virions are icosahedral
particles, about 39 nm in diameter, which replicate in vivo
by a conservative mechanism. Synthesis of plus and minus
RNA strands also occurs in viro, i.e. within isolated viral
particles (Fig. 2 ; reviewed in [21]). After transcription of
the double-stranded RNA template, the viral plus strand is
extruded from the particle into the cytoplasm where it
subsequently executes two essential functions. Firstly, it
serves as mRNA template which is translated in vivo
into the viral proteins Gag and Gag-Pol. Secondly the
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(+)ssRNA molecule is packaged into new viral particles.
Once this viral coat assembly has been completed, the
RNA-dependent RNA polymerase (Gag-Pol) acts as replicase, synthesizes a new minus strand and ¢nally generates
the complete dsRNA genome of the mature virus (Fig. 2)
[8].
The toxin-coding M viruses are satellites of L-A, parasitizing Gag and Gag-Pol for replication [5,15]. It is not
surprising, therefore, that their replication cycle is widely
analogous to that of L-A, di¡ering only in one respect :
not all plus-strand M transcripts are extruded from the
viral particle because a mature M virion can contain up
to two copies of the smaller M dsRNA genome instead of
just a single copy in the case of L-A [15]. This phenomenon, which is also true for ¢lamentous viruses, has been
called ‘headful replication’ to distinguish it from the ‘headful packaging model’ as occurs in some bacteriophages
[21]. The (+)ssRNAs of both L-A and M viruses contain
speci¢c packaging signals (so-called viral binding sites
(VBS)) within their 3P-terminal region, which are essential
for replication and RNA packaging (Fig. 3). These cisactive sequences consist of a stem loop structure whose
stem is interrupted by an unpaired, protruding A residue
[27,34,35]. Two sequence elements were found to be essen-
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tial for the replication of L-A. An internal replication
enhancer (IRE), almost indistinguishable from the VBS,
and a 3P-terminal recognition element (3P-TRE) consisting
of a small stem loop structure 5 bases from the 3P-terminus
(Fig. 3). In vivo, these cis elements are recognized by GagPol and are responsible for both (+)ssRNA binding and
RNA packaging into new virions [35]. Mutational analysis
of a full-length L-A cDNA clone demonstrated that both
the 5 bases of the consensus loop sequence (GAUYC)
(Y = pyrimidine) and the unpaired A in its stem sequence
were essential for VBS function, while the stem sequence
was only necessary to provide secondary structure [31]. In
contrast to L-A, the toxin-coding transcripts of ScV-M1
and ScV-M28 virions contain two potential VBS elements.
Both have 5-base loops, the second identical to that in M1
(GAUUC) and the ¢rst di¡ering by 1 base (GGUUC; Fig.
3). Each is separated from a bulged A by the identical 3bp stem, resulting in an 11- or 12-bp repeat. The last 9 bp
of this repeat are reiterated at 3375; the ¢rst but not the
second VBS in M1 also overlaps a 10-bp imperfect direct
repeat that includes the VBS loop (Fig. 3) [36,37]. The
stem of the L-A VBS also overlaps a direct repeat, not
including the loop; however, the signi¢cance of these repeats is still unknown. A correct 3P-end sequence and
structure is required for in vitro (3)ssRNA strand synthesis; L-A, M1 and M28 3P-ends are not substantially
di¡erent [38]. For conservative in vivo transcription of
the (+)ssRNA from the dsRNA template, correct recognition of its very 5P-terminal sequence by Gag-Pol is required. Besides the 5P-sequence 5P-GAAAAA, there is little or no homology immediately downstream from this
sequence in L-A, M28 or other dsRNAs. Therefore, this
terminal recognition element (5P-TRE) seems to be su⁄cient for the e¡ective initiation of transcription [36].
2.3. Preprotoxin processing and toxin secretion
Among the virally encoded killer toxins of S. cerevisiae,
K1 and K28 are the best-studied proteins. Although both
toxins di¡er signi¢cantly in their amino acid composition
263
and their molecular mode of action, they share striking
homologies with respect to their synthesis, processing
and secretion. Each toxin is translated as preprotoxin
(pptox) showing similar structures, subsequently undergoing post-translational modi¢cations via the endoplasmic
reticulum, Golgi apparatus and secretory vesicles, ¢nally
resulting in the secretion of a mature K/L heterodimeric
protein toxin (Fig. 4).
Sequencing of a K28-coding M28 cDNA revealed a single ORF encoding the 345 amino acid preprotoxin with a
predicted size of 37.6 kDa. In vivo expression of this
cDNA under transcriptional control of the strong and
constitutive PGK promoter resulted in killer toxin secretion and functional (i.e. protective) toxin immunity [13,39],
as previously demonstrated for M1 [17]. Mutational analysis further indicated that the toxin precursor (pptox) enters the yeast endoplasmic reticulum via a highly hydrophobic N-terminal secretion signal which precedes the K
and L subunits of the mature toxin (10.5 and 11 kDa,
respectively) which are separated from each other by a
potentially N-glycosylated Q sequence [40]. Screening for
mutants of K1 and/or K28 killers that failed to secrete
any biologically active toxin while remaining immune resulted in the identi¢cation of two gene loci, KEX1 and
KEX2 [41]. Kex2p, the gene product of KEX2, is a subtilisin-like endoprotease which preferentially cleaves proteins after Lys^Arg or Arg^Arg dipeptides [42^44], while
Kex1p, the product of KEX1, is a serine carboxypeptidase
that removes the C-terminal basic dipeptide exposed by
Kex2p action [45]. Both enzymes are membrane-anchored
and reside in a late trans-Golgi compartment, having their
active sites localized in the organelle lumen [46,47]. Substrates such as prepro-K-factor and the protoxins of K1,
K2 and K28 are processed as they pass through the Golgi
during secretion in a way that is highly homologous to
proinsulin processing and prohormone conversion in higher eukaryotic cells (Fig. 5) [40] : in the case of the K28
toxin precursor, Kex2p/Kex1p-mediated pptox processing
removes the N-glycosylated Q sequence, trims the carboxytermini of K and L and ¢nally results in the secretion of a
Fig. 5. Analogy in K28 preprotoxin processing in yeast and proinsulin maturation in mammalian cells (h-Kex2p, human homologue to yeast endopeptidase Kex2p).
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Fig. 6. Receptor-mediated mode of action of the yeast K1 and K28 viral toxins. Killing of a sensitive yeast cell is envisaged in a two-step process involving initial toxin binding to receptors at the level of the cell wall (R1) and the cytoplasmic membrane (R2). After interaction with the plasma membrane, K1 is acting from outside the cell and disrupts cytoplasmic membrane function, while K28 enters the cell by endocytosis in order to reach its ¢nal target, the yeast cell nucleus (note that the cell surface receptors R1 and R2 are di¡erent for both toxins; see text).
21-kDa K/L heterodimeric protein toxin whose K and L
subunits are covalently linked through a single disul¢de
bond between K-Cys56 and L-Cys340 [40]. Most importantly, the C-terminus of L contains a four amino acid
epitope which represents a classical endoplasmic reticulum
(ER) retention signal (HDELR). Since this signal is initially masked by a carboxy-terminal arginine residue, ER
retention of the toxin precursor is e¡ectively prevented
until the protoxin enters a late Golgi compartment in
which the Kex1p carboxypeptidase removes this residue
and uncovers the toxin’s intracellular targeting signal
(see below) [40].
In striking analogy to K28 pptox processing, the K1
viral toxin is likewise processed from a 316 amino acid
precursor, resulting in an K/L heterodimer of 21 kDa
whose subunits are covalently linked by three disul¢de
bonds [48]. In contrast to the well established processing
pathways of K1 and K28, it is not yet clear if in vivo
processing of K2 pptox results in the secretion of a monomeric or a heterodimeric protein toxin; the K2 sequence is
completely unrelated to the sequence of either K1 or K28
and, although its toxic mechanism is similar to that of the
ionophoric K1 toxin, K2 immunity seems to be distinct
[49]. Since the published data on K2 are insu⁄cient for
detailed comparison, and the K1 system has been intensively dealt with in previous reviews [7,50], the following
chapter will only emphasize the recently described and
unique K28 killer toxin.
2.4. Toxicity of the killer proteins
Even though the killer toxins posses di¡erent modes of
action, they do have one thing in common: all viral toxins
(K1, K2, K28) kill a sensitive yeast cell in a receptormediated two-step process (Fig. 6): the ¢rst step involves
a fast and energy-independent binding to a toxin receptor
within the cell wall of a sensitive target cell. In case of K1
and K2, this primary receptor has been identi¢ed as L-1,6D-glucan, whereas the cell wall receptor for K28 is a high
molecular mass K-1,3-mannoprotein [51,52]. It was specu-
FEMSRE 748 1-8-02
lated, however, that toxin binding to the primary cell wall
receptor either concentrates the toxin at the level of the
cell wall or mediates close contact between the toxin and
the target cell membrane [53]. Susceptible strains can become toxin resistant by chromosomal mutations in any of
a set of genes that are involved in the structure and/or
biosynthesis of yeast cell wall components; in fact, a
whole set of toxin resistant yeast kre mutants (for killer
resistant) has been identi¢ed for K1, and several yeast mnn
mutants (for mannoprotein) have been shown to be highly
resistant to K28 at the cell wall level [54,55].
The second, energy-dependent step involves toxin translocation to the cytoplasmic membrane and interaction
with a secondary membrane receptor. While the membrane receptor for K28 is still unknown, the K1 membrane
receptor was recently identi¢ed as Kre1p, an O-glycosylated yeast cell surface protein which is initially GPI-anchored to the plasma membrane and which is also involved in L-1,6-glucan biosynthesis and K1 cell wall
receptor assembly [56]. After having reached the cytoplasmic membrane, the toxin (K1) exerts its lethal e¡ect by ion
channel formation and disruption of cytoplasmic membrane function [56^59].
In contrast to the ionophoric mode of action in which
K1 acts from outside the cell, K28 represents the ¢rst viral
killer toxin for which it was demonstrated that it enters a
sensitive target (yeast) cell by endocytosis [37]. After receptor-mediated entry into the cell, the toxin traverses the
secretion pathway in reverse (via Golgi and ER), subsequently enters the cytosol, and ¢nally transduces its toxic
signal into the yeast cell nucleus where the lethal events
occur : inhibition of DNA synthesis and cell cycle arrest at
the G1 /S boundary [37,40,60]. Interestingly, this unique
retrograde K28 toxin transport critically depends on a
short, four amino acid motif (HDEL) at the very C-terminus of L which acts as intracellular targeting signal directing the toxin from an early endosomal compartment back
to the trans-Golgi network and thereby preventing toxin
degradation in the vacuole (Fig. 7) [37,40]. Just as in higher eukaryotic cells, resident yeast ER proteins like the
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265
Fig. 7. Retrograde K28 toxin transport in S. cerevisiae. Left panel: Simpli¢ed model of how the K/L heterodimeric K28 virus toxin is entering a sensitive yeast cell in order to reach the cytosol. In vivo toxicity of K28 involves endocytotic uptake, retrograde toxin transport via Golgi apparatus and ER
and ER exit into the cytosol through the Sec61 complex (CM, cytoplasmic membrane ; EV, endosomal vesicle). Right panel : Schematic drawing of the
subunit structure of selected microbial protein toxins carrying a carboxy-terminal ER targeting signal (modi¢ed according to [37,64]).
chaperones Kar2p (BiP) and/or the protein disul¢de-isomerase carry a C-terminal H/KDEL motif which facilitates Golgi-to-ER recycling of the proteins and their ¢nal
location within the ER lumen [61].
Interestingly, the K28 virus toxin as well as certain bacterial or plant toxins likewise carry C-terminal H/KDELlike sequences which are responsible for the intracellular
targeting and the retrograde transport of the toxins, a
strategy that is quite common among microbial protein
toxins with enzymatic activity on intracellular targets
[37,62]. What all these toxins do have in common is the
capacity to ¢rst bind to cell surface receptors, then to enter
the cell by endocytosis and, ¢nally, to kill the eukaryotic
target cell by modifying some essential cellular components within the cytosol. Some of these protein toxins
(like Pseudomonas exotoxin A, cholera toxin and yeast
K28 virus toxin) carry a carboxy-terminal H/KDEL (or
H/KDEL-like) sequence which can facilitate retrograde
toxin transport all the way from the cell surface to the
Golgi apparatus and the ER. Consequently, mutations
within this C-terminal targeting sequence dramatically reduce cytotoxicity by preventing correct interaction of the
toxin’s C-terminus with the K/HDEL receptor of the target cell [40,63]. In this respect it is also interesting to note
that some of these toxins contain disul¢de bonds at or
near their C-termini whose in vivo function is predicted
to ensure correct access of the toxin’s K/HDEL signal to
the K/HDEL receptor of the corresponding target cell [64].
FEMSRE 748 1-8-02
This situation is also true for the K28 toxin since it was
recently shown that the cysteine residue right next to the
L C-terminus (Cys340 ) is part of the disul¢de bond that
covalently joins K and L (Fig. 8). It, therefore, has been
postulated that correct disul¢de bond formation is required to ensure accessibility of the toxin’s HDEL signal
in vivo [40]. However, a noticeable di¡erence between
H/KDEL-carrying protein toxins produced and secreted
by di¡erent bacteria to that produced by a virus-infected
yeast cell is that the K28 toxin itself is expressed in a
eukaryotic cell, and therefore the toxin’s C-terminal ER
targeting signal has to be masked as long as the toxin
resides within the early secretory pathway. Once it has
reached a trans-Golgi compartment, the C-terminal arginine residue is removed by Kex1p cleavage, and the toxin’s
intracellular targeting signal is uncovered. Because of this
‘intelligent’ strategy, toxin-producing K28 killers are able
to prevent toxin retention in the ER lumen [37,40].
Once the toxin has reached the ER lumen of a sensitive
yeast cell, it is retranslocated into the cytosol by passing
the yeast Sec61 translocon which represents the major ER
export channel in yeast [37]. The Sec61 complex itself,
which consists of the transmembrane ER protein Sec61p
and the two smaller subunits Sbh1p and Sss1p [65], has
also been shown to be involved in the export and removal
of misfolded ER proteins from the secretory pathway
and their subsequent proteasomal degradation in the
cytosol [66,67]. However, in contrast to the majority of
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Fig. 8. Interaction of the toxin’s L-C-terminus with the cellular HDEL receptor Erd2p ensures retrograde toxin transport via endosomes and Golgi to
the ER. Subsequent toxin translocation into the cytosol is mediated by the Sec61 complex which represents the major ER export channel in yeast. The
indicated ERAD components Cue1p, Ubc7p, Der3p ( = Hrd1p), Ubc6p, and Der1p are not involved in ER-to-cytosol export of the toxin and, therefore,
ER exit of K28 should be mechanistically di¡erent from the export of misfolded ER proteins [37]. Due to the reducing environment of the yeast cell cytosol, the toxin dissociates into its two subunits: while L is ubiquitinated and targeted for proteasomal degradation, K is somehow transducing the toxic
signal into the nucleus. Previously described data [37,38] have been incorporated into this model.
H/KDEL-carrying microbial toxins, ER-to-cytosol translocation of the viral K28 killer toxin does not depend on
the ER-associated degradation pathway (ERAD), and
yeast mutants defective in ERAD components such as
Der3p/Hrd1p and/or Der1p show wild-type sensitivity
[37]. It therefore can be predicted that toxin export from
the ER should be mechanistically di¡erent from ER-tocytosol export of misfolded proteins.
After the heterodimeric K28 toxin has reached the cytosol, it dissociates into its two subunits K and L because
of the highly reducing environmental conditions of the
yeast cell cytosol. Subsequently after exit from the ER,
the toxin’s L subunit is ubiquitinated and targeted for
proteasomal degradation, while K is stably transducing
the toxic signal into the nucleus where the lethal events
¢nally occur: DNA synthesis is rapidly inhibited, cell viability is lost more slowly and cells arrest in early 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
[37,60,68]. So far, it is not known if K is entering the
nucleus (either by passive di¡usion due to its small size
or by an importin-mediated active transport mechanism)
FEMSRE 748 1-8-02
or if it is interacting in the cytosol with one or more yeast
proteins to exhibit its lethal e¡ect in the nucleus. Currently, two-hybrid screens are being performed to identify
yeast proteins that are potentially interacting with the toxin’s K subunit.
2.5. Toxin immunity
Besides the fact that the primary toxin target has yet to
be identi¢ed, the question of how immunity occurs in vivo
has still not been answered. Although the precise molecular basis for toxin immunity is still unknown, it has been
speculated that it might be conferred by the toxin precursor itself. This might act as competitive inhibitor of the
mature toxin by saturating or causing elimination of a so
far unidenti¢ed plasma membrane receptor that normally
mediates toxicity [69^71]. It was also shown that the unprocessed toxin precursor is su⁄cient to confer immunity
since in vivo expression of either K1 or K28 cDNAs in a
vkex2 null mutant lacking the ability of pptox processing
and thus incapable of releasing K and L from the intervening Q sequence results in immune non-killer yeast trans-
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formants [17,72,73]. Based on all these observations, a
model for K1 toxin immunity had been proposed in which
either loss or modi¢cation of the toxin’s secondary plasma
membrane receptor would cause immunity. When it was
demonstrated that expression of a cDNA copy of the preprotoxin was su⁄cient to confer normal immunity, it was
postulated that the central Q component of this precursor
might not only act as intramolecular chaperone ensuring
proper secretory pptox processing, but also by providing
some sort of a masking function by protecting membranes
of toxin-producing cells against damage by the hydrophobic components within the K subunit of K1 [48]. Later on,
a more plausible model was proposed, in which it was
speculated that interaction of the secreted protoxin with
the receptor, through the same toxin domain involved in
lethality, resulted in diversion of the complex to the vacuole and destruction [17]. This model was further strengthened by phenotypic analyses of various mutant pptox derivatives which clearly indicated that the K toxin was the
lethal component and that its secretion in the mature form
caused severe growth inhibition while secretion of K fused
to just an N-terminal fragment of Q was su⁄cient to confer
immunity [74]. Dependence of immunity on diversion of
the putative membrane receptor to the vacuole is consistent with the defect in immunity observed in many vps
mutants (Tipper and Sturley, personal communication).
One class of killer resistant kre mutants, therefore, should
show similar resistance in spheroplasts. More recently, a
chromosomal kre1-12 mutation was described which
caused K1 resistance in yeast cell spheroplasts without a¡ecting toxin binding to the cell wall, and the wildtype gene product, Kre1p, was identi¢ed as being the
long-sought membrane receptor for the K1 viral toxin
[56,75].
Although the precise mechanism for K1 immunity is
still obscure, a completely di¡erent mechanism must be
postulated for K28 killers, since it has been shown that
K28 killer yeasts take up their own toxin after it has been
secreted. Furthermore, the toxin subsequently re-enters the
secretory pathway of a killer cell and reaches the cytosol,
just as in a sensitive target cell. However, in contrast to the
latter, the killer cell is not killed but rather protected
against the lethal e¡ect of the toxin. This means that
K28 immunity is likely to a¡ect some unknown step either
within the yeast cell cytosol or eventually downstream
from that, possibly within the nucleus. Future experiments
will hopefully shed more light onto this interesting and so
far unique mode of toxin action and immunity.
2.6. Virus^host cell interactions
For maintenance and expression of a stable killer phenotype, chromosomal genes of the host cell play an important, sometimes an essential role. Besides the chromosomal SEC genes required for general secretion of
extracellular proteins and glycoproteins, the KEX-encoded
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267
proteases Kex2p and Kex1p are necessary for preprotoxin
processing and precursor maturation of the yeast pheromone K-factor [70,76^79]. Furthermore, there is a whole
set of chromosomal yeast genes which either directly or
indirectly a¡ect dsRNA virus propagation. As recently
reviewed by Wickner [8,22] and Polonelli [7], these genes
can be classi¢ed into two major groups, the maintenance
of killer genes, MAK, and the superkiller genes, SKI.
Among more than 30 chromosomal MAK genes that
have been identi¢ed so far, only three genes (MAK3,
MAK10, PET18) are required for stable maintenance of
both yeast dsRNA viruses, L-A and M; all other MAK
genes are only necessary for maintaining either of three
known toxin-coding M satellites [73,80]. MAK3 encodes
an N-acetyltransferase which is responsible for the N-acetylation of the Gag protein required for the assembly of
L-A and M [81,82]. Without this acetylation, the Gag
proteins are not able to self-assemble, resulting in a rapid
loss of all dsRNA viruses. In addition, Mak3p acetylates
at least three additional mitochondrial proteins explaining
the slow growth of mak3 mutants on a non-fermentable
carbon source. Like Mak3p, Mak10p is also needed for
cell growth on a non-fermentable carbon source, and it
was further shown to stabilize viral particles, in particular
those containing dsRNA, since in mak10 mutants, virions
containing single-stranded (+)ssRNA only remain stable
while mature viruses containing a complete copy of the
viral dsRNA genome are unstable. More recently, physical
interaction of Mak3p, Mak10p, and Mak31p was demonstrated and it therefore has been proposed that these Mak
proteins might also function as a complex in vivo [83,84].
The gene product of PET18 likewise contributes to the
overall virion stability, and Pet18p itself is thought to be
particle-associated [85^87]. For stable maintenance of the
toxin-coding M killer virions more than 30 MAK genes
have been described which, when mutated, all result in
an inability to propagate any toxin-coding M satellite virus. While some of the MAK gene products ful¢l completely di¡erent functions within the cell, like Mak1p as
DNA topoisomerase I [88], Mak11p as essential membrane-associated protein with L-transducin sequence patterns [89] and the nuclear protein Mak16p which is essential for G1 exit within the cell cycle [90], the majority of
the MAK genes are encoding ribosomal proteins and a¡ect
60S ribosomal subunit assembly [91^95]. Correspondingly,
most mak mutations either lead to a dramatic decrease in
the number of free 60S subunits or weaken ribosomal subunit joining and association [96]. Presumably, L-A is only
providing its M satellites with Gag and Gag-Pol when the
intracellular concentration of both viral proteins is su⁄cient. Therefore, any diminished Gag to Gag-Pol ratio
(either caused by a reduced e⁄ciency of L-A(+)ssRNA
translation or due to a limited availability of free 60S ribosomal subunits) would immediately result in a selective
loss of the viral M dsRNA genome [97^99].
Mutations in any of six chromosomal SKI genes pheno-
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Fig. 9. Model of the in vivo functions of the yeast SKI gene system. A: The indicated Ski protein complex Ski2cp (consisting of Ski2p, Ski3p, and
Ski8p) is involved in translation repression of poly(A)3 RNA (left panel) and also proposed to act as a RNA helicase and/or cofactor for the exosomal
3P to 5P degradation of mRNA (right panel). This model is based on data mainly derived from the labs of Wickner [107,112,117], Parker [113,120] and
Johnson [116]. B: Agar di¡usion assay (left) and Western blot analysis (right) illustrating the phenotypic consequences of a ski8 mutation in meiotic
segregants derived from a cross of a K28 wild-type killer (SKI8) to a non-killer ski8 mutant. On sporulation of the heterozygous SKI8/ski8 diploid K28
killer, meiotic segregants from each tetrad show a 2:2 segregation of low and high killer activity (left) as well as low and high levels of secreted K28
toxin (right). (The Western blot shown to the right illustrates the amount of secreted K28 toxin present in the culture supernatant of the indicated ski8
and SKI8 segregants. Samples were separated by SDS^PAGE, electroblotted onto a PVDF membrane, and subsequently probed with a polyclonal antibody against the toxin’s L subunit. Arrows indicate the positions of the K/L heterodimeric protein toxin and of its tetrameric derivative (K/L)2 that
forms spontaneously under conditions of a non-reducing SDS^PAGE.)
typically suppress mak mutations in dsRNA-harboring
killer strains [100]. These so-called superkiller genes
(SKI) were initially identi¢ed from mutations that caused
overexpression of the killer toxin, indicating that in ski
mutants either M dsRNA copy number is increased or
translation of the toxin-coding M(+)ssRNA transcript is
more e¡ective [101,102]. Subsequent work on the yeast
SKI gene system demonstrated that ¢ve out of six genes
that have been characterized so far (SKI2, SKI3, SKI6,
SKI7 and SKI8) are required in vivo to repress translation
of poly- (A)3 RNAs [103,104], whereas SKI1 (which is
identical to XRN1) encodes a 5P-exoribonuclease involved
FEMSRE 748 1-8-02
in general RNA degradation and turnover [101,105^107].
SKI2 encodes a DEVH box protein of the RNA helicase
family, Ski3p is a nucleoprotein, and Ski8p shows L-transducin domains [103,108,109]. All three Ski proteins (Ski2p,
Ski3p, and Ski8p) are proposed to form a complex in vivo
whose function might be to act as a RNA helicase and/or
cofactor for the exosome complex through interaction
with Ski7p [110] (reviewed in [111]). Ski6p is the only
Ski protein for which it was directly shown that it is
part of the eukaryotic exosome complex which ^ in analogy to the Escherichia coli homolog RNase PH ^ is responsible for the 3P-5P degradation of mRNA [112,113].
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The precise in vivo function of the SKI genes in yeast
and in higher eukaryotes is still controversial. On one
hand SKI genes may represent an antiviral defence system
whose main function is to down-regulate dsRNA viral
copy number and repress expression of non-poly(A)
mRNAs, including L-A and M virus RNAs which both
lack a 3P poly(A) tract and a 5P cap structure [104,107,
114,115]. Although it has been shown that ski2, ski3
and/or ski8 mutations derepress translation of non-poly(A) mRNAs in vivo, it has also been demonstrated that
Ski proteins (like Ski6p/Rrp41p) can be part of the cytoplasmic exosome core complex and, therefore, function in
a 3P mRNA degradation pathway [116,117]. This indicates
that the yeast SKI genes have at least two important in
vivo functions: in addition to their antiviral activity, Ski
proteins are also involved in a more basic cell function like
3P mRNA degradation [103,104,113]. Furthermore, in addition to their activity in repressing translation of poly (A)
minus mRNAs, the identi¢cation of SKI2 homologous
genes in the human genome [118,119] further supports
the idea that some of the Ski proteins are indeed part of
a more general cellular system which is involved in the
overall control of mRNA stability, structure and degradation [113,120,121]. Thus, based on the currently available
data it can be concluded that the Ski proteins in yeast (and
possibly their homologs in higher eukaryotes as well) have
at least two important in vivo functions (Fig. 9): ¢rstly,
they block translation of non-poly(A) mRNA possibly by
a¡ecting 60S ribosomal subunit joining [104,117] and secondly, as part of the exosome complex they can also be
involved in the 3P to 5P degradation of RNA [113,122]. In
a superkiller yeast, loss of both functions of the SKI genes
leads to a dramatic increase in toxin production and secretion, ¢nally resulting in a superkiller phenotype that
originally gave the name to these genes.
2.7. Killer viruses in the non-conventional yeasts
Hanseniaspora uvarum and Zygosaccharomyces bailii
Besides the viral killer system in S. cerevisiae, toxin-coding mycoviruses have also been identi¢ed in killer strains
of the non-conventional yeasts H. uvarum and Z. bailii
[9,14,123]. Since the viral killer system in U. maydis has
recently been reviewed [7], the following section will focus
on the more recently identi¢ed killer viruses ZbV and HuV
in Z. bailii and H. uvarum (Table 2). With respect to their
structure and intracellular replication cycle, the dsRNA
viruses ZbV and HuV are analogous to the S. cerevisiae
viruses ScV-L-A and ScV-M, and it was shown that they
can be successfully transferred to S. cerevisiae non-killer
strains either by VLP transfection or by protoplast fusion,
resulting in yeast transfectants and/or heterokaryons that
stably express the corresponding killer phenotype [14,123].
However, in contrast to the S. cerevisiae toxins, killer toxins of non-Saccharomycetes ^ and in particular those secreted by virus-infected killer strains of the yeasts Z. bailii
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269
and H. uvarum ^ show a broad-spectrum antimycotic potential, being not only lethal to a great variety of wood
decay basidiomycetes and phytopathogenic fungi (like
Heterobasidium, Postia, Serpula, Fusarium and/or Colletotrichum), but also to human pathogenic strains of Candida
albicans and Sporothrix schenkii [4,9,124,125]. Lethality of
the Z. bailii virus toxin Zygocin resembles that of certain
bacteriocins and/or eukaryotic defensins, and involves disruption of cellular integrity by permeabilizing cytoplasmic
membrane function. In analogy to the virally encoded preprotoxins in S. cerevisiae killer strains, the toxin-coding
dsRNA genome (2.1 kb) in the yeast Z. bailii encodes
the unprocessed precursor of the secreted and biologically
active killer toxin. Due to its broad killing spectrum, Zygocin e¡ectively kills human pathogenic strains of C. albicans, Candida glabrata and Candida tropicalis, and the
toxin is equally e¡ective against the yeast-like and the
mycelial stages of the corresponding fungi after germ
tube induction through cultivation in the presence of fetal
calf serum. Furthermore, on a molar basis, Zygocin is
even more e¡ective in killing human pathogenic yeasts
than the frequently used antifungals clotrimazole or miconazole [125], indicating that it might be an attractive antifungal candidate, preferably for the treatment of topical
mycoses. Undoubtedly, protein toxins secreted by nonconventional killer yeasts are highly promising candidates
for use in the therapy of fungal infections, but further
pharmacological studies are needed to demonstrate their
antifungal potential in the future.
3. Applications
During the last two decades, secreted killer toxins and
toxin-producing killer yeasts have found several applications. For instance in the food and fermentation industries, killer yeasts have been used to combat contaminating
wild-type yeasts which can occur during the production of
wine, beer and bread [126^128]. Killer yeasts have also
been used as bio-control agents in the preservation of
foods [129], in the bio-typing of medically important
pathogenic yeasts and yeast-like fungi [130^133], in the
development of novel antimycotics for the treatment of
human and animal fungal infections [134,135], and ¢nally
in the ¢eld of recombinant DNA technology [136,137].
3.1. Killer yeasts in wine fermentation
Soon after the observation that toxin secreting killer
yeasts can be the causal agent of stuck and/or protracted
wine fermentations by antagonizing toxin-sensitive wine
yeasts and thus negatively a¡ecting the sensory quality
of the wine [138^141], many e¡orts were undertaken to
use natural or ‘constructed’ killer strains as starter culture
in beer and wine fermentations (for a recent review see
[142]). Thus, in several studies it was demonstrated that
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genetically modi¢ed wine yeasts producing di¡erent killer
toxins simultaneously have an increased antagonistic ability in mixed yeast fermentations, exhibit a signi¢cantly
broader killing activity and are thus capable of outcompeting potentially contaminating yeasts like Candida, Hanseniaspora, Kloeckera and Pichia in mixed culture [19,32,
127]. Thus, the use of recombinant DNA technology made
it feasible to design multiple killer yeasts which confer a
strong selective advantage on their yeast hosts; an interesting observation which might have application in industrial fermentations where contamination by killer yeasts is
to be avoided.
3.2. Killer toxins as potential antifungals
Antifungal proteins, peptides and their synthetic derivatives possess the potential for being used in the treatment
of human fungal infections which dramatically increased
during the last two decades, particularly in immunocompromised patients [143,144]. Powerful antifungal proteins
are naturally produced by a diverse group of organisms
including bacteria, fungi, insects, invertebrates and vertebrates, as well as plants (for a recent review see [145]).
Within this group, secreted killer toxins mainly produced
by non-Saccharomyces yeasts show a broad spectrum of
killing activity against a great number of human and plant
pathogens, including the anti-Pneumocystis carinii activity
in a recently described killer strain of the yeast Pichia
anomala [146]. In the search for novel and more selective
antifungals, yeast and fungal cell wall components represent attractive targets, since these structures are usually
restricted to yeasts and higher fungi and do not occur in
mammalian cells [147,148]. Yeast cell walls predominantly
consist of an outer layer of electron-dense mannoproteins,
an additional glucan skeleton consisting of linear and
branched L-1,3- and L-1,6-D-glucans, and ^ to a much
lesser extent ^ of chitin [149]. Since each of these components was shown to act as the primary binding site and cell
wall receptor for di¡erent yeast killer toxins [51^53,150],
antifungal research is currently focusing on the possible
use of yeast killer toxins as novel antifungals [125,145].
For killer toxins secreted by various strains of the yeast
genus Hansenula it was demonstrated that these toxins not
only bind to yeast cell wall components, but that they also
strongly inhibit de novo L-1,3-D-glucan biosynthesis in
yeast [151,152]. Therefore, killer toxin-based antifungals
that e¡ectively target cell wall components which are restricted to yeasts and fungi should have a comparable
selectivity as antibiotics inhibiting bacterial cell wall biosynthesis and/or structure. However, most yeast killer proteins exhibit their cytotoxic activity only within a narrow
pH range and at temperatures between 20‡C and 30‡C
and, therefore, yeast toxins are probably not suitable for
oral and/or intravenous administration [153], but topical
applications in the treatment of super¢cial lesions might
well be possible [125,134,154].
FEMSRE 748 1-8-02
3.3. Killer toxin expression in transgenic plants
As an attempt to engineer pathogen resistance in crop
plants, transgenic plants are being constructed which are
capable of producing substances such as polypeptides that
are toxic to disease-causing pathogens like herbivorous
insects [155]. In this respect, killer toxins which are naturally produced and secreted by virus-infected strains of the
fungal pathogen U. maydis have been shown to be an
attractive and unique model for the introduction of fungal
resistance into tobacco plants [156]. Certain strains of the
yeast U. maydis, which is also known as smut fungus that
infects corn plants and induces tumor-like galls which ¢nally result in a black-colored smut formation, are naturally infected by a dsRNA virus which contains the genetic
information for a secreted antifungal protein toxin [7,157^
159]. From the three di¡erent U. maydis killer toxins that
have been identi¢ed so far (KP1, KP4, and KP6), the K/L
heterodimeric protein toxin KP6 as well as the monomeric
KP4 toxin have been successfully expressed in tobacco
plant cells under transcriptional control of the constitutive
cauli£ower mosaic virus promoter [156,160]. In both cases
it was demonstrated that the transgenic plants are capable
of processing the corresponding virus toxin into its active
and mature form undistinguishable from the authentic Ustilago toxin. However, the expression level of the KP6
toxin was too low to be useful as biological control of
fungal pathogens. In contrast, the expression level of the
recombinant KP4 toxin was so high, that a small piece of
tobacco leaf secreted enough toxin to e¡ectively kill cells
of a KP4-sensitive phytopathogenic fungus [156]. These
data indicate that a systemic production of virally encoded
yeast toxins in crop plants might provide a novel strategy
to engineer biological control of fungal pathogens [161].
3.4. Heterologous protein secretion via pptox secretion and
processing signals
Bakers’ yeast (S. cerevisiae) as well as ¢ssion yeast
(Schizosaccharomyces pombe) are not only powerful systems for studying eukaryotic cell biology and cell cycle
control, they are also becoming increasingly interesting
in the fast growing ¢eld of heterologous protein production and secretion. Traditionally, expression and puri¢cation of heterologous proteins is accomplished using prokaryotic systems such as E. coli or Bacillus subtilis. However, when expression of eukaryotic proteins is desired,
bacterial systems often turn out to be ine¡ective hosts
because of their limited capacity to perform multi-step
post-translational modi¢cations such as protein N-glycosylation, phosphorylation and acetylation [162]. Therefore,
unicellular eukaryotes such as the yeasts S. cerevisiae,
Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, K. lactis and S. pombe have become attractive hosts
for the expression of heterologous proteins (for review see
[163]). In contrast to the great majority of foreign proteins
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271
Fig. 10. Schematic drawing of a preprotoxin-based DNA construct for the e⁄cient processing and secretion of heterologous proteins in the ¢ssion yeast
S. pombe. A: Structure of a chromosomally integrating K28 preprotoxin expression cassette for thiamine-regulated processing and secretion of foreign
proteins expressed under transcriptional control of the ¢ssion yeast nmt1 promoter (S/P, secretion and processing signal derived from K28 pptox; P/
Tnmt1 , nmt1 promoter and transcription termination sequence; modi¢ed after [137]). B: Western analysis (left panel) of secreted GFP in culture supernatants of ¢ssion yeast transformants expressing the processing and secretion vector shown in A. Fluorescence microscopy (right panel) of the same cells
expressing and secreting recombinant GFP.
expressed within the host cell cytosol, only a few proteins
have been successfully secreted. Secretory proteins contain
a hydrophobic, N-terminal signal sequence that directs
entry into the eukaryotic secretion pathway, with the
most critical step being protein import into the lumen of
the ER and subsequent sorting to the Golgi network. During the past decade, an increasing number of medically
and/or pharmaceutically interesting secretory proteins
(such as mouse K-amylase, human antithrombin III or
placental alkaline phosphatase) have been expressed as
extracellular proteins by using homologous secretion signals either derived from yeast invertase, acid phosphatase,
pheromone P-factor, or from the plasmid-driven killer toxin of K. lactis [164^166]. More recently it has also been
shown that the secretion and processing signal derived
from the S. cerevisiae ScV-M28 killer virus is fully functional in ¢ssion yeast (S. pombe) and can be used to target
foreign proteins for secretion into the extracellular medium [137]. Moreover, heterologous protein secretion driven by the K28 pptox processing and secretion signal is
highly e⁄cient as shown for the green £uorescent protein
(GFP; Fig. 10), making ¢ssion yeast an attractive host for
the processing and secretion of foreign proteins. There-
FEMSRE 748 1-8-02
fore, preprotoxin-based vectors might be an attractive
means for e⁄cient processing and high level secretion of
heterologous proteins in yeast [137].
4. Concluding remarks
Over the years much has been learned about eukaryotic
cell biology by studying killer strains of the yeast S. cerevisiae and their virally encoded protein toxins. The viral
killer system in yeast not only developed into a model
system to study general eukaryotic cell biology but also
to analyze more speci¢c aspects of yeast virology and virus^host cell interactions. Besides that, killer toxins and
toxin-producing killer yeasts have also found applications
in the food and fermentation industries, where they proved
to be e¡ective in combating contaminating wild-type
yeasts during wine, beer and bread fermentation. Toxinsecreting killer yeasts have also been used as bio-control
agents in the preservation of foods, in the bio-typing of
medically important pathogenic yeasts and fungi, in the
development of novel antimycotics for the treatment of
fungal infections, and in the ¢eld of recombinant DNA
Cyaan Magenta Geel Zwart
272
M.J. Schmitt, F. Breinig / FEMS Microbiology Reviews 26 (2002) 257^276
technology. All this had been possible by a detailed analysis of at least some of the toxin-secreting killer strains
that have been found among around 700 yeast species
described to date. Considering that most of the killer
yeasts have still not been thoroughly investigated, and
that the great majority of yeast and fungi have not yet
been discovered, there is a great potential of new yeast
species in natural habitats waiting to be characterized ^
let’s do it.
[15]
[16]
[17]
[18]
[19]
Acknowledgements
Continuous support of our killer research through
grants from the Deutsche Forschungsgemeinschaft is
greatly appreciated as well as the many contributions of
coworkers and collaborators. In particular, we wish to
thank Donald Tipper for critically reading the manuscript,
Reed Wickner, Howard Bussey, Roy Parker, Thomas
Sommer, Dieter Wolf, Ferdinand Radler, and all past
and present members of the Schmitt lab.
[20]
[21]
[22]
[23]
References
[1] Bevan, E.A. and Makower, M. (1963) The physiological basis of the
killer-character in yeast. Proc. Int. Congr. Genet. 11203.
[2] Young, T.W. and Yagiu, M. (1978) A comparison of the killer character in di¡erent yeasts and its classi¢cation. Antonie Van Leeuwenhoek 44, 59^77.
[3] Starmer, W.T., Ganter, P.F., Aberdeen, V., Lachance, M.A. and
Pha¡, H.J. (1987) The ecological role of killer yeasts in natural communities of yeasts. Can. J. Microbiol. 33, 783^796.
[4] Hodgson, V.J., Button, D. and Walker, G.M. (1995) Anti-Candida
activity of a novel killer toxin from the yeast Williopsis mrakii. Microbiology 141, 2003^2012.
[5] Bostian, K.A., Sturgeon, J.A. and Tipper, D.J. (1980) Encapsidation
of yeast killer double-stranded ribonucleic acids: dependence of M on
L. J. Bacteriol. 143, 463^470.
[6] Scha¡rath, R. and Breunig, K.D. (2000) Genetics and molecular
physiology of the yeast Kluyveromyces lactis. Fungal Genet. Biol.
30, 173^190.
[7] Magliani, W., Conti, S., Gerloni, M., Bertolotti, D. and Polonelli, L.
(1997) Yeast killer systems. Clin. Microbiol. Rev. 10, 369^400.
[8] Wickner, R.B. (1996) Double-stranded RNA viruses of Saccharomyces cerevisiae. Microbiol. Rev. 60, 250^265.
[9] Schmitt, M.J., Poravou, O., Trenz, K. and Rehfeldt, K. (1997)
Unique double-stranded RNAs responsible for the anti-Candida activity of the yeast Hanseniaspora uvarum. J. Virol. 71, 8852^8855.
[10] Wickner, R.B., Bussey, H., Fujimura, T. and Esteban, R. (1995) Viral
RNA and the killer phenomenon of Saccharomyces. In: The Mycota,
Genetics and Biotechnology (Ku«ck, Ed.), pp. 211^226. Springer Verlag, Berlin.
[11] Wickner, R.B. (1989) Yeast virology. FASEB J. 3, 2257^2265.
[12] El-Sherbeini, M. and Bostian, K.A. (1987) Viruses in fungi: infection
of yeast with the K1 and K2 killer virus. Proc. Natl. Acad. Sci. USA
84, 4293^4297.
[13] Schmitt, M.J. and Tipper, D.J. (1990) K28, a unique double-stranded
RNA killer virus of Saccharomyces cerevisiae. Mol. Cell. Biol. 10,
4807^4815.
[14] Schmitt, M.J. and Neuhausen, F. (1994) Killer toxin-secreting dou-
FEMSRE 748 1-8-02
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
ble-stranded RNA mycoviruses in the yeasts Hanseniaspora uvarum
and Zygosaccharomyces bailii. J. Virol. 68, 1765^1772.
Esteban, R. and Wickner, R.B. (1986) Three di¡erent M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae : in vitro
M1 double-stranded RNA synthesis. Mol. Cell. Biol. 6, 1552^1561.
Fujimura, T., Ribas, J.C., Makhov, A.M. and Wickner, R.B. (1992)
Pol of gag-pol fusion protein required for encapsidation of viral
RNA of yeast L-A virus. Nature 359, 746^749.
Sturley, S.L., Elliot, Q., LeVitre, J., Tipper, D.J. and Bostian, K.A.
(1986) Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin. EMBO J. 5, 3381^3389.
Young, T.W. (1987) In: The Yeasts (Rose, A.H. and Harrison, J.S.,
Eds.), pp. 131^164. Academic Press, New York.
Bussey, H., Vernet, T. and Sdicu, A.M. (1988) Mutual antagonism
among killer yeasts: competition between K1 and K2 killers and a
novel cDNA-based K1-K2 killer strain of Saccharomyces cerevisiae.
Can. J. Microbiol. 34, 38^44.
Baker, T.S., Olson, N.H. and Fuller, S.D. (1999) Adding the third
dimension to virus life cycles : three-dimensional reconstruction of
icosahedral viruses from cryo-electron micrographs. Microbiol.
Mol. Biol. Rev. 63, 862^922.
Cheng, R.H., Caston, J.R., Wang, G.J., Gu, F., Smith, T.J., Baker,
T.S., Bozarth, R.F., Trus, B.L., Cheng, N., Wickner, R.B., et al.
(1994) Fungal virus capsids, cytoplasmic compartments for the replication of double-stranded RNA, formed as icosahedral shells of
asymmetric Gag dimers. J. Mol. Biol. 244, 255^258.
Wickner, R.B. (1996) Prions and RNA viruses of Saccharomyces cerevisiae. Annu. Rev. Genet. 30, 109^139.
Caston, J.R., Trus, B.L., Booy, F.P., Wickner, R.B., Wall, J.S. and
Steven, A.C. (1997) Structure of L-A virus: a specialized compartment for the transcription and replication of double-stranded RNA.
J. Cell Biol. 138, 975^985.
Icho, T. and Wickner, R.B. (1989) The double-stranded RNA genome of yeast virus L-A encodes its own putative RNA polymerase
by fusing two open reading frames. J. Biol. Chem. 264, 6716^6723.
Bruenn, J.A. (1991) Relationships among the positive strand and
double-strand RNA viruses as viewed through their RNA-dependent
RNA polymerases. Nucleic Acids Res. 19, 217^226.
Hayman, G.T. and Bolen, P.L. (1991) Linear DNA plasmids of Pichia inositovora are associated with a novel killer toxin activity. Curr.
Genet. 19, 389^393.
Huan, B.F., Shen, Y.Q. and Bruenn, J.A. (1991) In vivo mapping of
a sequence required for interference with the yeast killer virus. Proc.
Natl. Acad. Sci. USA 88, 1271^1275.
Dinman, J.D., Icho, T. and Wickner, R.B. (1991) A 31 ribosomal
frameshift in a double-stranded RNA virus of yeast forms a gag-pol
fusion protein. Proc. Natl. Acad. Sci. USA 88, 174^178.
Fujimura, T. and Wickner, R.B. (1988) Gene overlap results in a viral
protein having an RNA binding domain and a major coat protein
domain. Cell 55, 663^671.
Lopinski, J.D., Dinman, J.D. and Bruenn, J.A. (2000) Kinetics of
ribosomal pausing during programmed 31 translational frameshifting. Mol. Cell. Biol. 20, 1095^1103.
Fujimura, T., Esteban, R., Esteban, L.M. and Wickner, R.B. (1990)
Portable encapsidation signal of the L-A double-stranded RNA virus
of S. cerevisiae. Cell 62, 819^828.
Schmitt, M.J. and Schernikau, G. (1997) Construction of a cDNAbased K1/K2/K28 triple killer strain of Saccharomyces cerevisiae.
Food Technol. Biotechnol. 35, 281^285.
Dawe, A.L. and Nuss, D.L. (2001) Hypoviruses and chestnut blight :
exploiting viruses to understand and modulate fungal pathogenesis.
Annu. Rev. Genet. 35, 1^29.
Radler, F., Herzberger, S., Schonig, I. and Schwarz, P. (1993) Investigation of a killer strain of Zygosaccharomyces bailii. J. Gen. Microbiol. 139, 495^500.
Ribas, J.C., Fujimura, T. and Wickner, R.B. (1994) Essential RNA
binding and packaging domains of the Gag-Pol fusion protein of the
Cyaan Magenta Geel Zwart
M.J. Schmitt, F. Breinig / FEMS Microbiology Reviews 26 (2002) 257^276
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
L-A double-stranded RNA virus of Saccharomyces cerevisiae. J. Biol.
Chem. 269, 28420^28428.
Ribas, J.C. and Wickner, R.B. (1992) RNA-dependent RNA polymerase consensus sequence of the L-A double-stranded RNA virus:
de¢nition of essential domains. Proc. Natl. Acad. Sci. USA 89, 2185^
2189.
Eisfeld, K., Ri¡er, F., Mentges, J. and Schmitt, M.J. (2000) Endocytotic uptake and retrograde transport of a virally encoded killer toxin
in yeast. Mol. Microbiol. 37, 926^940.
Schmitt, M.J. and Eisfeld, K. (1999) Killer viruses in S. cerevisiae and
their general importance in understanding eukaryotic cell biology.
Recent Res. Dev. Virol. 1, 525^545.
Schmitt, M.J. and Tipper, D.J. (1995) Sequence of the M28 dsRNA:
preprotoxin is processed to an alpha/beta heterodimeric protein toxin. Virology 213, 341^351.
Ri¡er, F., Eisfeld, K., Breinig, F. and Schmitt, M.J. (2002) Mutational analysis of K28 preprotoxin processing in the yeast Saccharomyces cerevisiae. Microbiology 148, 1317^1328.
Wickner, R.B. and Leibowitz, M.J. (1976) Two chromosomal genes
required for killing expression in killer strains of Saccharomyces cerevisiae. Genetics 82, 429^442.
Fuller, R.S., Brake, A.J. and Thorner, J. (1989) Intracellular targeting and structural conservation of a prohormone-processing endoprotease. Science 246, 482^486.
Fuller, R.S., Brake, A. and Thorner, J. (1989) Yeast prohormone
processing enzyme (KEX2 gene product) is a Ca2þ -dependent serine
protease. Proc. Natl. Acad. Sci. USA 86, 1434^1438.
Redding, K., Holcomb, C. and Fuller, R.S. (1991) Immunolocalization of Kex2 protease identi¢es a putative late Golgi compartment in
the yeast Saccharomyces cerevisiae. J. Cell Biol. 113, 527^538.
Dmochowska, A., Dignard, D., Henning, D., Thomas, D.Y. and
Bussey, H. (1987) Yeast KEX1 gene encodes a putative protease
with a carboxypeptidase B-like function involved in killer toxin and
alpha-factor precursor processing. Cell 50, 573^584.
Bryant, N.J. and Boyd, A. (1993) Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three
processing proteinases to a single Golgi compartment. J. Cell Sci.
106, 815^822.
Shilton, B.H., Thomas, D.Y. and Cygler, M. (1997) Crystal structure
of Kex1deltap, a prohormone-processing carboxypeptidase from Saccharomyces cerevisiae. Biochemistry 36, 9002^9012.
Bostian, K.A., Elliott, Q., Bussey, H., Burn, V., Smith, A. and Tipper, D.J. (1984) Sequence of the preprotoxin dsRNA gene of type I
killer yeast: multiple processing events produce a two-component
toxin. Cell 36, 741^751.
Dignard, D., Whiteway, M., Germain, D., Tessier, D. and Thomas,
D.Y. (1991) Expression in yeast of a cDNA copy of the K2 killer
toxin gene. Mol. Gen. Genet. 227, 127^136.
Bussey, H. (1991) K1 killer toxin, a pore-forming protein from yeast.
Mol. Microbiol. 5, 2339^2343.
Hutchins, K. and Bussey, H. (1983) Cell wall receptor for yeast killer
toxin: involvement of (1 leads to 6)-beta-D-glucan. J. Bacteriol. 154,
161^169.
Schmitt, M. and Radler, F. (1988) Molecular structure of the cell wall
receptor for killer toxin KT28 in Saccharomyces cerevisiae. J. Bacteriol. 170, 2192^2196.
Schmitt, M. and Radler, F. (1987) Mannoprotein of the yeast cell
wall as primary receptor for the killer toxin of Saccharomyces cerevisiae strain 28. J. Gen. Microbiol. 133, 3347^3354.
Boone, C., Sommer, S.S., Hensel, A. and Bussey, H. (1990) Yeast
KRE genes provide evidence for a pathway of cell wall beta-glucan
assembly. J. Cell Biol. 110, 1833^1843.
Schmitt, M.J. and Radler, F. (1990) Blockage of cell wall receptors
for yeast killer toxin KT28 with antimannoprotein antibodies. Antimicrob. Agents Chemother. 34, 1615^1618.
Breinig, F., Tipper, D.J. and Schmitt, M.J. (2002) Kre1p, the plasma
membrane receptor for the yeast K1 viral toxin. Cell 108, 395^405.
FEMSRE 748 1-8-02
273
[57] de la Pena, P., Barros, F., Gascon, S., Lazo, P.S. and Ramos, S.
(1981) E¡ect of yeast killer toxin on sensitive cells of Saccharomyces
cerevisiae. J. Biol. Chem. 256, 10420^10525.
[58] Martinac, B., Zhu, H., Kubalski, A., Zhou, X.L., Culbertson, M.,
Bussey, H. and Kung, C. (1990) Yeast K1 killer toxin forms ion
channels in sensitive yeast spheroplasts and in arti¢cial liposomes.
Proc. Natl. Acad. Sci. USA 87, 6228^6232.
[59] Ahmed, A., Sesti, F., Ilan, N., Shih, T.M., Sturley, S.L. and Goldstein, S.A. (1999) A molecular target for viral killer toxin: TOK1
potassium channels. Cell 99, 283^291.
[60] Schmitt, M.J., Klavehn, P., Wang, J., Schonig, I. and Tipper, D.J.
(1996) Cell cycle studies on the mode of action of yeast K28 killer
toxin. Microbiology 142, 2655^2662.
[61] Schmitt, M.J. (1995) Cloning and expression of a cDNA copy of the
viral K28 killer toxin gene in yeast. Mol. Gen. Genet. 246, 236^246.
[62] Yoshida, T., Chen, C.C., Zhang, M.S. and Wu, H.C. (1991) Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of
ricin, modeccin, and Pseudomonas toxin. Exp. Cell Res. 192, 389^395.
[63] Chaudhary, V.K., Jinno, Y., Gallo, M.G., FitzGerald, D. and Pastan, I. (1990) Mutagenesis of Pseudomonas exotoxin in identi¢cation
of sequences responsible for the animal toxicity. J. Biol. Chem. 265,
16306^16310.
[64] Pelham, H.R.B., Roberts, L.M. and Lord, J.M. (1992) Toxin entry:
how reversible is the secretory pathway ? Trends Cell Biol. 2, 183^185.
[65] Beswick, V., Brodsky, J.L., Kepes, F., Neumann, J.M., Sanson, A.
and Garrigos, M. (1998) Expression, puri¢cation, and characterization of Sss1p, an essential component of the yeast Sec61p protein
translocation complex. Protein Expr. Purif. 13, 423^432.
[66] Pilon, M., Schekman, R. and Romisch, K. (1997) Sec61p mediates
export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation. EMBO J. 16, 4540^4548.
[67] Plemper, R.K., Bohmler, S., Bordallo, J., Sommer, T. and Wolf,
D.H. (1997) Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. Nature 388, 891^895.
[68] Schmitt, M., Brendel, M., Schwarz, R. and Radler, F. (1989) Inhibition of DNA synthesis in Saccharomyces cerevisiae by yeast toxin
K28. J. Gen. Microbiol. 135, 1529^1535.
[69] Tipper, D.J. and Schmitt, M.J. (1991) Yeast dsRNA viruses: replication and killer phenotypes. Mol. Microbiol. 5, 2331^2338.
[70] Bussey, H., Saville, D., Greene, D., Tipper, D.J. and Bostian, K.A.
(1983) Secretion of Saccharomyces cerevisiae killer toxin: processing
of the glycosylated precursor. Mol. Cell. Biol. 3, 1362^1370.
[71] Bussey, H., Sacks, W., Galley, D. and Saville, D. (1982) Yeast killer
plasmid mutations a¡ecting toxin secretion and activity and toxin
immunity function. Mol. Cell. Biol. 2, 346^354.
[72] Boone, C., Bussey, H., Greene, D., Thomas, D.Y. and Vernet, T.
(1986) Yeast killer toxin: site-directed mutations implicate the precursor protein as the immunity component. Cell 46, 105^113.
[73] Schmitt, M.J. and Tipper, D.J. (1992) Genetic analysis of maintenance and expression of L and M double-stranded RNAs from yeast
killer virus K28. Yeast 8, 373^384.
[74] Zhu, Y.S., Kane, J., Zhang, X.Y., Zhang, M. and Tipper, D.J. (1993)
Role of the gamma component of preprotoxin in expression of the
yeast K1 killer phenotype. Yeast 9, 251^266.
[75] Schmitt, M.J. and Compain, P. (1995) Killer-toxin-resistant kre12
mutants of Saccharomyces cerevisiae : genetic and biochemical evidence for a secondary K1 membrane receptor. Arch. Microbiol.
164, 435^443.
[76] Wickner, R.B. (1993) Host control of yeast dsRNA virus propagation
and expression. Trends Microbiol. 1, 294^299.
[77] Douglas, C.M., Sturley, S.L. and Bostian, K.A. (1988) Role of protein processing, intracellular tra⁄cking and endocytosis in production
of and immunity to yeast killer toxin. Eur. J. Epidemiol. 4, 400^408.
[78] Lolle, S.J. and Bussey, H. (1986) In vivo evidence for posttranslational translocation and signal cleavage of the killer preprotoxin of
Saccharomyces cerevisiae. Mol. Cell. Biol. 6, 4274^4280.
[79] Zhu, Y.S., Zhang, X.Y., Cartwright, C.P. and Tipper, D.J. (1992)
Cyaan Magenta Geel Zwart
274
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
[91]
[92]
[93]
[94]
[95]
[96]
[97]
[98]
M.J. Schmitt, F. Breinig / FEMS Microbiology Reviews 26 (2002) 257^276
Kex2-dependent processing of yeast K1 killer preprotoxin includes
cleavage at ProArg-44. Mol. Microbiol. 6, 511^520.
Sommer, S.S. and Wickner, R.B. (1982) Yeast L dsRNA consists of
at least three distinct RNAs; evidence that the non-Mendelian genes
[HOK], [NEX] and [EXL] are on one of these dsRNAs. Cell 31, 429^
441.
Tercero, J.C. and Wickner, R.B. (1992) MAK3 encodes an N-acetyltransferase whose modi¢cation of the L-A gag NH2 terminus is necessary for virus particle assembly. J. Biol. Chem. 267, 20277^20281.
Tercero, J.C., Dinman, J.D. and Wickner, R.B. (1993) Yeast MAK3
N-acetyltransferase recognizes the N-terminal four amino acids of the
major coat protein (gag) of the L-A double-stranded RNA virus.
J. Bacteriol. 175, 3192^3194.
Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M. and
Seraphin, B. (1999) A generic protein puri¢cation method for protein
complex characterization and proteome exploration. Nat. Biotechnol.
17, 1030^1032.
Polevoda, B. and Sherman, F. (2001) NatC Nalpha-terminal acetyltransferase of yeast contains three subunits, Mak3p, Mak10p, and
Mak31p. J. Biol. Chem. 276, 20154^20159.
Lee, Y.J. and Wickner, R.B. (1992) MAK10, a glucose-repressible
gene necessary for replication of a dsRNA virus of Saccharomyces
cerevisiae, has T cell receptor alpha-subunit motifs. Genetics 132, 87^
96.
Fujimura, T. and Wickner, R.B. (1987) L-A double-stranded RNA
viruslike particle replication cycle in Saccharomyces cerevisiae : particle maturation in vitro and e¡ects of mak10 and pet18 mutations.
Mol. Cell. Biol. 7, 420^426.
Fujimura, T., Esteban, R. and Wickner, R.B. (1986) In vitro L-A
double-stranded RNA synthesis in virus-like particles from Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 83, 4433^4437.
Thrash, C., Voelkel, K., DiNardo, S. and Sternglanz, R. (1984) Identi¢cation of Saccharomyces cerevisiae mutants de¢cient in DNA
topoisomerase I activity. J. Biol. Chem. 259, 1375^1377.
Icho, T. and Wickner, R.B. (1988) The MAK11 protein is essential
for cell growth and replication of M double-stranded RNA and is
apparently a membrane-associated protein. J. Biol. Chem. 263, 1467^
1475.
Wickner, R.B. (1987) MKT1, a nonessential Saccharomyces cerevisiae
gene with a temperature-dependent e¡ect on replication of M2 double-stranded RNA. J. Bacteriol. 169, 4941^4945.
Carroll, K. and Wickner, R.B. (1995) Translation and M1 doublestranded RNA propagation: MAK18 = RPL41B and cycloheximide
curing. J. Bacteriol. 177, 2887^2891.
Ohtake, Y. and Wickner, R.B. (1995) KRB1, a suppressor of mak7-1
(a mutant RPL4A), is RPL4B, a second ribosomal protein L4 gene,
on a fragment of Saccharomyces chromosome XII. Genetics 140,
129^137.
Wickner, R.B., Ridley, S.P., Fried, H.M. and Ball, S.G. (1982) Ribosomal protein L3 is involved in replication or maintenance of the
killer double-stranded RNA genome of Saccharomyces cerevisiae.
Proc. Natl. Acad. Sci. USA 79, 4706^4708.
Wickner, R.B. (1988) Host function of MAK16: G1 arrest by a
mak16 mutant of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci.
USA 85, 6007^6011.
Wickner, R.B. (1992) Double-stranded and single-stranded RNA viruses of Saccharomyces cerevisiae. Annu. Rev. Microbiol. 46, 347^
375.
Ohtake, Y. and Wickner, R.B. (1995) Yeast virus propagation depends critically on free 60S ribosomal subunit concentration. Mol.
Cell. Biol. 15, 2772^2781.
Valle, R.P. and Wickner, R.B. (1993) Elimination of L-A doublestranded RNA virus of Saccharomyces cerevisiae by expression of
gag and gag-pol from an L-A cDNA clone. J. Virol. 67, 2764^2771.
Dinman, J.D. and Wickner, R.B. (1992) Ribosomal frameshifting
e⁄ciency and gag/gag-pol ratio are critical for yeast M1 doublestranded RNA virus propagation. J. Virol. 66, 3669^3676.
FEMSRE 748 1-8-02
[99] Dinman, J.D. and Wickner, R.B. (1994) Translational maintenance
of frame: mutants of Saccharomyces cerevisiae with altered 31 ribosomal frameshifting e⁄ciencies. Genetics 136, 75^86.
[100] Wickner, R.B. and Toh-e, A. (1982) [HOK], a new yeast non-Mendelian trait, enables a replication-defective killer plasmid to be maintained. Genetics 100, 159^174.
[101] Toh, E.A., Guerry, P. and Wickner, R.B. (1978) Chromosomal
superkiller mutants of Saccharomyces cerevisiae. J. Bacteriol. 136,
1002^1007.
[102] Ridley, S.P., Sommer, S.S. and Wickner, R.B. (1984) Superkiller
mutations in Saccharomyces cerevisiae suppress exclusion of M2
double-stranded RNA by L-A-HN and confer cold sensitivity in
the presence of M and L-A-HN. Mol. Cell. Biol. 4, 761^770.
[103] Widner, W.R. and Wickner, R.B. (1993) Evidence that the SKI
antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA. Mol. Cell. Biol. 13, 4331^4341.
[104] Masison, D.C., Blanc, A., Ribas, J.C., Carroll, K., Sonenberg, N.
and Wickner, R.B. (1995) Decoying the cap-mRNA degradation
system by a double-stranded RNA virus and poly(A)3 mRNA surveillance by a yeast antiviral system. Mol. Cell. Biol. 15, 2763^2771.
[105] Toh, E.A. and Wickner, R.B. (1980) ‘Superkiller’ mutations suppress chromosomal mutations a¡ecting double-stranded RNA killer
plasmid replication in Saccharomyces cerevisiae. Proc. Natl. Acad.
Sci. USA 77, 527^530.
[106] Johnson, A.W. and Kolodner, R.D. (1995) Synthetic lethality of
sep1 (xrn1) ski2 and sep1 (xrn1) ski3 mutants of Saccharomyces
cerevisiae is independent of killer virus and suggests a general role
for these genes in translation control. Mol. Cell. Biol. 15, 2719^
2727.
[107] Benard, L., Carroll, K., Valle, R.C., Masison, D.C. and Wickner,
R.B. (1999) The ski7 antiviral protein is an EF1-alpha homolog that
blocks expression of non-poly(A) mRNA in Saccharomyces cerevisiae. J. Virol. 73, 2893^2900.
[108] Rhee, S.K., Icho, T. and Wickner, R.B. (1989) Structure and nuclear localization signal of the SKI3 antiviral protein of Saccharomyces cerevisiae. Yeast 5, 149^158.
[109] Matsumoto, Y., Sarkar, G., Sommer, S.S. and Wickner, R.B. (1993)
A yeast antiviral protein, SKI8, shares a repeated amino acid sequence pattern with beta-subunits of G proteins and several other
proteins. Yeast 9, 43^51.
[110] Araki, Y., Takahashi, S., Kobayashi, T., Kajiho, H., Hoshino, S.
and Katada, T. (2001) Ski7p G protein interacts with the exosome
and the Ski complex for 3P-to-5P mRNA decay in yeast. EMBO J.
20, 4684^4693.
[111] Butler, J.S. (2002) The yin and yang of the exosome. Trends Cell
Biol. 12, 90^96.
[112] Benard, L., Carroll, K., Valle, R.C. and Wickner, R.B. (1998) Ski6p
is a homolog of RNA-processing enzymes that a¡ects translation of
non-poly(A) mRNAs and 60S ribosomal subunit biogenesis. Mol.
Cell. Biol. 18, 2688^2696.
[113] Jacobs, J.S., Anderson, A.R. and Parker, R.P. (1998) The 3P to 5P
degradation of yeast mRNAs is a general mechanism for mRNA
turnover that requires the SKI2 DEVH box protein and 3P to 5P
exonucleases of the exosome complex. EMBO J. 17, 1497^1506.
[114] Thiele, D.J., Hannig, E.M. and Leibowitz, M.J. (1984) Genome
structure and expression of a defective interfering mutant of the
killer virus of yeast. Virology 137, 20^31.
[115] Searfoss, A.M. and Wickner, R.B. (2000) 3P poly(A) is dispensable
for translation. Proc. Natl. Acad. Sci. USA 97, 9133^9137.
[116] Brown, J.T., Bai, X. and Johnson, A.W. (2000) The yeast antiviral
proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo. RNA 6,
449^457.
[117] Searfoss, A., Dever, T.E. and Wickner, R. (2001) Linking the 3P
poly(A) tail to the subunit joining step of translation initiation:
relations of Pab1p, eukaryotic translation initiation factor 5b
(Fun12p), and Ski2p-Slh1p. Mol. Cell. Biol. 21, 4900^4908.
[118] Dangel, A.W., Shen, L., Mendoza, A.R., Wu, L.C. and Yu, C.Y.
Cyaan Magenta Geel Zwart
M.J. Schmitt, F. Breinig / FEMS Microbiology Reviews 26 (2002) 257^276
[119]
[120]
[121]
[122]
[123]
[124]
[125]
[126]
[127]
[128]
[129]
[130]
[131]
[132]
[133]
[134]
[135]
[136]
[137]
[138]
(1995) Human helicase gene SKI2W in the HLA class III region
exhibits striking structural similarities to the yeast antiviral gene
SKI2 and to the human gene KIAA0052: emergence of a new
gene family. Nucleic Acids Res. 23, 2120^2126.
Lee, S.G., Lee, I., Park, S.H., Kang, C. and Song, K. (1995) Identi¢cation and characterization of a human cDNA homologous to
yeast SKI2. Genomics 25, 660^666.
van Hoof, A. and Parker, R. (1999) The exosome: a proteasome for
RNA? Cell 99, 347^350.
Van Hoof, C., Janssens, V., De Baere, I., de Winde, J.H., Winderickx, J., Dumortier, F., Thevelein, J.M., Merlevede, W. and
Goris, J. (2000) The Saccharomyces cerevisiae homologue YPA1
of the mammalian phosphotyrosyl phosphatase activator of protein
phosphatase 2A controls progression through the G1 phase of the
yeast cell cycle. J. Mol. Biol. 302, 103^120.
Wang, Z. and Kiledjian, M. (2001) Functional link between the
mammalian exosome and mRNA decapping. Cell 107, 751^762.
Zorg, J., Kilian, S. and Radler, F. (1988) Killer toxin producing
strains of the yeasts Hanseniaspora uvarum and Pichia kluyveri.
Arch. Microbiol. 149, 261^267.
Radler, F., Schmitt, M.J. and Meyer, B. (1990) Killer toxin of Hanseniaspora uvarum. Arch. Microbiol. 154, 175^178.
Theisen, S., Molkenau, E. and Schmitt, M.J. (2000) Wicaltin, a new
protein toxin secreted by the yeast Williopsis californica and its
broad-spectrum antimycotic potential. J. Microbiol. Biotechnol.
10, 547^550.
Young, T.W. (1981) The genetic manipulation of killer character
into brewing yeast. J. Inst. Brew. 87, 292^295.
Boone, C., Scidu, A.M., Wagner, J., Degre, R., Sanchez, C. and
Bussey, H. (1990) Integration of the yeast K1 killer toxin gene
into the genome of marked wine yeasts and its e¡ect on vini¢cation.
Am. J. Enol. Vitic. 41, 37^42.
Bortol, A., Nudel, C., Fraile, E., de Torres, R., Giuletti, A.,
Spencer, J.F.T. and Spencer, D. (1986) Isolation of the yeast with
killer activity and its breeding with an industrial brewing strain by
protoplast fusion. Appl. Microbiol. Biotechnol. 24, 414^416.
Palpacelli, V., Ciani, M. and Rosini, G. (1991) Activity of di¡erent
‘killer’ yeasts on strains of yeast species undesirable in the food
industry. FEMS Microbiol. Lett. 68, 75^78.
Morace, G., Manzara, S., Dettori, G., Fanti, F., Conti, S., Campani, L., Polonelli, L. and Chezzi, C. (1989) Biotyping of bacterial
isolates using the yeast killer system. Eur. J. Epidemiol. 5, 303^310.
Boekhout, T. and Scorzetti, G. (1997) Di¡erential killer toxin sensitivity patterns of varieties of Cryptococcus neoformans. J. Med. Vet.
Mycol. 35, 147^149.
Buzzini, P. and Martini, A. (2001) Discrimination between Candida
albicans and other pathogenic species of the genus Candida by their
di¡erential sensitivities to toxins of a panel of killer yeasts. J. Clin.
Microbiol. 39, 3362^3364.
Buzzini, P. and Martini, A. (2001) Large-scale screening of selected
Candida maltosa, Debaryomyces hansenii and Pichia anomala killer
toxin activity against pathogenic yeasts. Med. Mycol. 39, 479^482.
Polonelli, L., Lorenzini, R., De Bernardis, F. and Morace, G. (1986)
Potential therapeutic e¡ect of yeast killer toxin. Mycopathologia 96,
103^107.
Polonelli, L., Conti, S., Gerloni, M., Magliani, W., Castagnola, M.,
Morace, G. and Chezzi, C. (1991) ‘Antibiobodies’ : antibiotic-like
anti-idiotypic antibodies. J. Med. Vet. Mycol. 29, 235^242.
Meinhardt, F., Kempken, F., Kamper, J. and Esser, K. (1990) Linear plasmids among eukaryotes: fundamentals and application.
Curr. Genet. 17, 89^95.
Heintel, T., Zagorc, T. and Schmitt, M.J. (2001) Expression, processing and high level secretion of a virus toxin in ¢ssion yeast.
Appl. Microbiol. Biotechnol. 56, 165^172.
van Vuuren, H.J.J. and Wing¢eld, B.D. (1986) Killer yeasts: cause
of stuck fermentations in a wine cellar. S. Afr. J. Enol. Vitic. 7, 113^
118.
FEMSRE 748 1-8-02
275
[139] Longo, E., Vela¤zques, J.B., Cansado, J., Calo, P. and Villa, T.G.
(1990) Role of killer e¡ect in fermentations conducted by mixed
cultures of Saccharomyces cerevisiae. FEMS Microbiol. Lett. 71,
331^336.
[140] Carrau, F.M., Neirotti, E. and Gioia, O. (1993) Stuck wine fermentations: e¡ect of killer/sensitive yeast interactions. J. Ferment. Bioeng. 76, 67^69.
[141] Franken, D.B., Ariatti, M., Pretorius, I.S. and Gupthar, A.S.
(1998) Genetic and fermentation properties of the K2 killer yeast,
Saccharomyces cerevisiae T206. Antonie Van Leeuwenhoek 73, 263^
269.
[142] Vondrejs, V., Janderova, B. and Valasek, L. (1996) Yeast killer
toxin K1 and its exploitation in genetic manipulations. Folia Microbiol. 41, 379^393.
[143] Fox, J.L. (1993) Fungal infection rates are increasing. ASM News
59, 515^518.
[144] Herbrecht, R. (1996) The changing epidemiology of fungal infections: are the lipid-forms of amphotericin B an advance? Eur. J.
Haematol. Suppl. 57, 5712^5717.
[145] Selitrenniko¡, C.P. (2001) Antifungal proteins. Appl. Environ. Microbiol. 67, 2883^2894.
[146] Seguy, N., Polonelli, L., Dei-Cas, E. and Cailliez, J.C. (1998) E¡ect
of a killer toxin of Pichia anomala to Pneumocystis. Perspectives in
the control of pneumocystosis. FEMS Immunol. Med. Microbiol.
22, 145^149.
[147] Hector, R.F. (1993) Compounds active against cell walls of medically important fungi. Clin. Microbiol. Rev. 6, 1^21.
[148] Kurtz, M.B. (1998) New antifungal drug targets: A vision for the
future. ASM News 64, 31^39.
[149] Cabib, E., Drgon, T., Drgonova, J., Ford, R.A. and Kollar, R.
(1997) The yeast cell wall, a dynamic structure engaged in growth
and morphogenesis. Biochem. Soc. Trans. 25, 200^204.
[150] Jablonowski, D., Fichtner, L., Martin, V.J., Klassen, R., Meinhardt,
F., Stark, M.J. and Scha¡rath, R. (2001) Saccharomyces cerevisiae
cell wall chitin, the Kluyveromyces lactis zymocin receptor. Yeast 18,
1285^1299.
[151] Yamamoto, T., Hiratani, T., Hirata, H., Imai, M. and Yamaguchi,
H. (1986) Killer toxin from Hansenula mrakii selectively inhibits cell
wall synthesis in a sensitive yeast. FEBS Lett. 197, 50^54.
[152] Kimura, T., Kitamoto, N., Kito, Y., Iimura, Y., Shirai, T., Komiyama, T., Furuichi, Y., Sakka, K. and Ohmiya, K. (1997) A novel
yeast gene, RHK1, is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits beta-1,3-glucan synthesis. Mol. Gen. Genet. 254, 139^147.
[153] Kandel, J.S. (1988) Killer systems in pathogenic fungi. In: Viruses
of Fungi and Simple Eukaryotes (Koltin, Y. and Leibowitz, M.,
Eds.), pp. 243^263. Marcel Dekker, New York.
[154] Walker, G.M., MacLeod, A.M. and Hodgson, V.J. (1995) Interactions between killer yeasts and pathogenic fungi. FEMS Microbiol.
Lett. 127, 213^222.
[155] Gibbons, A. (1991) Moths take the ¢eld against biopesticide. Science 254, 646.
[156] Kinal, H., Park, C.M., Berry, J.O., Koltin, Y. and Bruenn, J.A.
(1995) Processing and secretion of a virally encoded antifungal toxin
in transgenic tobacco plants: evidence for a Kex2p pathway in
plants. Plant Cell 7, 677^688.
[157] Koltin, Y. and Day, P.R. (1976) Inheritance of killer phenotypes
and double-stranded RNA in Ustilago maydis. Proc. Natl. Acad.
Sci. USA 73, 594^598.
[158] Koltin, Y. and Day, P.R. (1976) Suppression of the killer phenotype
in Ustilago maydis. Genetics 82, 629^637.
[159] Park, C.M., Bruenn, J.A., Ganesa, C., Flurkey, W.F., Bozarth, R.F.
and Koltin, Y. (1994) Structure and heterologous expression of the
Ustilago maydis viral toxin KP4. Mol. Microbiol. 11, 155^164.
[160] Kinal, H., Tao, J.S. and Bruenn, J.A. (1991) An expression vector for the phytopathogenic fungus, Ustilago maydis. Gene 98,
129^134.
Cyaan Magenta Geel Zwart
276
M.J. Schmitt, F. Breinig / FEMS Microbiology Reviews 26 (2002) 257^276
[161] Park, C.M., Berry, J.O. and Bruenn, J.A. (1996) High-level secretion
of a virally encoded anti-fungal toxin in transgenic tobacco plants.
Plant Mol. Biol. 30, 359^366.
[162] Romanos, M.A., Scorer, C.A. and Clare, J.J. (1992) Foreign gene
expression in yeast: a review. Yeast 8, 423^488.
[163] Giga-Hama, Y. and Kumagai, H. (1999) Expression system for foreign genes using the ¢ssion yeast Schizosaccharomyces pombe. Biotechnol. Appl. Biochem. 30, 235^244.
[164] Bro«ker, M., Ragg, H. and Karges, H.E. (1987) Expression of human
anti-thrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Biochim. Biophys. Acta 908, 203^213.
[165] Tokunaga, M., Kawamura, A., Yonekyu, S., Kishida, M. and Hishinuma, F. (1993) Secretion of mouse alpha-amylase from ¢ssion
yeast Schizosaccharomyces pombe: presence of chymostatin-sensitive
protease activity in the culture medium. Yeast 9, 379^387.
[166] Sambamurti, K. (1997) Expression and secretion of mammalian
proteins in Schizosaccharomyces pombe. In: Foreign Gene Expression in Fission Yeast: Schizosaccharomyces pombe (Giga-Hama, Y.
and Kumagai, H., Eds.), pp. 149^158. Springer, Berlin.
[167] Ostergaard, S., Olsson, L. and Nielsen, J. (2000) Metabolic engineering of Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev.
64, 34^50.
[168] Pfei¡er, P. and Radler, F. (1982) Puri¢cation and characterization
FEMSRE 748 1-8-02
[169]
[170]
[171]
[172]
[173]
[174]
of extracellular and intracellular killer toxin of Saccharomyces cerevisiae strain 28. J. Gen. Microbiol. 128, 2699^2706.
Gunge, N., Tamaru, A., Ozawa, F. and Sakaguchi, K. (1981) Isolation and characterization of linear deoxyribonucleic acid plasmids
from Kluyveromyces lactis and the plasmid-associated killer character. J. Bacteriol. 145, 382^390.
Worsham, P.L. and Bolen, P.L. (1990) Killer toxin production in
Pichia acaciae is associated with linear DNA plasmids. Curr. Genet.
18, 77^80.
Suzuki, C. and Nikkuni, S. (1994) The primary and subunit structure of a novel type killer toxin produced by a halotolerant yeast,
Pichia farinosa. J. Biol. Chem. 269, 3041^3046.
Sawant, A.D., Abdelal, A.T. and Ahearn, D.G. (1989) Puri¢cation
and characterization of the anti-Candida toxin of Pichia anomala
WC65. Antimicrob. Agents Chemother. 33, 48^52.
Hanes, S.D., Burn, V.E., Sturley, S.L., Tipper, D.J. and Bostian,
K.A. (1986) Expression of a cDNA derived from the yeast killer
preprotoxin gene: implications for processing and immunity. Proc.
Natl. Acad. Sci. USA 83, 1675^1679.
Tao, J., Ginsberg, I., Banerjee, N., Held, W., Koltin, Y. and
Bruenn, J. (1990) Ustilago maydis KP6 toxin: structure, expression
in Saccharomyces cerevisiae and relationship to other cellular toxins.
Mol. Cell. Biol. 10, 1373^1381.
Cyaan Magenta Geel Zwart