Introduction to Fungi, Third Edition

SACCHAROMYCES (SACCHAROMYCETACEAE)
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no longer required, and the removal of mating
type receptors, e.g. after the fusion of two
haploid cells.
10.2.6 The yeast vacuole
The vacuole is the central destination of membrane
trafficking in S. cerevisiae. It receives input
directly from the secretory route in the form
of most vacuolar proteins which are separated
at the Golgi stage from those bound for secretion.
Material also reaches the vacuole from
the endocytotic route (see above), and from
the cytoplasm, especially during starvation.
Cytoplasmic material may be engulfed directly
by the tonoplast (microautophagy) or redundant
material may first be surrounded by a double
membrane to form an autophagosome whose
outer membrane then fuses with the tonoplast
(macroautophagy). Details of these processes
have been described by Klionsky (1997) and
Thumm (2000). Degradation of protein is a
major function of the vacuole in starvation
situations, and about 40% of the total protein
content of a yeast cell can be degraded
within 24 h (Teichert et al., 1989). Not surprisingly,
the vacuole contains a large set of powerful
hydrolytic enzymes, especially proteases
(Klionsky et al., 1990).
When nitrogen is abundant, it is stored in
the vacuole as arginine at concentrations of
up to 400 mM, and this can be re-released into
the cytoplasm if nitrogen becomes limiting
(Kitamoto et al., 1988). Likewise, phosphate can
be stored and released (Castro et al., 1999), as
can many other ionic nutrients (Jennings, 1995).
Toxic ions and metabolites may be stored in
the vacuole (e.g. Ramsay & Gadd, 1997). Vacuoles
thus fulfil a crucial function in maintaining
the homeostasis of the yeast cytoplasm against
changing external conditions. In order to fulfil
such functions, the vacuolar morphology
can change dramatically, e.g. by fragmentation
of one large central vacuole into numerous small
ones (Çakar et al., 2000).
10.2.7 Killer yeasts and killer toxins
Killer yeasts are strains which produce toxins
capable of killing other strains belonging to the
same or to closely related species. Toxin producers
are resistant against their own toxin, but may
be susceptible to toxins produced by other
strains. Three important virus-encoded killer
toxins (K1, K2, K28) are known to exist in
S. cerevisiae; all three are polypeptides and are
encoded by double-stranded RNA encapsulated
in virus-like particles (VLPs). Another group of
double-stranded viruses (the L-A viruses) belonging
to the genus Totivirus is necessary for the
replication of the killer toxin VLPs. The subject
of killer yeasts has been reviewed by Magliani
et al. (1997) and Marquina et al. (2002).
The best-researched killer toxin is K1. It is
encoded by a single open reading frame and
is synthesized as a single polypeptide which is
initially localized in the ER membrane. As the
membrane-bound polypeptide travels the secretory
route, it is modified by glycosylation and
proteolytic cleavage, much like other secretory
proteins. In the Golgi system, the polypeptide is
cleaved into two parts which are held together
by disulphide bonds, and a third part, the glycosylated
region, which is not part of the active
toxin. The active toxin is secreted and diffuses
into the growth medium. The two parts of the
active molecule fulfil two different functions;
the b-chain binds the molecule to its receptor
site which is the b-(1,6)-glucan component of
the cell wall. Following binding to the wall, the
toxin is thought to be transferred to the
plasma membrane where the a-chain forms
a trans-membrane pore. Death of the target cell
occurs because the trans-plasma membrane
proton and ionic gradients are disrupted. The
toxin can bind to the wall of the producing
cell but not to its plasma membrane; presumably
a membrane receptor is altered, masked
or destroyed. Self-immunity is conveyed by a
precursor molecule of the mature toxin (Boone
et al., 1986).
The K1 toxin has been an important instrument
in elucidating the processing of proteins
along the secretory route, and the mechanism
of cell wall synthesis in S. cerevisiae. Additionally,
there are biotechnological implications. The
possession of a killer toxin conveys a selective
advantage upon a yeast strain, and killer yeasts
are particularly common (25% of all isolates)