J. Biol. Macromol., 3(2), 41-46, 2003

Yeast Prions
Giman Jung1* and Gary Jones2
1

Pioneering Research Laboratories, Toray Industry Corporation, 111 Tebiro, Kamakura, Kanagawa 248-8555, JAPAN.

2

Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National

Institutes of Health, Bethesda, Maryland 20892-0830 USA.

Introduction
The term “PRION” stands for “protein only” and means“infectious protein.” In mammals, prions cause lethal
neurodegenerative diseases (transmissible spongiform
encephalopathies, TSEs) such as Scrapie in sheep, bovine
spongiform encephalopathies (BSE; mad cow disease) of
cattle and Creutzfeldt-Jakob disease (CJD) in humans. The
first prion disease Scrapie, was first described in the early
18th century, its human variety in the 1920s. The nature of theTSE disease-causing agent remained a mystery until thesuggestion by Prusiner (45) of the involvement of novel"proteinaceous particles", which he termed prions. It should
be noted that the initial idea that protein may be the causative
scrapie agent was made in 1967 (2, 20).
Wickner (56) proposed that the genetic behavior of the
Saccharomyces cerevisiae non-mendelian genetic elements[PSI+] (10) and [URE3] (1) could be explained if they wereprions of the Sup35 and Ure2 proteins respectively. Since
then much evidence has accumulated to support this
proposal. There are currently four confirmed prions in yeast,[PSI+], [URE3], [RNQ+] (12) and [NU+] (42) and genetic
evidence suggests there may be many more (12). The factthat prions exist in yeast provides an ideal environment for
detailed genetic analysis of factors affecting prion
propagation and maintenance.
What is a prion?
When proteins are synthesized on the ribosome, theyusually adopt only one final 3-dimensional conformation.
Prion proteins somehow adopt an altered conformation fromits normal form (by a mechanism that is not understood, but
is thought to involve some kind of nucleation event, 46) a
state referred to as the prion conformation. In the case ofyeast prions, once present the prion form of the protein iscapable of recruiting newly synthesized protein into theprion form and thus deplete the cell of functional protein (Fig.
1). We should emphasize here that there is no difference inthe genome between cells carrying or lacking a prion, theonly difference is the conformational state of the particular
protein in question. It is therefore reasonable to state that
proteins can indeed behave as genes, in that they can containinheritable information.
The prion conversion does not occur in the whole protein
but appears to occur in a so-called prion domain. Typicalprion domains consist of 60 ~ 150 amino acids and theconformational change within the prion domain takes thecredit for prion conversion (27, 34-36). The prion
conformation is rich in β-sheets and is prone to aggregationin which amyloid fibers are formed (17, 18, 28, 52).
Amyloid is a type of protein aggregate, which forms
self-sustaining orderly fibers. Amyloid fibers are foundassociated with many chronic diseases (amyloidosis) such asprion diseases, Alzheimer disease, polyglutamine expansion
diseases and type II diabetes (4). The difference between
prion proteins and other amyloids is that prions aretransmissible and infectious. Thus, yeast prions have beenused as a genetic model to study aspects of amyloidosis andprion propagation in mammals.
Prions are not always disease associatedIn mammals, prions cause incurable brain diseases.
However, the prion killer image doesn’t apply in yeast.
Although, [URE3] causes slower growth of yeast, there
appears to be no general toxicity of prion aggregates in yeast.
To the contrary it has been suggested that the [PSI+] prion
may be beneficial to yeast under certain circumstances. The
presence of [PSI+] can provide protection to some yeast
strains against various stresses such as heat shock (14). It has
also been suggested that the possible production of
C-terminally extended proteins due to nonsense suppression
by [PSI+] could act as an aid to evolution (53). Moreover the[Het-s] prion of the filamentous fungus Podospora anserina*

To whom reprint requests should be addressed. E-mail:

Giman_Jung@nts.toray.co.jp

42

Yeast prions

Prion
induction

Replication

ReplicationFormamyloid
fibersIntensify
amyloid
fibersFigure 1. Illustration of the prion propagation.

is clearly carrying out a normal cellular function (9). The
possible existence of many eukaryotic prions has been
postulated by computer aided methods (37) and in genetic
screens (12). It therefore seems highly likely that manydifferent prions will exist naturally and will probably carryout normal cellular functions.

behavior has been demonstrated to varying degrees for[PSI+] (51) and [HET-s] (33).
Yeast prion dependence of the Hsp104 protein and theeffects of protein chaperones on prion maintenance also add
credibility to the protein-only nature of these non-mendelian
genetic elements.

Confirming the protein-only hypothesis?
Yeast prion research has addressed a key feature of theprion theory, that is, prion phenomena occur not by classicalgenetic elements (virus, plasmid or other nucleic acidreplicon) but by infectious proteins. The genetic criteria
applied by Wickner (56) to suggest that [PSI+] and [URE3]
are prions is in itself compelling evidence for protein-only
inheritance. Firstly, he showed [URE3] appears
spontaneously in certain ratio. This experiment was possible
because yeast prions are not lethal in yeast and could be
cured simply by treating them in extremely low
concentrations (1 ~ 5 mM conc.) of guanidine hydrochloride(Gdn-HCl). He found that [URE3] appears spontaneously ata low frequency (1/106) cells, and when cured could
spontaneously appear at a similar frequency [URE3] (Fig. 2).
This was termed “reversible curability.” Such behavior
would not be expected for a nucleic acid replicon. Secondly,
when he overproduced the Ure2 protein the ratio of prionappearance was increased (1/104). The idea of thisexperiment is that the frequency with which a prion arises
should increase if the cellular content of the normal form isincreased, regardless of the mechanism of prion generation.
However, a nucleic acid replicon generally does not arise denovo regardless of what proteins in the cell overproduced.
Yeast and filamentous fungi have provided the only
environments were the actually infective nature of the prion
form of a protein have been directly assessed. Infectious

The role of chaperones in prion propagation
A major contribution of yeast prion research has been to
identify essential roles for protein chaperones in prionappearance and propagation. Chaperones are proteins thataid and regulate the correct folding of other proteins. Many
types of chaperones have been described but only a subset
exert effects upon yeast prions. The finding that chaperones
can cure or enhance prion propagation may provide a new
approach to the treatment of prion/amyloid diseases.
Chaperones are usually induced by cellular insults such asheat shock. Indeed, many protein chaperones have been
identified in screens to identify heat-shock proteins and thus
are termed Hsp's. At least 6 groups of Hsp's have been
identified and are grouped together by molecular weight,(Hsp100, 90, 70, 60, 40 and 20). Some Hsp groups havesub-family members, for instance, in yeast the Hsp70 family
consists of 14 different proteins classed together by aminoacid homology. These Hsp70's function in various
compartments of the cell and carry out functions such as
preventing aggregation of denatured or incorrectly folded
proteins, and aiding protein transport across cell membranes.
The first reports describing the effects of a protein
chaperone on prion propagation identified a critical role for
the Hsp104 protein (6, 8). Hsp104 is a member of the ClpB
family of Hsp100's and is a "dis-aggregase". It cannot
prevent unfolded proteins aggregating but it can resolubilize
an aggregate once it has formed (19, 24, 25, 43). An interme-Jung and Jones

43

Reversible Curing[psi-]
clone

10-6
Select[PSI+]
clone[PSI+]
clone
1

10-6

Curing withguanidine

Select[PSI+]
clone

Sup35p Overproduction → higher frequency of [PSI+]
10-6

10-4
[psi-] strain overproducing Sup35p

Figure 2. Genetic criteria for a prion.

diate level of Hsp104 is required for efficient maintenance of[PSI+]. Depletion of Hsp104 activity in the cell by either
gene deletion or protein inhibition causes the curing of all
known yeast prions (12, 13, 38, 50). It is postulated thatHsp104's ability to break up prion aggregates into smaller
particles or "seeds" is responsible for efficient transfer of theprion from cell to cell (55). Over-expression of Hsp104 cancure [PSI+] but has no effect on [URE3] or [RNQ+] (12, 13,38). This finding suggests structural differences exist
between these prions. A single amino acid substitution in
Hsp104 protein causes dramatic weakening of two prions,[PSI+] and [URE3] (24). Also, alterations in the AAA
protease motif of Hsp104 can affect [PSI+] stability (21).
Cytosolic Hsp70's have also been implicated inmaintenance of [PSI+]. Yeast has two sub-classes of cytosolic
Hsp70's, the Ssa family comprising the Ssa1-4 proteins andalso the Ssb family comprising the Ssb1 and 2 proteins (11).
The Ssa1 protein has been implicated in [PSI+] propagation
both as an antagonist of prion curing by over-expression of
Hsp104 (41) and in general prion stability (22, 26). Although
the Ssa1 and Ssa2 proteins are 97% identical they can havedifferent effects upon yeast prions. Over-expression of Ssa1
was capable of efficiently curing [URE3] whereas
over-expression of Ssa2 was not (49). Deletion of the SSB
genes caused an increase in spontaneous appearance of[PSI+] (7) which can be explained by these ribosomeassociated protein chaperones having a role in folding of
newly synthesized polypeptides (39, 44). Over-expression of
Ssb1 can cure certain "strains" of [PSI+] (5).
The functions of Hsp70's in protein folding are usually
carried out in conjunction with an Hsp40 co-chaperone
partner. This also seems to be the case for Hsp70 effects onprions. Over-expression of the Ydj1 protein can cure weakforms of [PSI+] (30) and can also cure [URE3] (38). The
Hsp40 protein Sis1 has been shown to be important for
maintenance of [RNQ+] prion (32, 50), and theuncharacterized Apj1 protein, when over-expressed can curesome "strains" of [PSI+] (29). The importance of a directinteraction between Hsp70 and Hsp40 in [PSI+] propagation
is demonstrated by suppression of the prion destabilizing
SSA1-21 mutation by a second site suppressor that disrupts
interaction with Hsp40's (24).
Recently co-chaperones of the tetratricopeptide repeat(TPR) family have been implicated in yeast prion propagation. Over-expression of the Sti1 protein can cure "strains"of [PSI+] (29) while mutations in the C-terminal Hsp70
motif EEVD, required for interaction with TPR proteins arecapable of suppressing the SSA1-21 mutation (22).
Amyloid- a "special" type of chaperone substrate?
Because amyloid is formed into a regular orderedstructure, this raises the possibility that recognition ofamyloid and prion aggregates by protein chaperones may bedifferent from recognition of heat-denatured amorphousaggregates. Genetic evidence for this proposal has arisen
from the array of Hsp104 and Hsp70 mutants available inyeast that affect prion propagation. Many mutations exist in
Hsp70 (22, 26) and Hsp104 (24) that affect prion
propagation but have no effect on cell growth or cellular
thermotolerance. This suggests that either amyloidaggregates are more sensitive to the effects of thesechaperones or that they are recognized differently thandenatured amorphous aggregates.
Prions can affect the appearance of other prions

44

Yeast prion

The increased appearance of [PSI+] due to overexpressionof the Sup35 protein was shown to be dependent on thepresence of another suspected prion called [PIN+] (13). A
genetic screen revealed the identity of [PIN+] as the [RNQ+]
prion and also identified an array of putative yeast prions that
could also behave in a [PIN+]-like manner (12). Similar
results were observed for strains carrying the less wellcharacterized [NU+] prion (42).
An antagonistic effect on prion strength and induction has
also been reported for the [PSI+] and [URE3] prions (49).
The mechanism for how prions can affect each others
induction and stability is unknown. Possible models include
established prion aggregates providing a template for newprion aggregates to initiate or due to the sequestration ofprotein chaperones providing an environment favorable for
new prion formation.
Mechanism of prion curing by guanidine hydrochloride
Tuite et al (54) showed that an array of chemicals or stresstreatments were capable of curing [PSI+] to some degree.
One potent and widely used prion-curing agent is guanidinehydrochloride (Gdn-HCl). Medium containing 1-5mM
Gdn-HCl efficiently cures yeast of all confirmed prions by aprocess that inhibits replication of the prion (15). Yeast cells
grown in the presence of 1mM Gdn-HCl in liquid culture
have been estimated to have intra-cellular Gdn-HCl
concentrations around 20mM (23). The mechanism of
curing by Gdn-HCl has recently been shown to involve theinhibition of Hsp104, effectively creating conditions
mimicking a deletion of Hsp104 (16, 24, 25, 40). The most
likely nature of this inhibition is to disrupt the ATPase
activity of the Hsp104 protein. Although it is beyond
contention that Gdn-HCl is curing prions predominantly
through an Hsp104 dependent mechanism, there is someevidence that other proteins or pathways capable of exerting
effects on [PSI+] may also be affected by Gdn-HCl (31, 55).
The elucidation of this curing mechanism further
emphasizes the importance of Hsp104 in prion propagation.
Fungal and plant homologues of Hsp104 have been
identified, but as yet no mammalian counterpart has been
isolated. It seems reasonable to assume that such a mecha-

nism as protein dis-aggregation will have been conserved
through evolution and a mammalian protein analogous to
Hsp104 will eventually be identified. Inhibition of such a
protein may be a potential treatment for prion and amyloid
diseases.
Conclusion and future prospects
In almost a decade since Wickner (56) proposed that
prions exist in yeast, researches in this area have gone a long
was in proving the protein-only hypothesis. Many cellularfactors (mostly protein chaperones) have been identified that
affect yeast prion propagation, most of these factors areconserved in mammals. It is through the genetic andbiochemical analysis of the existing chaperone mutants andother cellular factors, in relation to their known cellularfunctions and prion phenotypes, that will allow us to unravel
the complex nature of prion propagation in yeast. Due to thehigh degree of conservation in protein chaperones, findings
are likely to be directly relevant to amyloid and prion
formation in higher eukaryotes.
Researchers are also exploring some novel uses of prions.
Prion domains appear modular and transferable. Proteins thatare not natural prions can be made to behave like prions by
the addition of the Sup35 prion domain (47). This raises thepossibility of protein engineering using prion domains. Most
recently prion fibres have been coated with metal particles
and been shown to conduct electricity (48). Others havefound forming prion aggregates does not cause a total loss of
enzymatic function (3). It may therefore be possible todevelop immobilized functional proteins using prion fibres
that will have industrial applications.
The relatively youthful area of yeast prion research has
already produced many significant findings and willcontinue to do so for many years to come. The relevance ofprions in biology is not yet realized and it is through researchon yeast prions were most of our understanding of thisrelevance has been and will continue to be based upon.
We thank D.C. Masison, C. Schwimmer, R. D. Wegrzyn and
R. B. Wickner.

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Communicated by Hiroshi Ueno