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Prions remodel gene expression in yeast

Mick F. Tuite and Brian S. Cox

Epigenetic mechanisms participate in the regulation of gene transcription in eukaryotes. Two studies in yeast have revealed an
additional mechanism for controlling global gene transcription that is based on an inherited self-perpetuating change in the
conformation of two different components of key transcriptional regulatory complexes.

The epigenetic regulation of eukaryotic gene
transcription is mediated by a combination of
DNA modification and chromatin remodelling1.
DNA modification involves the methylation of
CpG sequences, and chromatin remodelling is
based on post-translational modifications of
histones, for example acetylation and phospho-
rylation. These changes can lead to an inherited
change in phenotype without any underlying
change in DNA sequence. Two recent studies,
one by Li and colleagues2 and one by Patel et
al.3 on page 344 of this issue, point to a quite
different epigenetic mechanism that can regu-
late the transcription of a set of genes in yeast.
Remarkably, it exploits an inherited self-perpet-
uating change in protein conformation, other-
wise known as a prion.
Prions were first identified in mammals
as protein-only infectious agents associated
with several devastating and ultimately fatal
brain diseases, the transmissible spongiform
encephalopathies (TSEs), such as human
CreutzfeldtJakob disease and bovine spongi-
form encephalopathy. The infectious prion
in each case is a self-propagating, alternative
conformational form of the cellular protein
PrP (ref.4). Once established, the prion form
of PrP can capture and convert the normal
soluble form of PrP into the transmissible
prion conformation.
Mick F. Tuite and Brian S. Cox are in the Kent Fungal
Group, Department of Biosciences, University of
Kent, Canterbury, Kent CT2 7NJ, UK.
e-mail: m.f.tuite@kent.ac.uk
The existence of prions in fungi emerged during mitosis or meiosis. In other words,
from studies on [URE3] and [PSI+], two non- the determinants of [URE3] and [PSI+] are
Mendelian traits in the yeast Saccharomyces prions. The protein associated with [URE3]
cerevisiae5. In both cases, the inheritance of prion is Ure2 and regulates the transcription
the particular trait is not accompanied by of the GLN3 gene, whose product is involved
any change in DNA sequence but rather is in nitrogen catabolite repression; the [PSI+]
due to the transmission of alternative confor- prion-associated protein is a translation ter-
mational forms of specific cellular proteins mination factor Sup35 (eRF3). In both cases
Cyc8 Tup1
Invertase
+ Lactate
levels
SUC2 CYC7 CYC1 +
Lac +
SUC2 CYC7 CYC1 +
Lac
[OCT+]
+++++
SUC2 CYC7 CYC1
Lac+
Figure1 Genetic screen for the yeast [OCT+] prion. The CYC1 gene encodes iso-1-cytochromec and
CYC7 encodes iso-2-cytochromec. To utilize lactate, (Lac+) yeast cells require cytochromec and this is
provided mainly by the CYC1 gene, although high levels of CYC7 gene product can compensate for the
absence of CYC1. Disruption of the CYC1 gene leads to an inability to utilize lactate (Lac). The Cyc8
Tup1 complex represses transcription of the CYC7 gene. The inactivation of Cyc8 either by mutation
or the formation of the [OCT+] prion leads to derepression of the CYC7 gene, resulting in elevated
levels of iso-2-cytochromec that are sufficient to permit the utilization of lactate in the cyc1 strain.
Transcription of the SUC2 gene encoding invertase is also repressed by the Cyc8Tup1 complex.

nature cell biology volume 11 | number 3 | MARCH 2009 241

2009 Macmillan Publishers Limited. All rights reserved.
news and views
1 465
Cyc8
1 202
Tup1
1 524
Swi1
(Gln)n Gln-rich
(Gln-Ala)n Asn-rich
Figures2 Glutamine/asparagine-rich regions in Cyc8, Swi1 and Tup1. The locations of poly-glutamine
tracts (red), glutamine-rich tracts (blue), asparagine-rich tracts (brown) and poly-(Gln-Ala) tracts (green)
in these proteins are indicated, together with their locations (residue numbers).
the inherited prion phenotype is due to a
dominant-negative effect of the correspond-
ing protein. For example, [PSI+] cells have a
translation termination defect. Interestingly,
the only phenotype associated with [PIN+], a
third well-studied yeast prion, is to facilitate
the de novo formation of other prions.
The report2 last year that a component of the
SWISNF yeast chromatin remodelling com-
plex, Swi1, can also switch to a metastable prion
form called [SWI+] first raised the possibility
that yeast prions might regulate gene transcrip-
tion at a global level6. The new study by Patel et
al.3 shows that the transcriptional co-repressor
Cyc8 can also form a prion called [OCT+]. Cyc8
together with the Tup1 protein forms a tran-
scriptional repressor complex, which together
with the SWISNF remodelling complex is
responsible for regulating the transcription of
400 or more genes7. Functional inactivation
of the Cyc8Tup1 or SWISNF complex by
prion formation would therefore be expected
to have profound effects on global gene expres-
sion and, in turn, on a variety of cellular pheno-
types. Several quite distinct [OCT+]-associated
phenotypes were indeed observed by Patel and
colleagues3, including defects in mating, sporu-
lation and constitutive invertase activity.
Given the implications of their finding for
global gene expression regulation, Patel et al.3
had to establish beyond reasonable doubt that
242
996
713
1314
[OCT+] has all the properties expected of a
prion. The first clue that Cyc8 might form a
prion came from an earlier study from the
same laboratory8 in which Cyc8 was identi-
fied in a screen for yeast proteins that facilitate
de novo formation of the [PSI+] prion when
overexpressed, as shown for the prion [PIN+].
Cyc8 was one of eleven proteins identified in
the screen. The spontaneous conformational
change leading to prion formation in yeast
as in mammals, occurs more readily when
the relevant gene is overexpressed, and this
property has indeed in the past facilitated the
identification of prions.
To fully validate the hypothesis that
[OCT+] is the prion form of Cyc8, Patel and
colleagues3 systematically tested all known
properties of yeast prions. In doing so, they
cleverly exploited the versatile genetic system
of this eukaryotic model organism. Luckily,
the detection of the formation of the [OCT+]
prion was greatly facilitated by a simple phe-
notypic assay associated with the loss of the
co-repressor function of Cyc8 (Fig.1). This
assay allowed the authors to demonstrate that
overexpression of CYC8 induced at high fre-
quency the ability to grow on lactate (Lac+),
a phenotype diagnostic of a non-functional
Cyc8, but which, in contrast with classical cyc8
gene mutations associated with this pheno-
type, was dominant in genetic crosses.
nature cell biology volume 11 | number 3 | MARCH 2009
2009 Macmillan Publishers Limited. All rights reserved.
The [OCT+] phenotype is, like all yeast prion
phenotypes, inherited in a non-Mendelian
fashion; this was shown by cytoduction, the
efficient transmission of [OCT+] by cytoplas-
mic transfer in the absence of nuclear transfer.
Furthermore, Patel et al.3 demonstrated that
maintenance of the [OCT+] prion and its asso-
ciated phenotype is, in a similar manner to all
other native yeast prions, dependent on the
activity of the molecular chaperone Hsp104.
Another characteristic of all yeast prions is the
presence of a structurally distinct prion-form-
ing domain (PrD) typically located at either
the amino or carboxy terminus of the protein9.
Yeast PrDs have an unusually high content of
Asn and Gln residues, a feature that promotes
amyloid formation, which in turn is required
for the propagation of the prion state. The puta-
tive PrD domain of Cyc8 is located within the
C-terminal half of the protein, contains 52%
of Gln residues and includes both a (Gln/
Ala)32 repeat and a homopolymeric stretch of
31 Gln residues (Fig.2). Interestingly, several
transcription factors have stretches rich in Gln
and/or Asn including Tup1, a binding partner
of Cyc8 (Fig.2). Patel et al.3 demonstrated that
overexpression of a truncated version of the
CYC8 gene that is lacking this domain in the
dominant [OCT+] mutant restored Cyc8 co-
repressor function. Furthermore, expression
of a fusion between the putative Cyc8PrD
and the fluorescent tag green fluorescent pro-
tein (GFP) in an [OCT+] strain resulted in the
formation of distinct aggregates in the cyto-
plasm, whereas when expressed in prion-free
[oct] cells, the fusion protein remained soluble,
indicating that [OCT+] was able to induce the
aggregated form of Cyc8PrDGFP.
Two additional experiments were performed
to confirm that Cyc8 is responsible for the
[OCT+] prion3. First, in the absence of cyc8,
yeast strains could not mutate to the dominant
[OCT+] form. Second, Patel and colleagues3
showed that cell-free extracts derived from
[OCT+] cells transformed about 5% of [oct]
cells to [OCT+] by using a recently developed in
vivo infectivity assay10. These crucial experi-
ments, in conjunction with the non-Mendelian
behaviour of the [OCT+] trait in genetic crosses,
leave little doubt that Cyc8 does indeed give
rise to the [OCT+] prion.
The elaborate tests conducted by Patel et al.3
amount to a functional definition of a prion: it
must form spontaneously from a specific protein
with a concentration-dependent probability; it
must template the conformational change in

cognate molecules that promotes aggregate for-
mation and growth and on which the phenotypic
change depends; aggregates must increase in
numbers by fragmentation, a phenomenon that
may or may not require the help of chaperones
such as Hsp104.
[OCT+] thus becomes the fifth genuine
yeast prion, and Cyc8 the second example of
a component of a global transcription factor
chromatin complex that can form a prion.
Yet although there is strong genetic evidence
that both Swi1 and Cyc8 can re/misfold into
a transmissible prion conformation, there is a
lack of direct physical evidence that these two
proteins in their native form are able to switch
to an alternative amyloid-like state in vivo in
the nucleus. It is nevertheless unlikely that the
phenotypes observed could be due to a phe-
nomenon that does not involve the generation
of a self-propagating conformational change,
although at least one self-perpetuating prion-
like state has been reported in yeast that does
not involve an alternative amyloid-like confor-
mation: the [] prion phenotype is associated
with the self-activation of a vacuolar protease,
which involves the processing of an inactive
precursor form by its mature form11.
How to grow a bud:
an importin acts in asymmetric division
David S. Goldfarb
The growth of daughter cells in budding yeast is a classic model for investigating mechanisms involved in asymmetric cell
division. An unexpected collaboration between the DEAD-box protein Dbp5 and the nuclear transport receptor Kap104 controls
localized protein synthesis at the bud tip during mitosis.
The localization of mRNAs is common to
asymmetric cell division in many eukaryo-
tes1. For example, during asymmetric cleav-
ages in Ilyanassa obsoleta embryos, mRNAs
that encode conserved developmental pattern-
ing factors first localize to interphase centro-
somes, and then move to a region of the cell
cortex that will be inherited by only one of the
daughter cells2. The means by which mRNAs
localize at the site of asymmetric growth
David S. Goldfarb is in the Department of Biology,
University of Rochester, Rochester, NY 14627, USA.
e-mail: dasg@mail.rochester.edu
nature cell biology volume 11 | number 3 | MARCH 2009 243
Does Swi1 and/or Cyc8 prion formation
provide a novel mode of global gene expres-
sion regulation in yeast? For [OCT+], Patel et
al.3 provide evidence for changes in expression
of target genes such as SUC2 (which encodes
invertase; Fig.1) and suggest that such changes
may have a beneficial effect on the host cell in
certain environments. The potential benefi-
cial effects of a yeast prion have already been
reported12,13, yet spontaneous prion switches in
yeast are rare events (frequencies are typically
between 105 and 107) but may be induced
by certain environmental triggers14. It is nev-
ertheless worth noting that frequencies of
prion switching are not greatly different from
the frequencies of nuclear gene mutations.
It has been suggested that the [PSI+] prion
may represent a Darwininan adaptation13,
although there have been arguments for the
[URE3] and [PSI+] prions being detrimental
disease-like states15. However, with one notable
exception, in the bacterium Escherichia coli16,
no amyloid seems to be formed by anything
but (concentration-dependent) chance and
none shows any suitability for exploitation in
physiological adaptation. Having said that, if
the only phenotype associated with the [OCT+]
is elaborated in budding yeast, where cis-
elements within certain mRNAs direct their
transport along actin filaments into the bud
by the She2She3Myo4 complex3. Protein
synthesis from these mRNAs is induced in
the bud by membrane-associated kinases that
phosphorylate translation repressors3. On
page 350 of this issue, van den Bogaart etal.4
describe a mechanism that requires the impor-
tin Kap104 (transportin in vertebrate cells)
and which could preferentially increase levels
of protein synthesis in the growing bud.
The nuclear transport of macromolecules,
including mRNAs, is mediated by soluble
2009 Macmillan Publishers Limited. All rights reserved.
news and views
prion had been its inability to activate the
mating type gene MAT, [OCT+] might have
been viewed as an epigenetic regulator of a key
developmental process in yeast.
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receptors, most of which belong to the karyo-
pherin family of importins and exportins.
Each member binds to a set of cargoes bearing
distinct nuclear localization signal (NLS) or
nuclear export signal motifs. Kap104 medi-
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Hrp1). Data from an earlier study, now worth
revisiting, indicated that Kap104 might also
have a direct role as a remodelling factor in
the removal of Nab2 from newly exported
mRNAs5. However, a more recent study
concluded that the RNA-dependent DEAD
box protein Dbp5 is instead responsible for