To: BSE-L@uni-karlsruhe.de
Nature 428, 323 - 328 (18 March 2004); doi:10.1038/nature02392
Conformational variations in an infectious protein determine prion
strain differences
MOTOMASA TANAKA1, PETER CHIEN1,2, NARIMAN NABER3, ROGER COOKE3 &
JONATHAN S. WEISSMAN1,2
1 Howard Hughes Medical Institute, Department of Cellular and Molecular
Pharmacology, University of California-San Francisco, San Francisco,
California 94143, USA
2 Graduate Group in Biophysics, University of California-San Francisco,
San Francisco, California 94143, USA
3 Department of Biochemistry and Biophysics, University of
California-San Francisco, San Francisco, California 94143, USA
Correspondence and requests for materials should be addressed to J.S.W.
(jsw1@itsa.ucsf.edu).
A remarkable feature of prion biology is the strain phenomenon wherein
prion particles apparently composed of the same protein lead to
phenotypically distinct transmissible states1-4
.
To reconcile the existence of strains with the 'protein-only' hypothesis
of prion transmission, it has been proposed that a single protein can
misfold into multiple distinct infectious forms, one for each different
strain1-3
,
5
.
Several studies have found correlations between strain phenotypes and
conformations of prion particles6-10
;
however, whether such differences cause or are simply a secondary
manifestation of prion strains remains unclear, largely due to the
difficulty of creating infectious material from pure protein3
,
5
.
Here we report a high-efficiency protocol for infecting yeast with the
[PSI+] prion using amyloids composed of a recombinant Sup35 fragment
(Sup-NM). Using thermal stability and electron paramagnetic resonance
spectroscopy, we demonstrate that Sup-NM amyloids formed at different
temperatures adopt distinct, stably propagating conformations. Infection
of yeast with these different amyloid conformations leads to different
[PSI+] strains. These results establish that Sup-NM adopts an infectious
conformation before entering the cellfulfilling a key prediction of the
prion hypothesis5
and
directly demonstrate that differences in the conformation of the
infectious protein determine prion strain variation.
The yeast prion state [PSI+]11
results from self-propagating aggregation of the translation termination
factor protein Sup35, leading to a nonsense suppression phenotype. In
yeast containing a nonsense mutation in the ade1 gene, [PSI+] colonies
are white or pink and grow on media lacking adenine, whereas [psi-]
colonies are red and require adenine. Transient overexpression of the
Sup35 amino-terminal, glutamine/asparagine-rich (prion) domain leads to
Sup35 aggregation and de novo appearance of [PSI+]. In vitro, this prion
domain forms self-seeding amyloid fibres12
,
13
.
As with mammalian prions and other yeast prions, [PSI+] exhibits a range
of heritable phenotypic strain variants14
that differ in mitotic stability14
,
dependence on the cellular chaperone machinery15
and in the solubility and activity of Sup35 (refs 14
,
1618
),
leading to differences in the ade1 colour phenotype. Sup35 aggregates
obtained from different [PSI+] strains also vary in their ability to
seed polymerization of purified protein in vitro18
.
In addition, [PSI+] strains can have a major role in determining the
specificity of prion transmission9
,
10
,
19
.
This link between prion strains and transmission barriers is likely to
be general, as the ability of mammalian prions to cross species barriers
also differs substantially among mammalian prion strains2
,
4
.
Here we present a high-efficiency protocol for infecting yeast with
preformed prion particles. Earlier studies demonstrated that
introduction by liposome fusion of bacterially produced Sup-NM (Sup35
N-terminal residues 1254) induces [PSI+]20
;
however, this liposome-based infection strategy was poorly suited to
test the role of prion conformations in strain diversity as only a small
fraction (12%) of yeast were converted to [PSI+]. More fundamentally,
Sup-NM amyloid formation occurs after encapsulation within liposomes,
making it difficult to control for or evaluate the conformation of the
infectious form. To overcome these difficulties, we have developed a
modified transformation protocol in which preformed Sup-NM amyloid
fibres along with a URA3-marked plasmid are introduced into yeast
spheroplasts by treatment with polyethylene glycol (PEG) (Fig. 1a
).
The inclusion of the URA3 plasmid allows us to select for the small
fraction of yeast that have successfully taken up material from
solution. After spheroplast transformation, cells are grown on plates
that are devoid of uracil and contain trace amounts of adenine. On such
plates, [psi-] yeast form small, intensely red colonies, whereas [PSI+]
yeast form large white colonies. Transformation of [psi-] yeast with
plasmid alone or with soluble Sup-NM did not lead to detectable
formation of [PSI+] colonies. In contrast, inclusion of preformed Sup-NM
amyloid seeds led to significant production of large, white (Ade+)
colonies (Fig. 1b
).
As expected for a prion, infectivity was sensitive to proteinase but not
nuclease treatment (Supplementary Fig. S1
).
Increasing the concentration of Sup-NM fibres resulted in a
dose-dependent increase in conversion to Ade+ colonies, with the
fraction of Ade+ colonies among the Ura+ colonies approaching 100% at
high Sup-NM concentrations (Fig. 1c
).
These converted Ade+ colonies exhibited the hallmarks of the [PSI+]
prion state: the Ade+ trait was inherited in a non-mendelian manner, the
trait was readily cured by transient growth on medium containing
guanidine hydrochloride (GuHCl) and was associated with the formation of
large Sup35 aggregates, that were pelletable by high-speed
centrifugation (Supplementary Figs S2 and S3
).
Sup35 overexpression only induces [PSI+] in yeast harbouring a second
prion, termed [PIN+]21
(Supplementary Fig. S4
).
In contrast, we found that the efficiency of infection was independent
of [PIN+] (Fig. 1c
),
consistent with the requirement for [PIN+] in the initial conversion in
vivo of Sup35 to the prion state but not for the subsequent propagation
of [PSI+]. Our ability to infect [pin-] cells at high efficiency
indicates that [PSI+] induction was not simply a by-product of
increasing cellular prion protein levels, a possibility for which it was
difficult to control for in previous protein infection experiments5
,
20
,
22
,
rather, conversion to [PSI+] was a consequence of introducing Sup-NM in
an infectious (prion) conformation.
Figure 1 Induction of the [PSI+] prion by in-vitro-converted Sup-NM
amyloid fibres. Full legend
High resolution image and legend
(79k)
We examined the specificity of our infectious particles. Whereas the
prion domain of Sup35 is conserved across a range of budding yeast,
[PSI+] 'infectivity' is often limited by transmission barriers23-25
analogous to the species barriers in mammalian prions. For example,
prion domains of Sup35 from Saccharomyces cerevisiae and Candida
albicans are both capable of forming heritable conformations but do not
cross-seed each other in vivo or in vitro23
.
Our infection experiments faithfully reproduced this specificity in
prion transmission. Saccharomyces cerevisiae fibres were unable to
infect yeast that had the genomic SUP35 prion domain replaced by the C.
albicans sequence (Fig. 1d
).
Similarly, C. albicans could infect C. albicans yeast, albeit at low
efficiency, but were unable to infect yeast that had the wild-type (S.
cerevisiae) SUP35. Finally, crude extracts from [PSI+] S. cerevisiae or
C. albicans could be used as a source of infectious material and
displayed the same self-specificity as recombinant prions (Fig. 1e
).
Induction of [PSI+] by Sup35 overexpression leads to a range of prion
strains that can be distinguished readily by differences in their colony
colour on rich medium (YEPD)14
.
To determine whether infection with pure Sup-NM protein led to a similar
range of strains, we plated PEG-transformed cells on to medium rich in
adenine (SD-URA), which allowed yeast to grow robustly regardless of the
[PSI+] state (Fig. 2a
,
top). Randomly chosen colonies were then streaked on YEPD to reveal
their colour phenotype, which indicated whether they remained [psi-]
(red) or had converted to a weak [PSI+] (pink) or strong [PSI+] (white)
state. As expected, mock-infected yeast or yeast infected with soluble
Sup-NM yielded only red [psi-] colonies. In contrast, infection with
preformed fibres led to a range of [PSI+] strains (Fig. 2a
,
bottom). Importantly, when we used extracts or partially purified Sup35
proteins from strong or weak [PSI+] strains, we exclusively observed
strong or weak [PSI+] transformants, respectively (Fig. 2b
;
see also Supplementary Fig. S5
).
Thus, the different prion strains observed in yeast transformed with
pure protein are not a consequence of the transformation procedure but
rather arose from intrinsic heterogeneity in the Sup-NM prions formed in
vitro.
Figure 2 Generation of multiple [PSI+] strains by Sup-NM amyloid fibres
converted in vitro. Full legend
High resolution image and legend
(82k)
Earlier studies have shown that, as with other amyloid-forming proteins4
,
26
,
Sup-NM spontaneously adopts a mixture of distinct fibre types12
,
27
,
suggesting that these different amyloid conformations may be responsible
for the different [PSI+] strains generated in the above infection
experiments. To explore the relationship between prion strains and
amyloid conformations, we prepared Sup-NM amyloids with different
conformations in vitro. We generated Sup-NM amyloids at different
temperatures (4 °C, 23 °C and 37 °C), as previous studies have found
that for some proteins polymerization temperature can influence the
range of preferred amyloid conformations4
,
10
.
Both resistance to thermal denaturation in the presence of SDS and
limited proteolysis (Supplementary Fig. S6
)
indicated that fibres formed at 4 °C had markedly different physical
properties than those formed at 23 °C or 37 °C (Fig. 3a
).
In particular, 4 °C fibres showed a relatively low melting temperature
(Tm = 62 4 °C) and broad melting transition (W = 29 6 °C) compared with
23 °C (Tm = 78 3 °C, W = 18 3 °C) or 37 °C (Tm = 79 2 °C, W = 12 2 °C)
fibres.
Figure 3 Different conformations of Sup-NM amyloid fibres are formed at
different temperatures. Full legend
High resolution image and legend
(67k)
Electron paramagnetic resonance (EPR) spectroscopy on spin labels
introduced at specific positions within Sup-NM revealed that there are
general structural features common to all Sup-NM fibres as well as
localized differences characteristic of particular fibre conformations.
We prepared Sup-NM mutants in which a single cysteine residue was
substituted into wild-type Sup-NM (which lacks cysteines) at
approximately every tenth residue. The mutants were then labelled with a
cysteine-specific spin probe28
.
For each labelled protein, we measured spectra of soluble Sup-NM and of
fibres formed under various conditions. Regardless of the polymerization
state, residues in the carboxy-terminal region of Sup-NM (residue 158
and beyond) displayed sharp EPR spectra indicative of highly mobile spin
probes29
(Fig. 3b
,
c
).
In contrast, residues in the middle of the prion domain (especially
those between residues 36 and 76) in amyloid forms, but not in the
unpolymerized state, showed broad peaks at low (P1) and high (P3)
fields, indicative of highly immobilized spin probes29
.
Amyloid seeds from a Sup-NM mutant (S17R) have been shown to convert
wild-type Sup-NM into a conformation distinct from that produced by
wild-type seeds10
.
We found that in S17R-seeded fibres the maximal position of the P1 peak
is at residue 63 compared with the maximum seen at residue 46 in the 4
°C or 37 °C fibre forms (Fig. 3c
).
We also observed significant, site-specific differences in the
intensities of the P1 peak between fibres formed at 4 °C or 37 °C (Fig.
3c
).
Our ability to produce distinct Sup-NM amyloid forms solely by varying
the polymerization temperature allowed us to test directly the role of
prion conformation in determining strain properties in vivo. We found
marked differences in the [PSI+] strains generated by prion infections
depending on the conformation of the infectious material used. Fibres
formed at 4 °C had a high efficiency of infection with the large
majority of colonies showing a strong (white) [PSI+] strain phenotype
(Fig. 4a
,
b
),
whereas fibres formed at 23 °C or 37 °C had lower infectivity and
produced almost exclusively weak (pink and/or sectored) [PSI+] strains
(Fig. 4a
,
b
).
The weak strains showed increased levels of soluble Sup35 and were more
readily cured by Hsp104 overexpression (Supplementary Figs S3 and S7
).
In principle, these differences in prion strains could be a consequence
of differences in the prion titre of 4 °C and 37 °C fibre preparations
(for example, introduction of multiple infectious particles could
promote strong strains). We excluded this possibility by demonstrating
that strain phenotypes do not depend on the concentration of seed or the
infection efficiency: 4 °C fibres yielded strong strains even when
diluted tenfold (infection rate 20%) and 37 °C fibres yielded weak
strains even when the concentration was increased tenfold (infection
rate 80%) (Fig. 4c
).
Once formed, the 37 °C fibre conformation is stable and can be
transmitted by subsequent polymerization even at 4 °C. Polymerization of
Sup-NM at 4 °C, induced by seeding with a small amount of 37 °C fibre,
leads to an amyloid with the properties of 37 °C fibres; that is, a high
melting temperature (Tm = 77 4 °C) and a yield of predominantly weak
strains in infection experiments (Fig. 4d
).
Together, the above data demonstrate that a single protein can adopt
multiple distinct, self-propagating (infectious) conformations and that
these conformational differences underlie heritable differences in prion
strains.
Figure 4 Induction of distinct [PSI+] strains by Sup-NM amyloid fibres
formed at different temperatures. Full legend
High resolution image and legend
(118k)
The existence of multiple distinct heritable strains has been difficult
to explain in the context of the protein-only hypothesis of prion
inheritance2
,
3
.
Strains now seem to be a nearly universal feature of prions: a wide
range of different strains have been found for mammalian prions, for the
three known naturally occurring yeast prions, and for a variety of
artificial yeast prions4
.
Cellular factors such as chaperones probably have an important role in
the manifestation of strain phenotype and promoting stable strain
propagation18
.
Nonetheless, the ubiquitous nature of strains argues that they arise
from a common physical property shared by all prions rather than from a
specific feature of a given prion, such as the sequence of the
infectious protein. All naturally occurring prions appear to be based on
self-propagating, beta -sheet-rich aggregates often referred to as
amyloids1
,
12
,
13
,
22
,
26
,
30
.
Many amyloid-forming proteins, including those involved in
non-infectious diseases, can misfold into more than one form, and these
differences can subsequently be propagated by templated conversion4
.
Together with previous studies correlating strains with prion
conformation, our work, by directly demonstrating that different amyloid
conformations lead to distinct [PSI+] strains, argues that the ability
of proteins to misfold into multiple amyloid forms constitutes the
physical foundation of the strain phenomenon.
Note added in proof: We have found that prion particles purified from
weak [PSI+] strains show increased resistance to thermal denaturation in
the presence of SDS (Tm = 74 6 °C, W = 17 4 °C) compared with prion
particles purified from strong [PSI+] strains (Tm = 59 5 °C, W = 27 5
°C). This result indicates that conformational differences in infectious
prion particles created in vitro are faithfully propagated in vivo.
Methods
Construction of plasmids and yeast strains Plasmid vectors encoding
Sup-NM single cysteine mutants for bacterial protein expression were
generated using QuikChange
mutagenesis (Stratagene). Throughout we used isogenic [psi-] and [PSI+]
derivatives of 74D-694 [MATa, his3, leu2, trp1, ura3; suppressible
marker ade1-14(UGA)]23
.
With the exception of Fig. 1c
,
all strains were [PIN+]. Similar results were obtained with a
W303-derived strain. A yeast strain expressing the prion domain from C.
albicans SUP35 was generated by replacing the wild-type chromosomal
locus in the parental 74-D694 strain, as previously described10
.
Preparation of spheroplasts for transformation Yeast strains were grown
in YEPD media to an optical density of 0.5 at 600 nm and successively
washed with sterile H2O, 1 M sorbitol and SCE buffer (1 M sorbitol, 10
mM EDTA, 10 mM dithiothreitol, 100 mM citrate, pH 5.8). Cells were
spheroplasted with lyticase ( 250 µg for yeast cells of 50 ml YEPD) in
SCE buffer at 30 °C for 30 min (see Supplementary Information
for a description of lyticase preparation). Spheroplasts were washed
with 1 M sorbitol and STC buffer (1 M sorbitol, 10 mM CaCl2, 10 mM Tris,
pH 7.5). Pelleted cells were resuspended in STC buffer and mixed with
sonicated amyloid fibres, URA3-marked plasmid (pRS316) (20 µg ml-1) and
salmon sperm DNA (100 µg ml-1). Fusion was induced by addition of 9
volumes of PEG buffer (20% (w/v) PEG 8000, 10 mM CaCl2, 10 mM Tris, pH
7.5) for 30 min. Cells were centrifuged, resuspended in SOS buffer (1 M
sorbitol, 7 mM CaCl2, 0.25% yeast extract, 0.5% bacto-peptone),
incubated at 30 °C for 30 min and plated on synthetic media lacking
uracil overlaid with top agar (2.5% agar)20
.
For experiments in Fig. 1b
,
adenine (20 mg l-1) was included only in the top agar.
Preparation of infectious in vitro particles and yeast extracts Amyloid
fibres of Sup35-NM were formed spontaneously at 4 °C, 23 °C or 37 °C or
with indicated 5% (w/w) seed by dilution of GuHCl-denatured proteins
into 5 mM potassium phosphate buffer (pH 7.4) containing 150 mM NaCl10
.
Fibres were collected by centrifugation at 20,000g for 20 min,
resuspended with phosphate buffer, and sonicated. The final
concentration of amyloid fibres in infection experiments was 2.5 µM
unless otherwise indicated. For preparation of yeast extracts, cells
were spheroplasted with lyticase and sonicated. Concentration of total
protein in the yeast extracts was measured by Bradford assay. For
transformations involving extracts, the final concentration of total
protein of the crude yeast extract was 200 µg ml-1.
In vivo analysis of prion strains and species specificity After growth
on SD-URA ( 7 days), the efficiency of conversion to prion state and
phenotypic strength of prion strains was examined by the following
colour assay. Positive URA3 transformants were randomly selected and
streaked onto YEPD plates containing one-quarter of the standard amount
of yeast extract to enhance the colour phenotype. Control strong [PSI+]
and [psi-] yeast colonies were also streaked onto each plate for
comparison. After 35 days, colonies were classified as strong [PSI+]
(white), weak (pink) [PSI+] or [psi-] (red) strains. In quantification
experiments at least three measurements of 56 colonies from independent
transformations were made. All quantification experiments used the
SD-URA followed by streaking assay rather than direct plating on SD-URA
trace ADE.
In vitro analysis of amyloid fibre Sup-NM proteins C-terminally tagged
with 7 times -histidine were purified as reported previously23
,
except in the case of the cysteine mutants where 1 mM beta
-mercaptoethanol was included in all purification buffers. The thermal
stability of amyloid fibres was examined by SDSpolyacrylamide gel
electrophoresis (PAGE) as performed previously10
.
The Coomassie-stained band intensities were fitted to a sigmoidal curve
with IgorPro3.0 (WaveMetrics)
, using the
following equation: y = A + B/(1 + exp((C - x)/D)), where x, y, C, D
indicate temperature, band intensity, melting (Tm) and transition (W)
temperatures, respectively. For EPR spectroscopy, single cysteine
mutants were labelled by 4-maleimido-2,2,6,6-tetramethyl-1-piperidinoxy
(5
equivalents) (Sigma) in 6 M GuHCl containing 25 mM sodium phosphate
buffer (pH 8.0) overnight with 80% efficiency, and purified on Ni-NTA
agarose (Qiagen)
. Absence
of labelling at non-cysteine residues was confirmed using wild-type
Sup-NM. Amyloid fibres were prepared by dilution of the denatured
proteins into 5 mM potassium phosphate buffer containing 150 mM NaCl in
the presence of 5% (w/w) seed. Fibres were formed at 4 °C and 37 °C with
5% (w/w) seed of spontaneously formed amyloids at 4 °C and 37 °C,
respectively. S17R-seeded fibres were formed by polymerization of
wild-type Sup-NM at 23 °C with 5% (w/w) seed of spontaneously formed
S17R mutant fibres10
.
Fibre formation was monitored in parallel by thioflavin T fluorescence10
.
After polymerization, fibres were collected by centrifugation at 20,000g
for 20 min. Pelleted fibrillar or soluble Sup-NM ( 50 µg) was loaded
onto a flatcell and EPR spectra were measured in an ER/200D EPR
spectrometer (IBM Instruments)
at room
temperature with 25 mW microwave power and a modulation of 2.0 G at 100
KHz over a scan range of 100 G.
Supplementary information
accompanies this paper.
Received 25 November 2003; accepted 5 February 2004
------------------
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Acknowledgements. We thank S. Collins, J. Newman, L. Osherovich, K.
Tipton and members of the Weissman laboratory for discussion and reading
of the manuscript. M.T. was supported by JSPS postdoctoral fellowships
for research abroad. P.C. was supported by National Science Foundation
Graduate Fellowships and the ARCS foundation. Funding was also provided
by Howard Hughes Medical Instititute, The David and Lucile Packard
Foundation and the National Institutes of Health.
Competing interests statement. The authors declare that they have no
competing financial interests.
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=================================
Nature 428, 265 - 267 (18 March 2004); doi:10.1038/428265a
Cell biology: The strain of being a prion
MICK F. TUITE
Mick F. Tuite is in the Department of Biosciences, University of Kent,
Canterbury, Kent CT2 7NJ, UK.
e-mail: M.F.Tuite@kent.ac.uk
Prions are remarkable infectious agents associated with certain brain
diseases. But they also occur in fungi, experiments with which now
provide plausible answers to some critical questions about prion biology.
A widely (but not universally) accepted dogma about the agents known as
prions is that they are protein-based entities that are
self-perpetuating, 'infectious' and devoid of any transmissible nucleic
acids. Yet despite intensive research into this 'protein only'
hypothesis1
,
two crucial challenges have remained unanswered. Can infectivity with
purified prion protein be demonstrated? And how can different prion
'strains' be generated without any underlying change in the amino-acid
sequence of the prion protein or in the genetic make-up of the host?
Papers in this issue by King and Diaz-Avalos2
and Tanaka et al.3
(pages 319
and 323
)
address these challenges. They provide the most dramatic demonstration
to date of the validity of the protein-only hypothesis.
Infection of the host by a prion can, over time, lead to the replication
and subsequent transmission of an aggregated fibrous form of the
infectious protein known as an amyloid. In mammals, the only known prion
protein (PrP) is associated with a group of neurodegenerative diseases
that include CreutzfeldtJakob disease in humans and bovine spongiform
encephalopathy in cows4
.
Prion proteins have also been linked with stable, heritable traits in
two fungi (Saccharomyces cerevisiae and Podospora anserina), although
none of the four known fungal prions can be considered 'disease-causing'
some may in fact be beneficial to the host5
.
King and Diaz-Avalos2
,
and Tanaka et al.3
,
have exploited the prion properties of the Sup35p protein of S.
cerevisiae. Sup35p is an essential factor required by the
protein-synthesizing organelles the ribosomes to terminate synthesis
of a polypeptide chain. Cells in which most of the Sup35p protein
molecules have been converted to their prion form ([PSI+] cells) are
defective in this crucial termination phase.
[PSI+] cells can be readily detected in a population of 'normal'
genetically marked [psi-] cells by a simple screen involving cell colour
and protein analysis (Fig. 1
,
overleaf). Using this screen, King and Diaz-Avalos, and Tanaka et al.,
independently developed what are essentially protein-only transformation
systems and used them to demonstrate that [psi-] cells can be 'infected'
that is, turned into [PSI+] cells with an amyloid form of part of
the Sup35p protein. The part they use is one end of the protein, the N
region, which contains all the structural information needed for
maintaining the [PSI+] state in vivo and can readily assume an amyloid
form in vitro5
.
By generating amyloid forms of the protein that are free from any other
yeast protein, and by showing that the resulting infectivity is not
affected by treatment with agents that destroy nucleic acids, the
authors' findings leave little doubt that a single protein species can
act as an infectious agent based on its conformation. Moreover, previous
work with Sup35p in S. cerevisiae6
and with a different fungal prion protein (the HET-s protein of P.
anserina)7
,
did indeed provide some evidence in favour of the view that it is the
amyloid form of the protein that transmits infectivity8
.
That evidence fell short of being compelling, but the gap is now filled
by the new results2
,
3
.
Figure 1 Prion behaviour in the yeast S. cerevisiae. Full legend
High resolution image and legend
(46k)
The existence of different prion 'strains' has always cast a shadow on
the protein-only hypothesis. In mice, for example, at least 20 prion
strains have been described which produce different disease
characteristics but are not the result of a change in host genetic
make-up9
.
It has been proposed that prion strains could arise through the
existence of distinct, self-propagating conformers of otherwise
identical PrP polypeptide chains. But no direct experimental evidence
was forthcoming9
until now.
There are at least two distinct [PSI+] strains (or 'variants'), as
defined by the degree of suppression of a host nonsense mutation
(ade1-14), and biochemically by the proportion of Sup35p that remains
soluble and hence free to participate in the termination process (Fig. 1
).
During the de novo appearance of the [PSI+] prion, both types of strain
can emerge within a population of otherwise genetically identical [psi-]
cells. Once a prion strain has emerged, it is stably propagated and
efficiently transmitted to daughter cells during cell division. Evidence
already exists that Sup35p may be in a different conformational state in
different strains; for example, Sup35p from different [PSI+] strains has
different amyloid seeding propensities in vitro10
.
King and Diaz-Avalos2
,
and Tanaka et al.3
,
provide the first direct evidence that conformationally distinct,
amyloid forms of Sup35p underlie the transmissible differences in the
[PSI+] strains. They show that at least two distinct conformers of
Sup35p can be generated in vitro as defined by various biophysical
measurements. By demonstrating that the 'infection' of [psi-] cells with
the different conformers each generates different [PSI+] variants, both
groups firmly establish the link. Furthermore, these conformational
differences are propagated in vivo3
.
At least for yeast prions, attention can now turn to the baffling matter
of how the amyloid form is propagated, thereby ensuring stable
transmission to daughter cells. Two basic mechanisms, known as
template-directed refolding and seeded nucleation, have been widely
touted11
.
But what is the molecular nature of the propagating unit (or seed)? Is
it a conformationally altered monomeric form of the protein, or the
highly aggregated amyloid form? These questions can now be tackled using
the new prion transformation assays, and there are already candidates.
For example, high-molecular-weight Sup35p aggregates in [PSI+] cells can
be broken down into smaller, detergent-insoluble Sup35p-based polymers,
and the size of the polymer depends on the [PSI+] strain12
.
Could these detergent-insoluble polymers be the active seeds? We now
have a powerful way of approaching the remaining mysteries of prion
propagation in yeast, and one that could equally be applied to the cells
of higher organisms.
------------------
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