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Colloquium
Changes in the miciclle region of Sup35 profoundly
alter the nature of eDiaenetic inheritance for
the yeast prion [PSi+]
. _
Jia-Jia Liu*t, Neal Sondheimertt, and Susan L. Lindquist
Department of Molecular Genetics and Cell Biology, Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637
The yeast prion [PSI+] provides an epigenetic mechanism for the
inheritance of new phenotypes through self-perpetuating changes
in protein conformation. lPSI+] is a nonfunctional, ordered aggre-
gate of the translation termination factor Sup35p that influences
new Sup35 proteins to adopt the same state. The N-terminal region
of Sup35p plays a central role in prion induction and propagation.
The C-terminal region provides translation termination activity.
The function of the highly charged, conformationally flexible
middle region (M) is unknown. An M deletion mutant was capable
of existing in either the prion or the nonprion state, but in either
case it was mostly insoluble. Substituting a charged synthetic
polypeptide for M restored solubility, but the prions formed by this
variant were mitotically very unstable. Substituting charged flex-
ible regions from two other proteins for M created variants that
acquired prion states (defined as self-perpetuating changes in
function transferred to them from wild-type [PSI+] elements), but
had profoundly different properties. One was soluble in both the
prion and the nonprion form, mitotically stable but melotically
unstable, and cured by guanidine HCI but not by alterations in heat
shock protein 104 (Hsp104p). The other could only maintain the
prion state in the presence of wild-type protein, producing Men-
delian segregation patterns. The unique character of these M
variants, all carrying the same N-terminal prior-determining re-
gion, demonstrate the importance of M for [PSI+] and suggest that
a much wider range of epigenetic phenomena might be based on
self-perpetuating, prior-like changes in protein conformation than
suggested by our current methods for defining prion states.
Yeast prions represent a fundamentally different mechanism
for the transmission of genetic information than DNA based
inheritance. With priors, heritable changes in phenotype are
produced by self-perpetuating changes in protein conformation
rather than by any changes in nucleic acids (1-34. tPSI+] and
other genetic elements of this type are called prions because of
conceptual similarities between their modes of transmission and
that postulated for the infectious agent of the mammalian prion
diseases (1, 44. However, yeast prions play a different role in the
biology of cells that harbor them. They are not generally
pathogenic. Rather, they modify metabolism in an epigenetic
manner that can be beneficial to the organism under certain
circumstances (S. 6~.
The protein determinant of ~PSI+] is Sup35, a subunit of the
translation termination factor (7~. In [psi-] cells, which lack the
prion, Sup35 protein (Sup35p) is soluble and functional. In
[PSI+] cells, most Sup35p is found in self-perpetuating, ordered
aggregates. In this state, the protein is nonfunctional. The
reduced concentration of functional translation-terminator fac-
tor causes ribosomes to occasionally read-through stop codons
(2, 3~. Thus, the presence of [PSI+] is routinely monitored by
suppression of nonsense-codon mutations in auxotrophic mark-
ers (8~. The phenotype is heritable because Sup35p in the [PSI+]
16446-16453 1 PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4
state influences newly synthesized Sup35p to adopt the same
state, and because the protein is passed from mother cell to
daughter during mitosis. When the daughter cell starts to make
her own Sup35 proteins, they are influenced by preexisting
[PSI+I complexes (inherited from the mother's cytoplasm) to
undergo conformational conversion. Thus, the change in Sup35p
function is inherited cytoplasmically.
Sup35p can be divided into three regions based on sequence
analysis and functional investigations. The C-terminal region (C,
amino acids 254-685) is responsible for the translation termi-
nation activity and is essential for viability (9-12~. The N-
terminal region (N. amino acids 1-123) is required for the
induction and maintenance of tPSI+] (11-13~. Deletion of N
eliminates IPSI+ l, whereas even transient over expression of N
induces [PSI+] (12~. N is also responsible for the species barrier:
in chimeric Sup35 proteins created from different species, the
prion state is efficiently transferred only between proteins that
share the same critical region of N (14-16~. The role of the
region between N and C (M, amino acids 124-253) remains
unclear.
In inter-specific comparisons of Sup35p amino acid sequences,
N and M are less conserved than C (7, 17~. However, general
features of these regions have been retained for long periods of
evolution (14-16, 18~. N regions from even distantly related
Hemiascomycetes are rich in glut amine and asparagine residues
(16, 19~. M regions are highly charged, and their sequences are
heavily biased toward a subset of charged amino acids (9, 16, 18,
19~. In Saccharomyces cerevisiae, 42% of the residues in M are
charged. All positively charged residues are lysines, and these
cluster at the N-terminal end of M. The negatively charged
residues, mostly glutamates, are concentrated at the C-terminal
end.
Although tPSI+] is inherited in an orderly way, both mitoti-
cally and meiotically, it is metastable. tPSI+] cells occasionally
give rise to ~?si-] cells and vice versa as the tPSI+] conformation
is lost or gained (20~. The rate at which [PSI+] elements are lost
greatly increases during growth on media containing guanidine
hydrochloride (Gdn HCl) (21, 22~. The inheritance of [P.~+1 is
. ~ _ ~
This paper results from the Arthur M. Sackier Colioqulum of the Nationai Acaclemy of
Sciences, "Self-Perpetuating Structurai States in Biology, Disease, ancl Genetics," heicl
March 22-24, 2002, at the Nationai Acaclemy of Sciences in Washington, DC.
Abbreviations: Gcin.HCi, guanicline hycirochioricle; YPD, yeast extract/peptone/clextrose.
*Present aciciress: Department of Neurology ancl Neurologicai Sciences, Stanforcl University
Schooi of Medicine, MSES Bulicling, Room P259, 1201 Weich Roacl, MC5489, Stanforcl,
CA 94305.
t3.-~.L and N.S. contributecl equally to this work.
$Present aciciress: Department of Pecliatrics, Chiiciren's Hospitai of Philaclelphia, 34th ancl
Civic Center Bouievarcl, Phliacleiphia, PA ~ 9104.
To whom reprint requests shouicl be sent at the present aciciress: Whiteheacl insti-
tute of Biomeclicai Research, 9 Cambricige Center, Cambricige, MA 02142. E-maii:
iinclquisLacimin~wi.mit.eclu.
www.pnas.org/cgi/cloi/1 0.1 073/pnas.252652099
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Representative terms from entire chapter:
gdn hcl
also modulated by several protein chaperones (23-26~. This
effect is most striking with heat shock protein 104 (HsplO4p), a
protein remodeling factor. Both overexpression and deletion of
the HSP104 gene cure cells of tPSI+] (23~. HsplO4p must be
present at an intermediate concentration for tPSI+] propagation.
The ability of Sup35p to exist in two stable and heritable
conformations is fundamental to the conversion of cells from
.tpsi-] to tPSI+] and vice versa. It is known that the crucial
features of Sup35p required for this transition are confined to N
and M: transferring N and M to a heterologous protein is
sufficient to confer all of the prion behaviors of tPSI+] to that
hybrid protein (27~. Moreover, the transition from the nonprion
to the prion state in vivo has been modeled in vitro by using the
purified NM fragment of Sup35p. This polypeptide can exist for
extended periods in the soluble state with a high degree of
random coil and converts to a f-sheet-rich amyloid by seeded
polymerization (28-30~. To date, all of the critical elements for
prion induction and propagation have been mapped to N.
However, the M region (amino acids 124-253) is critical for
solubility of NM in vitro. M alone remains soluble for months and
cannot be seeded by preformed amyloids. On the other hand, N
alone is soluble only in denaturing buffers (29, 31~. This obser-
vation would suggest that M might play an important role in
prion biology but its function has not been investigated in vivo.
We created several alterations in the M region. They produced
very different effects, demonstrating that M plays crucial roles
in tPSI+] biology. In fact, the self-perpetuating prion states of
these altered Sup35 proteins are so strikingly different from
those of wild-type (WT) protein, they suggest that a much
broader range of behaviors might involve prior-like changes in
proteins than has previously been suspected.
Materials and Methods
Strains, Cultivation Procedures, and Genetic Analysis. The tPSI+] and
[psi-] isogenic strain pair used were 74-D694a [MATa, adel-
14(~UGA), trpl-289(
A cost
Asia]
SUP35^M
B
C
APSIS Nisi-] Sup35^M
S P S P S P
_ ~ C!
d~#~ .,, ._ ~ ~ ~ ~~ ~ ~ Pup
NM-GFP
GFP
NM-GFP
SC-ADE
SC-ADE
~ ~~ ~ ~ ~: ~~ ~__- Sup35AM
:: :: :: i:
SUP35
SUP35^M
Fig. 1. Sup357\Mp can convert to [PSI+]. (A) Five-fold serial dilution and
growth of SUP35AM cells compared with ~ [PSI+] and [psi-] cells on SC-ADE
plates (30C, 5 days). (B) Sup35p solubility assay of [PSI+], [psi-], and
Sup35AMp. After cell Iysis, high speed centrifugation and separation by
SDS/10% PAGE Sup35AMp was detected by immunoblotting with anti-
Sup35p antibody. S. supernatant fraction; P. pelleted fraction. (C) Induction of
heritable [PSI+] factors in WT (SUP35) [psi-] and SUP35AM [psi-] strains.
Five-fold serial dilutions of cells after 24-h induction of NM-GFP or GFP alone,
plated on SC-ADE (30C, 5 days).
gated to horseradish peroxidase (1:5,000), and immune com-
plexes were visualized with enhanced chemiluminescence (ECL)
reagent (Amersham Pharmacia).
Results
Sup35AMp Is Functional and Mostly Insoluble but Can Exist in Both
Prion and Nonprion States. Starting with tpsi-] cells, the WT copy
of SUP35 was replaced with a gene carrying a deletion of the
middle region (SUP35~. The strain contained a tPSI+~-
suppressible nonsense mutation in the ADE1 gene. In this
background, tPSI+] cells grow on synthetic media deficient in
adenine (SC-ADE) and are white on rich media. [psi-] cells do
not grow on SC-ADE and produce red colonies on rich media
because of the buildup of a colored byproduct of adenine
biosynthesis.
Recombinant strains containing the SUP35AM gene (n = 16)
at the SUP35 locus were red on rich medium but showed a faint
background of growth (dark red in color) on SC-ADE. Thus, at
least some Sup35AM protein is functional in translation termi-
nation, but the protein is not as active as WT Sun35D (Fig. 1 A
and C).
r r ~ c7
Differential centrifugation of cell lysates revealed that a much
smaller fraction of Sup357\Mp was present in the supernatant
after a 100,000 x g spin, compared with WT Sup35p in [psi-]
strains (Fig. 1B). Coomassie blue staining demonstrated equal
loading of total proteins in these fractions and revealed no
detectable changes in the solubility of other proteins (data not
shown). The partial insolubility of Sup357\Mp explains the weak
suppressor phenotype of SUP35AM cells because less Sup35p is
available for translational termination than in WT cells. Clearly,
the highly charged M region provides a solubilizing function for
16448 1 www.pnas.org/cgi/doi/10.1073/pnas.252652099
the Sup35 protein as a whole in vivo, as it does for the NM
fragment in vitro.
Insolubility is a characteristic of Sup35p in the tPSI+] state (2)
(Fig. 1B). The weak suppressor phenotype of SUP35AM could be
due to a weak tPSI+] variant (see below). However the pheno-
type was unaffected by growth on Gdn HCl, which efficiently
cures tPSI+] (data not shown), suggesting that the insolubility of
Sup357\Mp might not be caused by prion formation. Also
suggesting that the aggregates were not prior-like, the suppres-
sor phenotype was recessive when SUP35AM cells were mated
with WT Nisi-] cells (data not shown). Moreover, differential
centrifugation showed that aggregates of Sup352\Mp in diploid
cells did not cause WT Sup35p to fractionate into the pellet. That
is, the Sup35AM protein in these cells could not recruit WT
Sup35p to take on the tPSI+] state (data not shown). The
aggregates had none of the characteristics of a prion and were
more likely simply caused by loss of the solubilizing activity
normally conferred by the highly charged M region.
Sup357\Mp was, however, capable of acquiring a heritable
tPSI+~-like state when SUP35AM cells were mated to WT tPSI+]
cells. After mating, the strong suppressor phenotype of the
tPSI+] parent was invariably dominant (data not shown), indi-
cating that WT protein had converted Sup35AMp into a form
that reduced its ability to function in translation. Moreover, the
strong suppressor phenotype of the SUP35AM x tPSI+] diploid
was cured by growth on medium containing Gdn HCl (data not
shown), indicating that Sup352\Mp had acquired the tPSI+] state
from WT protein, and could be cured of this state with Gdn HCl,
as is the WT protein.
Next, we asked whether Sup35l`Mp could acquire the prion
state through another common mechanism, by transient over
expression of NM. In previous studies, we used NM fused to GFP
to monitor the formation and propagation of tPSI+] in living
cells (2~. Sup35p in the tPSI+] state has the capacity to capture
NM-GFP and induce it to adopt the same state, forming GFP
aggregates visible by fluorescence microscopy. Furthermore,
overexpression of NM-GFP induces new tPSI+] element forma-
tion in [psi-] cells (2~. This state is retained even when the
NM-GFP plasmid is lost.
SUP35AM cells were transformed with expression plasmids for
GFP alone or NM-GFP. After 4 h of induction, intense coales-
cent foci were observed in many cells expressing NM-GFP, but
never in cells expressing GFP alone (ref. 2 and data not shown).
When plated to copper-free medium without selection for the
plasmid, cells that had expressed NM-GFP produced colonies
with a tPSI+] phenotype at a much higher frequency than those
expressing GFP alone (Fig. 1C), suggesting that transient over
expression of NM had converted the Sup35l\Mp to the prion
state. This was confirmed by 4:0 segregation of the suppressor
phenotype in crosses to WT tpsi-] cells and curing by growth on
medium containing Gdn HCl (data not shown). Therefore, the
Sup352\Mp can exist in two different states (we designate them
PSI+ and Ski- that are genetically analogous to the
tPSI+] and [psi-] states of the WT Sup35 protein. Unlike WT
Sup35p, however, the protein is largely insoluble in both cases
(Figs. 1B and 2B).
Sup35AMp Can Maintain Different Prion Variants. Although haploid
PSI+ strains were readily obtained by overexpressing NM-
GFP, they were not readily obtained by other standard methods.
For example, in one case SUP35AMwas integrated at the site of
WT SUP35 (in tandem with it) in a tPSI+] strain (i.e., both
SUP35AM and SUP35 were present). These transformants re-
tained the tPSI+] phenotype, as expected because WT Sup35p
in the prion state converts Sup352\Mp to that state. However,
when one of the two genes was excised by selection against the
URA3 marker that had been used for transformation, clones
carrying only SUP35AM did not survive. SUP35AM derivatives
Liu et a/.
A
B
C DETACH AM
DETACH
Sip ~ ~
SC-ADE
T 1 23 4 5
Cal
[PSI~ [PSI~
~ _,,
T S P T S P
SC-ADE
_ HI - c. _ Su p35
~ ~ Sup35/\M
FETAL.
T 1 2 3
Cal
~ YPD
second
round
mating
PETALS
[psi l '1 [pX~
_ ~ _ . _
~ ~ _ lit ~ _
i. *O
SC-ADE
-
Fig. 2. Sup35AMp can maintain different [PSI+] variants. (A) Most SUP35AM
spores from SUP35/`M x WT [PSI+] crosses were not viable. (Left) Five repre-
sentative tetrads from spores of a [PSI+] diploid from a SUP35AM [psi-] x
SUP35 [PSI+] cross, dissected on YPD medium. (Right) Growth of one set of
tetrads with one surviving SUP35AM spore on YPD and SC-ADE. T. tetrad; S.
spore. (B) Sup35p solubility properties of the WT spore and the SUP35AM
spore (methods as described in Fig. 1). T. total protein; S. supernatant; P. pellet.
(C) Sup35~Mp can form and propagate [ETA+]. (Upper Left) Growth of
SUP35AM [psi-] haploid strain ([pSi-]~M), ~ [ETA+] haploid strain ([ETA+]~,
and the diploid from the cross of these two strains on SC-ADE medium. (Upper
Right) Three representative tetrads dissected from the diploid SUP35/`M[psi-]
x SUP35[ETA+] on YPD medium. (Lower Right) Grovvth on SC-ADE of haploid
parental strains and two diploid strains from a cross of a SUP35AM spore from
SUP35l`M [psi~] ([psi-]~M)/SUP35 [ETA+] ([ETA+]WT) diploid to WT [psi-].
were readily obtained when the initial insertion had been in a
tpsi-] background and other SUP35 [PSI+] variants are readily
obtained by this method (35~. Similarly, sporulating SUP35/
SUP35AM ~psi-] diploids yielded the expected number of viable
SUP35AM spores, but sporulating SUP35/SUP35AM tPSI+]
diploids yielded very few (Fig. 2A). Those that were recovered
grew very slowly, even on rich media (Fig. 2A), and by differ-
ential centrifugation, virtually all of their Sup351\M protein was
found in the pellet (Fig. 2B). Genetic crosses between these
slow-growing strains and the WT tpsi-] strain generated tPSI+]
diploids (data not shown). Thus, Sup357\Mp in the prion state
can transmit that state to WT protein. But [PSI+] cells in which
the only copy of Sup35p is Sup357\Mp can have unexpected
problems with viability.
One explanation is that Sup352\Mp, like WT Sup35p, can form
prion variants with different "strengths." The phenomenon of
different prion states (called prion "strains" or variants) that are
strong, moderate, weak, and very weak is well characterized (13,
Liu et a/.
38-40~. These variants are not caused by genetic differences, but
are caused by epigenetic differences in the rates that prion
variants capture and convert new Sup35p to the prion state. They
have different levels of soluble Sup35p and different rates of
translation termination (39-41~. Because Sup35AMp is inher-
ently less soluble than WT Sup35p, if it acquired a strong prion
state there might be too little translation termination activity to
keep cells viable. The haploid tPSI+~^M strains induced by
transient overexpression of NM-GFP might represent weak
variants, viable because a greater fraction of the Sup35AM
protein remains soluble and active.
To test this possibility, we mated sit- cells to the weak
tPSI+] variant fETA+~. Conversion of Sup351\Mp by this weak
variant should leave a greater fraction of Sup35AMp in solution
and produce more viable haploid SUP35AM PSI+ spores.
The diploid showed the same suppression of the adel-14 marker
as the fETA+] haploid parent (Fig. 2C Left), suggesting that the
Sup357\M protein had converted to a weak prion state. In
contrast to the poor viability of SUP35AM spores after mating to
strong tPSI+] strains (Fig. 2A), nearly all SUP35AM progeny
from the fETA+] were viable (Fig. 2C UpperRight). When these
progeny were mated to WT [psi-] tester strains, the diploids
exhibited the suppressor phenotype characteristic of fETA+]
strains (Fig. 2C Lower Right). Thus, Sup35~\Mp could acquire,
maintain, and transmit the fETA+] state to WT protein. The N
region is sufficient to form prion variants of different strengths.
The M Region Promotes Mitotic Stability of the [PSI+] State. On rich
media, tPSI+~-mediated nonsense suppression is not required for
growth, yet WT cells retain tPSI+] with high fidelity. In contrast
tPSI+~^M strains lost the prion at a high rate (Fig. 3A). We asked
whether we could restore stability simply by restoring solubility
to the protein. To do this, a DNA fragment encoding a highly
charged polypeptide rich in lysine and glut amic acid (6xKDG)
was inserted in place of M creating the replacement SUP35NKC.
As expected, when the WT SUP35 gene was replaced by
SUP35NKC in [psi-] cells, they retained a tpsi-] phenotype. The
solubility of the Sup35NKC protein in this state was comparable
to that of WT Sup35p (Fig. 3B). To determine whether this
protein could acquire the prion state, SUP135NKC mutants were
mated to a typical strong prion strain (Fig. 3C) and the diploid
strain showed the suppressor phenotype. Sporulation of this
diploid (data not shown) produced haploid SUP35NKC tPSI+]
cells (tPSI+]NKC). Most Sup35NKCp was soluble in ~si-]NKC
strains, and most of the protein became insoluble when it
adopted the tPSI+]NKC state (Fig. 3B). Unlike PSI+ cells,
tPSI+]NKC cells exhibited no growth defect when streaked on rich
medium (data not shown). However, even though Sup35NKCp
appeared to be as soluble as WT protein and produced no
general growth disadvantage, the tPSI+]NKC phenotype was
highly unstable (Fig. 3D). Thus, replacement of the M region
with a charged polypeptide that increases its inherent solubility
in vivo is not sufficient to restore stability to the prion state. M
provides more than a simple solubilizing function to Sup35p. It
also promotes the mitotic stability of tPSI+~.
The M region is highly charged and, in the soluble state,
circular dichromism spectroscopy shows it to have a highly
flexible structure (~60% random coil; A. Cashikar and T.
Scheibel, personal communication). Our next alterations were to
replace the M region with two naturally occurring polypeptides
that, like WT M, are highly charged and are known to have
conformational flexibility.
The Human Topoisomerase Linker Restores Mitotic Stability but
Causes Meiotic Instability. The human topoisomerase linker (T)
has a percentage of charged residues similar to Sup35Mp. The
linker has been characterized by x-ray crystallography and
contains both structured and unstructured regions that link
PNAS | December 10, 2002 | volt. 99 | supple. 4 | 16449
A [PSI~M
APSIS
B
CD
[PSI~ 1psi ]
T S P T S P
S u p 3 5 F ~ ~ ., D,
[no;-lN K\;
[PSI~NKC ~Sj-lNKC
T S P T S P
_ _ _ Sup35NKC
[PS/~
~ .2N
[PSI~NKC
a_
it_
-
-
-
-
-
-
Fig. 3. The M region promotes mitotic stability of strong [PSI+]. (A) Appear-
ance of red sectors ([psi-]) out of white colonies when [pS/+]^M cells were
streaked onto YPD and incubated for several days (Left). Growth of [PSI+] cel Is
on YPD shown for comparison (Right). (B) Sup35p solubility assay of the NKC
mutant protein compared with that of V\/T Sup35p (methods as described in
Fig. 1). (Left) WT [PSI+] and [psi~] strains. (Right) SUP35NKC [PSI+] and [psi~]
([PS/+]NKCand [pSi ]NKC) haploid strains. (C) Mating SUP35NKC[psi ] ([psi ]NKC)
to [PSI+] generated [PSI+] diploid. Growth of the two parental strains and a
diploid (2N) progeny on SC-ADE is shown. (D) [PSI+]NKC cells were mitotically
unstable. Cells were grown in liquid YPD for 16 h and plated onto YPD plates.
Most colonies were red/white sectored (Let). A close-up image of one of the
sectored colonies is shown (Right).
other functional domains of the protein (42~. When an M to T
replacement (SUP35NTC) was inserted in tandem with SUP35 in
[psi-] cells, strains retaining only SUP35NTC were obtained at
an equal frequency to WT. SUP35NTC strains were phenotyp-
ically identical to WT tpsi-] cells with respect to growth on rich
media and SC-ADE. Sup35NTCp could readily be converted to
the prion state, tPSI+]NTC, by matings to WT tPSI+] strains. The
haploid progeny of sporulation showed normal viability.
tPSI+iNTC strains were also obtained after transient overexpres-
sion of Sup35NTCp from an inducible plasmid in the SUP35NTC
background.
tPSI+]NTC was tested for other common prion properties
including curability, mitotic stability, and non-Mendelian inher-
itance during meiosis (see Table 1~. It was mitotically stable (Fig.
16450 1 www.pnas.org/cgi/doi/10.1073/pnas.252652099
4A), capable of growth on SC-ADE (Fig. 4B) and was cured by
growth on media containing Gdn HCl, but was not cured by
either the overexpression or the deletion of HSP104 (Table 1
Fig. 4C). To further characterize tPSI+]NTC and ~si-]NTC states,
we analyzed the solubility of the Sup35NTC protein. In both
tPSI+iNTC and [psi-]NTC cells, most Sup35NTCp was soluble
after a 100,000 x g spin (Fig. 4D). To test the aggregation of
Sup35p by using GFP, we expressed a plasmid with a fusion of
the N and T regions to the GFP marker (NT-GFP) in [PSI+ jNTC
and LDsi-iNTC cells. NT-GFP showed a diffuse fluorescence
pattern in both strain types confirming that the protein does not
form large aggregates (Fig. 4E). Therefore this protein can exist
in states that are genetically analogous to the prion states of
Sup35p, but in both states most of the protein remains soluble
after centrifugation at 100,000 x g for 20 min.
The tPSI+]NTC state was dominant in crosses to [psi-iNTC cells,
indicating that it readily converted soluble Sup35NTCp to the
prion state. When tPSI+]NTC homozygous diploids were sporu-
lated, the frequency of meiotic transmission of the suppressor
phenotype to offspring was not always 4:0, the ratio typical for
WT tPSI+] diploids, but it was clearly non-Mendelian (Fig. 4F
and Table 1~. This segregation pattern was similar to that of
another yeast prion, tURE3] (43~. These findings suggest that the
highly charged M region also influences the accurate propaga-
tion of tPSI+] elements through meiosis. The different effects
of M substitutions on mitotic and meiotic stability suggest that
the mechanisms for maintaining meiotic and mitotic stability
are, at least in part, distinct.
Substitution of the Hsp90p Linker for M Causes Another Distinct
Genetic Behavior. The other M substitution we tested was derived
from S. cerevisiae Hsp90 protein. This highly charged region
(amino acids 210-262 of the polypeptide sequence) connects the
two stably folded domains of Hsp90p and is degraded by even
very short treatments with proteases, suggesting it is not inher-
ently a tightly folded polypeptide (444. As with SUP35NTC, when
M was replaced by this portion of the HSP90 coding sequence
(~SUP35N9C), [psi-] cells retained a nonsuppressor state (data
not shown).
In contrast to SUP35NTC, SUP35AM, and SUP35NKC cells, a
suppressor state could not be induced in haploid SUP35N9C cells
by overexpression of polypeptides containing the N region (data
not shown). The protein could, however, acquire a tPSI+~-like
state when SUP35N9C cells were mated to WT tPSI+] cells (Fig.
5A). The diploid strain had many other characteristics of [PSI+]
strains, including a suppressor phenotype that was eliminated
by plating to media containing Gdn HCl (see Table 1~. It also
showed strong mitotic stability. But surprisingly, sporulation of
this diploid always produced two tPSI+] colonies with a SUP35
genotype and two SUP35N9C with the tpsi-] phenotype
(Fig. SA).
These observations suggested that Sup35N9Cp could enter a
tPSI+~-like state, but could only acquire that state from pre-
formed ~PSI+] elements and could not thereafter retain it on its
own (when separated by sporulation from the WT protein). To
more fully characterize these transitions we examined the solu-
bility of the Sup35N9C protein in the haploid SUP35N9C strain,
the diploid SUP35/SUP35N9C tPSI+] strain and the haploid
progeny of sporulation. The Sup35N9C protein was almost
entirely soluble in the SUP35N9C parent, but was insoluble (as
was WT Sup35p) in the heterozygous tPSI+] diploid (Fig. SD).
After sporulation, Sup35N9Cp became soluble once again in the
SUP35N9C haploid progeny, whereas the insoluble prion state
was maintained in SUP35 progeny (Fig. SD, right lanes). This
result was confirmed by the presence of small foci in the tPSI+]
diploid after 2 h of expression of an N9-GFP fusion protein (Fig.
SB). In contrast, N9-GFP fluorescence in the nonsuppressed
haploid SUP35N9C remained diffuse (Fig. SC). Thus,
~-- ~ r-~ ~ r
ciu et a/.
i
Table 1. M substitution mutants and their properties
Protein
[PSI+] inducibility . solubility Hsp104 curability
Region length; % charge; Segregation
no. of positive amino acid Sup35 N terminus Mate to pattern Stable in Gdn.HCI Hsp104 over
residues that are Iysine over express [PSI+] [PSl+]:[psi-] mitosis curable [PSI+] [psi-] Z\HSP104 express
WT; 130 aa; 42%; 24 of 24
AM
KDG6(NKC); 18 aa; 67%; 6 of 6
HuTop I (NTC); 79 aa;
44%; 17 of 21
+
+
+
+
Hsp90 (N9C); 53 aa; 77%; 17 of 17 ~ - +
N.T., not tested.
*Tested in heterozygous [PSI+] diploid (WT/N9C).
Sup35N9Cp can readily enter a tPSI+~-like state under the
influence of WT protein in that state, but it cannot maintain that
state on its own.
Discussion
We have demonstrated that the M region of Sup35p makes
important and diverse contributions to genetic and biochemical
properties of PSI+. Sup35p mutants with a deletion of the M
region or with substitutions in place of M can form priors, but
these states are strikingly distinct from WT tPSI+] and from each
other. A wide variety of prion states and behaviors can be
4:0
4:0
4:0
4:01 7% + +
3:1 56%
2:2 22%
1:3 4%
2:2*
+ + - +
+ +
+ + +
+ +
+ +
N.T. N.T.
conferred on the same C-terminal functional domain and N-
terminal prion domain by intervening "auxiliary" sequences.
Prion proteins such as PrP and Sup35p aggregate when
adopting the prion conformation (2, 37, 45, 46~. However,
large-scale aggregation is neither necessary nor sufficient for
entry into the prion state. (The former has also been suggested
by the analysis of certain URE3 prion variants, ref. 47.) We
have shown that M helps maintain Sup35p in the soluble state
and, as a result, Sup35AMp is found in the pellet after
centrifugation of cell lysates, regardless of its prion state. This
finding confirms the special nature of prion state protein.
A rpSl~NTc
~ [PSI~
p~NTC~
IS ~ ~S']
~ ! -
APSIS
c
E
PSI~NTC|
USE] I
[pS/~NTC~1 04
jlNTC^1 of
~ [p5l~NTC
SC-ADE
B YPD
SC-ADE
D
SilNTC [psl~NTC
T S P T S P
_ - Sup35NTC
1
F
Tetrad
Tetrad 2
Tetrad 3
Fig. 4. Substitution of human topoisomerase I linker forthe Sup35p M region causes meiotic instability. (A) [psi-] and [PSI+] strains containing the linker
from human topoisomerase I in place of the M linker of Sup35p were plated onto YPD; [PSI+] and [psi~] were plated for comparison. (B) Growth phenotype of
SUP35NTCcells on YPD and SC-ADE. (C) Deletion of HSP104 did not affectthe suppressors/ate of [PSI+]NTC cells. (D) Sup35p solubility assay of [PSI+]NTC and [psi-]NTC
(methods as described in Fig. 1). (E) Expression of NT-GFP in [psi~]NT~ and [PSI+]NTC did not induce aggregation. (F) Tetrad dissection of a [PSI+]NTC diploid shows
varying numbers of [PSI+]NTC and [psi~]NTC spores.
Liu et al.
PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16451
A
SUP35N9Cx[PSI~
SUP35N9C spore #1
SUP35 spore #1
SUP35 spare #2
SUP35N9C spore #2
D
B
N9C
~ [ASIA N9C [if spore spore_
T S P T S P T S P T S PT S P T S P
~ Sup35N9C
Fig. 5. Substitution of the Hsp90p linker for the Sup35p M region causes
distinct genetic behavior. (A) Diploid [PSI+] cells with the SUP35/N9C geno-
type grew white on YPD (top row). On sporulation of this diploid, two red
colonies and two white colonies were always obtained (following rows). (B)
Expression of N9-GFP in the SUP35/N9C diploid [PSI+] strain causes aggrega-
tion. (C) Expression of N9-GFP in haploid cells expressing only N9C did not
cause aggregation. (D) Sup35p solubility assay of N9C indicates that it pelleted
only in the presence of Sup35p in the [PSI+] state. The protein returned to the
soluble state after sporulation (methods as described in Fig. 1).
Differences between the Sup35AM protein in tPSI+~^M cells
and in ~5i-~M cells are not simply a difference between
aggregated and nonaggregated states. This point is also dem-
onstrated by our experiments with Sup35NTCp. No aggre-
gated state was detectable in tPSI+]NTC cells. The prion state
of Sup35NTCp may well involve higher-order complexes, but
if so, they are clearly different in character from the large
complexes of WT Sup35p in the tPSI+] state.
The M region is also important for the stabilization of tPSI+]
during cell division. Cells with either the SUP35AM or the
SUP35NKC replacement genotype could enter a prion state, but
this state was not well maintained during mitotic division. Cells
with the SUP35NTC genotype could also enter the prion state,
and tPSI+iNTC was mitotically stable. However, tPSI+]NTC was
not propagated after meiosis with the same fidelity as [PSI+~.
Thus, the propagation of prion elements is quite sensitive to
changes in the M region. Requirements for the maintenance of
the prion during mitotic and meiotic cell division are distinct and
M contributes to them both.
Altering the M region also had important consequences for
prion curing. Because HsplO4p function is sensitive to Gdn HCl,
it has been suggested that Gdn HCl treatment cures cells through
the inactivation of HsplO4p (48-51), but this hypothesis is
controversial (48, 50, 52~. We have identified a prion state,
tPSI+]NTC, which can be cured by growth on Gdn HCl but cannot
be cured by HSP104 deletion (Table 1~. This finding indicates
that curing by Gdn HCl is not solely caused by HsplO4p inacti-
vation. The results also suggest that some feature of M strongly
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PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16453
