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Colloquium
Conservation of a portion of the S. cerevisiae
Ure2p prion domain that interacts with
the full-length protein
Herman K. Edskes and Reed B. Wickner*
Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, MD 20892-0830
The [URE3] prion of Saccharomyces cerevisiae is a self-propagating
inactive amyloid form of the Ure2 protein. Ure2p residues 1-65
constitute the prion domain, and the remaining C-terminal portion
regulates nitrogen catabolism. We have examined the URE2 genes of
wild-type isolates of S. cerevisiae and those of several pathogenic
yeasts and a filamentous fungus. We find that the normal function of
the S. cerevisiae Ure2p in nitrogen regulation is fully complemented
by the Ure2p of Candida albicans, Candida glabrata, Candida kefyr,
Candida maltose, Saccharomyces bayanus, and Saccharomyces para-
doxus, all of which have high homology in the C-terminal nitrogen
regulation domain. However, there is considerable divergence of
their N-terminal domains from that of Ure2p of S. cerevisiae. lURE3s']
showed efficient transmission into S. cerevisiae ure2A cells if express-
ing a Ure2p of species within Saccharomyces. However, [URE3S'] did
not seed self-propagating inactivation of the Ure2p's from the other
yeasts. When overexpressed as a fusion with green fluorescent
protein, residues 5-47 of the S. cerevisiae prion domain are necessary
for curing the [URE3] prion. Residues 11-39 are necessary for an
inactivating interaction with the full-length Ure2p. A nearly identical
region is highly conserved among many of the yeasts examined in this
study, despite the wide divergence of sequences found in other parts
of the N-terminal domains.
the word "prior" means "infectious protein." Considerable
~ evidence supports a prion basis for the transmissible spon-
giform encephalopathies (TSEs) of mammals, with an amyloid
form of the PrP protein as the culprit (1~. The gene for PrP
controls the clinical and pathological features of the TSEs (2-7~.
The scrapie agent is far more radiation resistant than even small
genome viruses (8), and purification of the infectious agent
purifies an amyloid form of PrP (9~. PrP is clearly necessary for
and central to infectivity, but showing that it is sufficient has
been difficult.
LURES] (10) and tPSI+] (11) are nonchromosomal genes of S.
cerevisiae whose molecular basis was long obscure. Genetic evi-
dence first identified LURES] and tPSI+] as prions of the yeast
Ure2p and Sup35p, respectively (12~. Three criteria distinguishing
prions from viruses and plasmids are (i) after curing a prion, it can
arise again de novo in the cured strain, (ii) overexpression of the
protein increases the frequency with which the prion arises de nova,
and (iii) the prior's propagation depends on the chromosomal gene
encoding the protein, but the presence of the prion has a similar
phenotype to recessive mutation of the chromosomal gene (12~.
Both LURES] and tPSI+] satisfy all three criteria as prions of Ure2p
and Sup35p, respectively (reviewed in refs. 13-16)
Ure2p is a regulator of nitrogen catabolism, acting by binding to
the Gln-3 GATA transcription factor and thereby keeping the latter
in the cytoplasm when the medium contains a rich nitrogen source
such as NH3 or glutamine (17-22~. This prevents the transcription
of genes, such as DALS, encoding enzymes or transporters needed
to use poor nitrogen sources (23-25~. The N-terminal 65-90
16384-16391 1 PNAS 1 December 10, 2002 1 vol 99 1 suppl. 4
residues of Ure2p largely determine the prion properties of the 354
residue protein (26, 27), whereas the C-terminal 261 residues are
sufficient for nitrogen regulation (18, 26~. Overexpression of the
prion domain is sufficient to induce the de novo appearance of
LURES] at rates far higher than even the elevated rates observed on
overexpression of the full-length Ure2p protein (26, 27~. A similar
phenomenon has been observed for the Sup35p prion domain and
induction of tPSI+] appearance (28~. Moreover, expression of just
the prion domain is sufficient to maintain LURES] (29~.
The first biochemical evidence for the yeast prions and hint of
their molecular basis was the observation that Ure2p is protease-
resistant in extracts of LURES] cells, but not in extracts of normal
strains (26~. The similarity of this finding to the protease resistance
of PrP in scrapie brains (9) was, of course, highly suggestive.
Moreover, it is the prion domain of Ure2p that forms the protease-
resistant core of the prion form (26, 30, 314. Ure2p is aggregated
in vivo specifically in LURED strains, and this aggregation requires
the prion domain (32~.
Ure2p purified from yeast is a stably soluble dimer (30, 33), but
the synthetic prion domain, Ure2pl-65, spontaneously and rapidly
forms amyloid fibers in vitro (30~. Moreover, in the same way that
expression of the prion domain induces the de novo appearance of
the LURES] prion in viva, its presence in vitro induces the full-length
Ure2p to form amyloid filaments containing both the prion domain
fragment and the full-length molecule (30~. The self-propagation of
this reaction, the specificity for the Ure2p prion domain, and the
similarity of the pro/ease-resistance patterns of this in vitro amyloid
to that of Ure2p in LURES] cells strongly support the concept that
LURES] is a self-propagating inactive amyloid form of Ure2p.
Ure2p filaments have been directly observed in vivo specifically
in tURE3] cells (31~. These filaments were observed in large
networks, localized to limited areas of the cytoplasm, with generally
only a single network observed in a single cell section. Other areas
of the cytoplasm were depleted of Ure2p (31~.
Several lines of evidence suggest a structure for the Ure2p
amyloid in which the prion domain forms a central ,13-sheet-rich core
surrounded by the appended functional domain. Protease digestion
of the 400 rim wide amyloid filaments formed by full-length Ure2p
leaves narrow filaments morphologically similar to those formed by
the prion domain alone and composed of the prion domain (30~.
Aggregated Ure2p from LURES] cells shows little reaction with
This paper results from the Arthur M. Sackier Colloquium of the National Academy of
Sciences, "Self-Perpetuating Structural States in Biology, Disease, and Genetics," held
March 22-24, 2002, at the National Academy of Sciences in Washington, DC.
Abbreviations: TSE, transmissible bovine encephalopathy; GST, glutathione S-transferase;
GFP, green fluorescent protein; USA, ureidosuccinate; YPAD, yeast extract peptone adenine
dextrose.
Data deposition: The sequences reported in this paper have been deposited in the GenBank
database (accession nos. AF525165-AF525199).
*To whom reprint requests should be addressed. E-mail: wickner~helix.nih.gov.
www.pnas.org/cgi/doi/10. 1 073/pnas. 162349599
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antibody to the prion domain, but reacts well with anti-Ure2C (31~.
The same is true of Ure2p filaments seen by electron microscopy
specifically in LURES] cells (31~. This observation suggests that, in
this structure, the prion domain is inside and the C-terminal domain
is outside.
The Ure2p prion domain was fused to the N terminus of several
enzymes with small substrates (34), including glutatione S-
transferase (GST), which is similar to the C-terminal domain of
Ure2p (18, 35, 36~. Amyloid formed in each case, and the enzymatic
activity of the fusion proteins were essentially the same in the
amyloid form as in the soluble form, when suitable correction was
made for diffusion effects (34~. These results suggested that amyloid
formation does not inactivate Ure2p function by changing the
conformation of the C-terminal domain, but rather by either
sterically blocking interaction of Ure2p with Gln3p or by Ure2p
being diffusion-limited in its filament form (34~. Several of the
fusion proteins formed monofilaments with a helical form. The
helical repeat length was consistent within a given filament, but,
remarkably, varied dramatically from one filament to another, even
though the filaments were composed of the same fusion protein
(34~. This finding indicates that the geometry of Ure2p filament
formation is determined by some stochastic process that occurs
during filament initiation.
The epidemic of bovine spongiform encephalopathy in the
United Kingdom, followed by over 100 human TSE cases caused by
consumption of infected bovine material has highlighted the im-
portance of cross-species transmission of the mammalian TSEs. A
similar phenomenon has been demonstrated in variants of tPSI+]
in which the region of Sup35 of various yeasts corresponding to the
prion domain of Sup35Sc was fused to the S. cerevisiae C-terminal
domain of Sup35 (37-39~. These studies demonstrated that the N
termini of the Sup35 proteins of Pichia methanolica and Candida
albicans can act as prion domains. Moreover, the divergence of
these prion domains correlated with a "species barrier," much like
that long documented for the mammalian TSEs.
We previously showed that overexpression of fragments of Ure2p
or fusions of such fragments with green fluorescent protein (GFP)
could efficiently cure the PUREE prion (32~. Here we have defined
the portions of Ure2N and Ure2C required for this curing activity.
As one approach to the functional significance of the Ure2N curing
region, we examined homologs of Ure2p and found strong conser-
vation of this part of the otherwise rapidly evolving N-terminal
domain.
Materials and Methods
Yeast Strains and Media. Media were as described by Sherman (40~.
The ureidosuccinate (USA) uptake phenotype of ura2 strains was
tested on synthetic dextrose plates to which was added the required
amino acids and bases (except uracil) and 100 ,ug/ml of USA.
S. cerevisiae "wild-type" cultures were obtained from three
sources. Some were locally purchased; these include cultures sold
for making bread (SAF Perfect Rise yeast, Lesaffre, Bruxelles,
Belgium; Fleischmann Active Dry yeast, www.breadworld.com;
Peter McPhie's Sour Dough strain, National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda) as well as cultures sold for home beer and wine making
(Red Star Dry Wine Yeast, Premier cuvee; Wyeast catalog no.
1007, www.wyeastlab.com, German Ale; Wyeast catalog no.
2112xL, California Lager; White Labs catalog no. WLP002,
www.whitelabs.com, English Ale yeast; Boots home beer making
genuine brewers yeast). Yeast cultures from Centraalbureau voor
Schimmelcultures (CBS, www.cbs.knaw.nl) in the Netherlands
(CBS400, palm wine from Elaies guineensis, Ivory Coast; CBS405,
bill wine from Osbeckia grandiflora, West Africa; CBS429, ferment-
ing must of champagne grapes; CBS2087 flower of lychee, Tonkin,
China; CBS2247, grape must, Cape Province, South Africa;
CBS3093, alpechin, Spain; CBS4734, from juice of sugar cane;
CBS5112, grape must, Spain; CBS5287, grapes, Russia; CBS6216,
Edskes and Wickner
tap water, Rotterdam, The Netherlands; CBS7957, cassava flour,
Sao Paulo, Brazil). Clinical isolates of S. cerevisiae (41) were kindly
provided by J. McCusker (Duke University Medical Center,
Durham, NC). YJM145 is a segregant from YJM128 which was
cultured from the lung of a man with immune deficiency syndrome.
YJM413 is a segregant from clinical isolate YJM454. YJM280 is a
segregant from YJM273, which was cultured from peritoneal fluid
of a patient. YJM320 is a segregant from YJM309, which was
cultured from the blood of a patient. YJM326 is a segregant from
clinical isolate YJM310. YJM339 is a segregant from YJM311,
which was cultured from the bile tube of a patient.
Saccharomyces bayanus (YJM562) and Saccharomycesparadoxus
(YJM498) were kindly provided by J. McCusker. Candida glabrata
(37A; ref. 42), Candida kefi~r (telemorph is Klayveromyces ma~xia-
nus) (B4425) (43), Candida maltosa (B4430) (44), C. albicans
(Darlington strain) (45), and Candida lipolytica (telemorph is
Yarrowia lipolytica) (B3163) (46) were kindly provided by K. J.
Kwon-Chung (National Institute of Allergy and Infectious Disease,
National Institutes of Health, Bethesda). Ashbya gossypii (47) was
purchased from American Type Culture Collection (catalog
no. 8717~.
Plasmid Constructions. PCR used Pt' Turbo polymerase (Strat-
agene) unless otherwise stated.
Construction of yeast expression plasmids. pH7 (2,u LEU2 PADH1) and
pH317 (2,u LEU2 PGAT 1) have been described (32, 48~. pH199, a 2,u
LEU2 plasmid containing GFP under control of the ADH1 pro-
moter, has also been described (32~.
To create pH722 (LEU2 CEN PURE2), first the NheI-BamHI
bordered ADH1 promoter of pH124 (32~ was replaced by the
similarly bordered GAL1 promoter from pH250 (48), creating
pH316 (LEU2 CEN PGA[~- Then, a 413-bp URE2 promoter
fragment bordered byNheI and BamHI sites was amplified by PCR
from S. cerevisisue strain S288C using oligos HE194 (5'-
AGAGCTAGCTTAGTAGAGCTGTGTAGAG-3') and HE19Sb
(5'-TTGGGATCCAACTTAATTTGCAGCTTAAAAC-3') and
cloned into the EcoRV site of pBC KS + creating pH497. Replacing
the NheI-BamHI bordered GAL1 promoter of pH316 with the
similarly bordered URE2 promoter from pH497 resulted in pH722.
Likewise, replacing the NheI-BamHI bordered GAL1 promoter of
pH317 with the URE2 promoter from pH497 resulted in pH723
(~LEU2 2,u PURE21.
The HindIII and XbaI sites were removed from the TRP1 gene
in the 2,u TR~1 vector pRS424 (49) by site-directed mutagenesis
using oligos HE128 (5'-AAGAGAGCCCCGAAAGTTTA-
CATTTTATGTTAGCTG-3') and HE129 (5'-GGCCGCA-
GAATGTGCTCTTGATTCCGATGCTGACTTG-3'), respec-
tively, resulting in plasmid pH342. The ADH1 promoter was
amplified from pH7 by using oligos HE66 (5'-AGAGCTAGCAT-
TACGCCAGCAACTTCT-3') and HE67 (5'-ACAAGATCTTA-
ATGCAGCCGGTAGAG-3') and ligated into PvuII digested
pH342 creating pH401 (~TRP1 2,u PADH1) The TRP1 and ADH1
promoters are facing each other in this construct.
Truncations of the Ure2p C terminus fused to GFP. pH328 contains the
C-terminal part of URE2 starting at Asp-66 (32~. Further N-
terminal deletions were made by amplifying URE2-GFP fusions
from pH326 (32~. PCR products were cloned into the EcoRV site
of pBC KS+ (Stratagene), sequenced, and inserted as BamHI-
X7zoI fragments into the BamHI~ oI window of pH7.
Deletions in the C-terminal fragment of the URE2-GFP fusion
proteins were created through amplification of URE2 fragments
from pH13 (32), cloned into the EcoRV site of pBC KS+ and
sequenced. The truncated fragments bordered by BamHI and NotI
sites were fused to GFP through exchange with the BamHI-NotI
fragment from pVTG4 (32~.
Plasmids expressing truncations of the Ure2p N terminus fused to GFP.
pVTG4 containing a part of URE2 terminating at Arg-65 has been
described (32~. Further N-terminal deletions were made by ampli-
PNAS I December 10, 2002 | vol. 99 | suppl. 4 | 16385
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Table 1. Curing and Implementation by Ure2C-6FP fusion proteins
Complementation
Plasmid URE2 part* GFP signal Curing, USA-/Total of ure2A
pH328 1-2, 66-35~GFP + 40/40
pH409 1, 86-35 - GFP + 40/40
pH410 1, 106-35 - GFP + 40/40
pH550 1, 111-35~GFP + 40/100
pH495 1, 116-350GFP +/ - 0/100
pH411 1, 126-35 - GFP 0/40
pH445 1-2, 66-293-GFP + 0/40
pH444 1-2, 66-313-GFP + 0/40
pH443 1-2, 66-333-GFP + 0/40
pH494 1 -2, 66-34~GFP 0/100
pH760 1-2, 66-347-GFP + 98/100
pH 549 1 -2, 66-349-G FP + 100/100
pH199 -GFP +
*The portions of Ure2p are shown as residue numbers. After the Ure2p portion is the sequence GGR followed
by GFP.
tCuring was assayed in strain YHE64 (MA Tcx ura2 leu2 trpl [URE3]) as USA- transformants/total. USA complemen-
tation was assayed in strain YHE887 (MATrx ura2 /eu2 ure2).
fying URE2-GFP fusions from pVTG4. PCR products were cloned
into the EcoRV site of pBC KS+, sequenced, and transferred as
BamHI-X7zoI fragments into the BamHI~ oI window of pH7.
C-terminal truncations were made by amplifying the ADH1
promoter and parts of URE2 from pVTG4. PCR products were
cloned into the EcoRV site of pBC KS+ and sequenced. The
truncated URE2 fragments bordered byBamHI end NotI sites were
fused to GFP through exchange with the BamHI-NotI fragment
from pVTG4. In pH767, S33 is encoded by AGC instead of AGT,
and in pH548, T41 is encoded by ACT instead of ACA.
The URE2Nl-45-SGR-GFP fragment from pH547 was transferred
as a BamHI-X7zoI fragment into the BamHI-XhoI window of
pH401 creating pH792.
Cloning URE2 from Different S. cerevisiae Strains. Yeast strains were
grown on yeast extract peptone adenine dextrose (YPAD) to single
colonies. Colonies were resuspended in 50 ,ul H2O containing 3
mg/ml zymolyase and incubated at 37C for 30 min. One microliter
of this suspension was used to amplify the URE2 gene with oligos
HE252 (5'-CTGCAAATTAAGTTGTACACC-3') and HE253
(5'-TTCCTCCTTCTTCTTTCTTTC-3'). PCR products were
cloned into EcoRV digested pBC KS+ and sequenced.
Cloning URE2 Homologs from Different Fungi. All yeasts were grown
in liquid YPAD, harvested, and genomic DNA was extracted as
described (50~. A. gossypii was grown on solid YPAD, and genomic
DNA was extracted as described (51~. Degenerate PCR primers
were designed based on the alignment of S. cerevisiae URE2 and C.
albicans URE2 (strain SC5314 of C. albicans; http://www-
sequence.stanford.edu/group/candida/~. Two degenerate sense
strand primers, HE207 (5'-CCIAAYGGITTYAARGTIG-
CIATH-3'; Y = C + T.; R = A + G; H = A + C + T) and
HE208 (5'-GGICAYGCICCIATGATHGGICAR-3'), and three
degenerate antisense strand primers, HE209 (5'-RTAI-
GCIGCIGCGTTYTCIGTRTC-3'), HE210 (5'-RTCIACIACRT-
TRTTCCAIGGIAC-3'), and HE211 (5'-CATCATRTGYT-
TIGTCCAYTTRTA-3'), were used to amplify URE2 homologs by
using PCR supermix (GIBCO/BRL). PCR products were cloned
into pCR2.1/TOPO (Invitrogen) and sequenced. Based on these
sequences, nested organism-specific inverse primer sets were de-
signed. Genomic DNA was digested with one of a number of
restriction endonucleases and the generated fragments were self
ligated. The 5' and 3' regions of the URE2 homologs were amplified
by using PCR supermix and cloned into pCR2.1/TOPO. To ensure
16386 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 162349599
that full-length URE2 ORFs were identified, PCR primers were
designed that hybridized to the 5' and 3' untranslated regions. After
amplification of URE2, the PCR products were cloned into EcoRV-
digested pBC KS+ and sequenced. No PCR product could be
obtained that contained the 5' untranslated region of the URE2
gene of S. paradoxus. A PCR product could be obtained when
genomic DNA of S. paradoxus was used in a PCR reaction with the
S. bayanus-specific 5' oligo. Finally, the URE2 homologs were
amplified by PCR using oligos that created a BamHI site followed
by the nucleotides CAA upstream of the start AUG and aX7loI site
immediately downstream of the stop codon. C. Iipolytica was
amplified by using a PCR primer that created a HindIII site
immediately downstream of the stop codon of URE2 as it contains
an internal XhoI. PCR products were cloned into the EcoRV site
of pBC KS+ (Stratagene) and sequenced. The different URE2
ORFs were cloned as BamHI~ oI fragments into the different
expression vectors. Only C. Iipolytica URE2 was doned as aBamHI-
HindIII fragment into the different expression vectors. If URE2 was
expressed under the control of the S. cerevisiae URE2 promoter, the
ORFs were first cloned into expression vectors containing the
GAL1 promoter. Subsequently, the NheI-BamHI bordered GAL1
promoter was replaced by the similarly bordered URE2 promoter.
Results
Interaction Domains of Ure2p Based on Curing. We previously showed
that overexpression of parts of Ure2p or of their fusions with GFP
led to efficient curing of the tURE3] prion (32~. Ure2C (residues
66-354) fused to GFP could both complement the nitrogen regu-
lation function of Ure2p and cure. We now find that the N terminus
of the curing region of Ure2C (fused to GFP) is between residues
111 and 116, whereas its C terminus is between residues 333 and 349
(Table 1~. The amounts of fusion protein expressed from the
various constructs was checked by the level of green fluorescence
(Table 1~. Comparable amounts were expressed from most con-
structs, but some constructs showed decreased expression, making
the N terminus of the region needed for curing ambiguous. The
minimal portion of the fusion proteins for complementing a ure27\
mutation is residues 86-354.
Although overexpression of the N-terminal domain of Ure2p
induces tURE3] prion formation, this same domain, when overex-
pressed in a tURE3] strain, cures cells of the prion (32~. Curing also
takes place when the Ure2p N terminus fused to GFP is overex-
pressed in a tURE3] strain (32~. By making N- and C-terminal
deletions in the Ure2 domain of these GFP fusion proteins (Table
Edskes ancl Wickner
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Representative terms from entire chapter:
nitrogen regulation
Table 2. N-terminal domain interactions with Ure2p
Plasmid URE2 part GFPsignal*
Interference,$
Curing,t USA-/total USA+/106
pH 199 -G FP + cytopl. 15/718 13
pVTG4 M1, M2-R65-GFP + 439/440 68,000
pH545 M1, N3-R65-GFP + 200/200 93,000
pH486 M1, N0R65-GFP + 100/100 73,000
pH762 M1, N5-R65-GFP + 100/100 22,000
pH487 M1, G6-R65-GFP + 9/300 11,000
pH408 M1, N7-R65-GFP + 229/340 109,000
pH763 M1, V9-R65-GFP + 197/200 35,000
pH764 M1, N11-R65-GFP + 6/180 79,000
pH349 M1, S13-R65-GFP + 0/118 71
pH350 M1, R20R65-GFP + cytopl. 1/118 3
pH351 M1, S30R65-GFP + 0/118 8
pH352 M1, N45-R65-GFP + cytopl. 0/118 3
pH769 M1-S33, S63-R65-GFP + cytopl. 5/100 4
pH768 M1-135, S63-R65-GFP + cytopl. 4/100 1
pH767 M1-F37, S63-R65-GFP + mainly cytopl. 4/100 4,600
pH442 M1-F39, S63-R65-GFP + 0/240 130,000
pH484 M1-V43, S63-R65-GFP 0/200 66
pH548 M1-N44, S63-R65-GFP + 129/300 60,000
pH547 M1-N45, S63-R65-GFP + 101/200 57,000
pH546 M1-N46, S63-R65-GFP + 80/200 61,000
pH485 M1-N47, S63-R65-GFP + 295/300 39,000
pH766 M1-N49, S63-R65-GFP + 86/100 70,000
pH441 M1-N50, S63-R65-GFP + 139/140 63,000
pH765 M1-S53, S63-R65-GFP + 95/100 53,000
*The GFP signal of most fusion constructs transformed into the [URE3] strain was aggregated. Others showed an
even cytoplasmic distribution ('cytopl.').
tCuring was tested as in Table 1.
tInterFerence was measured as USA+ cells per 1 o6 cells. All USA+ clones tested became USA- on loss of the plasmid,
indicating that this is not [URE3] induction, but simply interference with Ure2p action.
2), we more accurately defined this tURE3] curing region. We find
that residues 5-47 are needed for this curing activity. Comparable
levels of protein were expressed for nearly all of the constructs as
judged by the level of GFP fluorescence.
Unstable Inactivation of Ure2p by Overexpressed Ure2N-GFP. Over-
expression of Ure2N-GFP from pVTG4 (
Table 3. Amino acid changes in natural isolates of S. cerevisiae
Strain
Changes
>1278b; YJM145; YJM413; YJM280; YJM326; SAF; Fleischmann; Boots home; None
McPhie sourdough; CBS2087 Lychee, China; CBS4734 Sugar cane; CBS7957
Cassava flour Brazil; German Ale
YJ M320
CBS400 Palm wine-ivory Coast; CBS405 Bili wine, West Africa
CBS3093 Alpechin, Spain; CBS5112 Grape must Spain
CBS5287 Grapes Russia
English Ale; California lager; CBS6216 tap water Roterdam
A224
A264
L231
V245
N23S
CBS429 grapes, France; Red Star Wine; CBS2247 Grape must, S. Africa N70Y V258
YJM339
N43b S10, E260, R344
N43b means insertion of an asparagine residue after amino acid 43. A224 means a change in codon 224 without
changing the amino acid encoded.
4~. The N-terminal regions show substantial divergence, although
they remain asparagine/glutamine rich (Fig. 1~. Interestingly, the
N-terminal region identified by deletion analysis as needed for
curing of tURE3] is conserved among S. cerevisiae, S bayanus, S.
paradoxus, C. glabrata, C. kefyr, and A. gossypii. This region is
missing from C. maltosa, C. albicans, and C. Iipolytica. As C. albicans
is an asexual organism, it was possible that this domain is missing
in the particular strain we examined. However, a C. stellatoidae
strain we examined (synonymous with C. albicans), and two addi-
tional C. albicans strains (present in the database) all had N-
terminal domains identical to the first C. albicans strain, and lacked
the conserved N-terminal region. The URE2 sequences of S.
bayanus and S. paradoxus are nearly identical, but differ in the
asparagine-rich domain between Ser-40 and Leu-81 of the S.
cerevisiae sequence, indicating a relative plasticity of this region.
Complementation of a S. cerevisiae ure2 Deletion by URE2 Homologs.
The URE2 ORFs of S. cerevisiae, S. bayanus, S. paradoxus, C.
glabrata, C. keJ5yr,A. gossypii, C. maltosa, C. albicans, and C. Iipolytica
were placed under control of the S. cerev~siae URE2 promoter in
pH722 (LEU2 CEN PURE21- Complementation was assayed as the
Table 4. Conservation of URE2 C-terminal domains
Identity with S. cerevisiae, %
Species Amino acid
DNA
Complementation
S. Paradoxus
S. Bayanus
C Glabrata
C Kefyr-1
C Kefyr-2
K. Lactis
A. gossypii
C Maltosa
C Albicans
C Lipolytica
100
99
92
91
91
91
89
82
80
78
94
85
78
79
79
77
71
73
73
66
t
_/+*
+
Comparison starts at Ser-100 of S. cerevisiae and equivalent positions in
other organisms. The sequence from K. Iactis was obtained from the GenBank
database (gi: 14009513). Complementation is based on expression of the
whole sequence with the S. cerevisiae URE2 promoter on a CEN plasmid in
YH E888.
*Not improved if the URE2 homolog is on a multicopy plasmid.
16388 1 www.pnas.org/cgi/doi/10.1073/pnas.162349599
inability to grow on USA plates. C. Iipolytica URE2 did not
complement, andA. gossypii URE2 complemented weakly. For both
the complementation was not improved significantly when ex-
pressed from the 2,u plasmid pH723 (also under control of the S.
cerevisiae URE2 promoter). All of the other homologs comple-
mented the ure2 deletion in strain YHE888 (MA Tc~ ura2 leu2::hisG
trpl::hisG ure2::G418), preventing USA uptake with ammonia,
while allowing it with proline as a nitrogen source.
Induction of [URE3] by Overproduced URE2 Homologs. The URE2
homologs were placed under control of the GAL1 promoter and
overexpressed in a strain with an intact chromosomal S. cerevisiae
URE2 gene. Induction of tURE3] was assayed as appearance of
USA+ colonies (Table 5~. Only S. paradoxus and, to a slight degree,
S. bayanus were able to induce the appearance of tURE3] at higher
than background rates. The lower than background rates for most
of the other homologs probably reflects their masking the sponta-
neous tURE3] events by complementing the functional deficit of
S. cerevisiae Ure2p and not being themselves inactivated by the
S. cerevisiae tURE3] (see below).
Curing of [URE3] by Overexpression of Ure2p Homologs. Each of the
Ure2p homologs was expressed from the GAL1 promoter on a
LEU2 CEN plasmid in strain YHE64 (MA Ta ura2 leu2 trpl
tURE3~. Transformants were confirmed to still have tURE3;
as judged by the USA+ phenotype (20 USA+ of 20 tested in
each case). This finding shows that homolog production is effi-
ciently repressed on glucose medium, a critical point for this
experiment. Transformants were grown to single colonies on
YPAGal2%Rafl% to overexpress the Ure2p homolog, and colo-
nies were then replicaplated to leucine dropout plates containing
dextrose. Leu+ colonies were spotted on a grid on dextrose media
lacking leucine, and replicaplated twice to allow growth and
dilution of any remaining heterologous Ure2p. Patches were then
replicaplated to USA plates to score retention or loss of tURE3~.
There was no curing by any of the Saccharomyces Ure2 or by the
nonfunctional C. Iipolytica Ure2p. However, each of the other
Ure2s completely cured tURE33. That this is curing, and not
masking of the USA+ phenotype, is shown by the fact that the
glucose-repressed transformants are USA+, and the cells are again
repressed by glucose after the expression of homolog.
Can Overexpressed Ure2N'~45-GFP Inactivate Ure2 Homologs? Strain
YHE888 (ure2) containing plasmids expressing URE2 homologs
Eciskes and Wickner
S. cerevis~ae
S. paradoxus
bayanus
glabrata
kefyr-1
kefyr-2
lactis
gossypii MQQEIRNSNTPNTGSe~Pe~~eSeeee~HL~ SIDYTSRA-------------------------
lipolytica
maltose
albicans
~c---interference domain--->~
~c----------curing domain------------~
1 10 20 30 40 50 60
MMNNNGNQVSN~SNAL~Q9NIGNRNSNTTTDQSNINFEFSTOV---NNNNNNNSSS-----NNNNVQN
~ e ~ A ---~ S NNNNN A
MGDSRaTGTI~~aS~ eSGQ-----~ K ~ Y ~ NGL---~eV D GNHNLVNTSEDee--
MQQDMHNG-----GT~ TI~ S~ L~ AID~NQQQLMEEV~Q~SMNAFNIQQQHQQQQE
MQQDMHNG-----GTe~TI~~eS~ aSLe~ AID~NQQQLMEEVeQ~SMNAFNIQQQHQQQQE~
MQQDMQNG-----GP~~TI~ S~ N~L~ SID~NQQQLLEEA~QGSINAYNAQ---QQQQEH
MMSTDQHIQ
70 80 90
X-ray structure ->
1>
S. c~revisiae NNSGRNGSQNNONENNIKNT-----hEQHRQQQQA-----------------------~ ------FSDMSHVEY
S. paradoxus ~ S ~ G~ D~----_~________ _________________________
S. bayanus S N T N S D -----I ~ .- ----------------------------------~.
C. glabrata eKD.SINTNMMSRQVP.QH.HGSQL.Q.E.MNE.Q---------------------------------- NP ---
C. kefyr-1 VQKQQEQQaQQLQQQQQQQQQQQQQQQaQQ~ QLQQQQQLQQHHHHQQRQQHPNNNVQAGTSQQQML~QGANSIDS
C. kefyr-2 VQK--~ QQLQQQQQQQQQQQQQQQ~QQ ~ ~Q-QQQQQLQQHHHHQQRQQHANNNVQAGTSQQQML~QGANSIDS
K. lactis LQ--QQAQ QQLHMQQLQQAQQQQAQQ AH. QVHQ----VQHQHVQQ------DHMPIGQSQQQAMYQGPNPIDS
A. gossypii ------------------------------P ..PLDELSRGAAGGPAAGGGPEGSNMVGASSAAQVTA.GGP.V.DS
C. lipolytica MSGAHTISHLSAGLRSVNIGDQQQNEANLNLLQQQLENEAT.QTQQ
C. maltose MeMeDQRIPQeTGDeSNNSNSN-NNNNNNNNTHTISNLSAGLKSVSLTDQQQNEVNLNLLQQQLHRESSNQQQQ
C. albicans MeDNS.NeN.SNeN.TNNeNNNQSVNVNVNNTNNNTQTISNLSAGLKSVSLTDQQQNEVNLNLLQQQLHQEASTQQQQ
100 llQ 120 130 140 150 160 170
6, cerevisiae SRIT~FFQEQPLEGYT~FSHRSAPNGFKVAIV~SELGFHYNTIFLDFN~GEHRAPEFVSVNPNARVPALIDHGMDNLS
S. paradoxus
Se bayanug
C. glabrata
C. kefyr-1
C. kefyr-2
K. lactis
A. gossypii
C. lipolytica
C. maltose
C. albicans
e ~ ~ e ~ eNe e
e e e ~ e e N ~M
N~~M
N~~M
L Q AQ SD .~. e ~ eI~ ~ aELPF. e e ~ ~ ~ e ~He eQ~ ~ e ~ e eTI~ oFN..T.
Q ~~N~ ~Ae ~ e ~ aI e ~NLPF.~. e ~ aN..Q. e e e ~ aTIe ~ ~ ~ e ~ ~ ~ e ~ eFNE~T
~ ~ ~ eQ~ ~ eNe eTe eFe e e e e e e e e e e e e e e eIe e ~ eNLpFe eFe e e ~ aN~eQ~T~ TI~~~ e e e ~ e eYNe ~T
Fig. 1. Alignment of Ure2p homologs from yeasts and a filamentous fungus by using GCG pileup. The "." indicates identity, whereas "-" indicates a gap. S and T
residues are red, and Q and N residues are green. The C-terminal (nitrogen regulation) domain begins at residue 100 in the S. cerevisiae sequence and continues to
residue 354, though the figure only shows through residue 174. The "interference domain" is the portion of Ure2N which when overexpressed as a GFP fusion interferes
with Ure2p activity. The "curing domain" is the portion that, fused to GFP, is needed for curing [URE3].
(LEU2 CEN PURE2URE2hm~g) was transformed with either
pH401 (TRP1 2,u PAHD1) or pH792 (TRP1 2~ PAHDJURE2N1 45
GFP), and transformants were plated at serial dilutions on USA
plates. Only the S. cerevisiae Ure2p was inactivated through over-
expression of URE2Ni-45-GFP.
Transformants containing the S. cerevisiae, C. glabrata, or C. kefyr
URE2 were also plated quantitatively giving the same result as
above. It is surprising that the nearly identical S. bayanus and S.
paradoxus Ure2s were not inactivated. The fact that the source of
Ure2p determined whether interference was observed indicates
that Ure2Ni~4s was not interfering with the action of another
component of the nitrogen control pathway.
Ability of Ure2 Homologs to Propagate 1URE3]. URE2 homologs were
expressed in strain YHE888 (ure2) under control of the S. cerevisiae
URE2 promoter from a centromeric LEU2 plasmid. [URE3] was
introduced into these strains by cytoduction from strain 4833-3B
(~MATa ura2 argl karl-1 tURE3-1]), and the cytoductants were
examined for the USA phenotype. Only the URE2 homologs from
S. bayanus (11 USA+ of 11 cytoductants) and S. paradoxus
Edskes and Wickner
(6 USA+ of 11 cytoductants) were able to propagate [URE3].
These are the same two Ure2s that can induce S. cerevisiae tURE3]
appearance when overexpressed. The Ure2s of C. kefyr (both
genes), C. albicans, C. maltosa, and C. glabrata gave only USA-
cytoductants. The Ashbya Ure2p could not be tested because of its
incomplete complementation ability.
Discussion
Curing of [URE3]. Because propagation of tPSI+] (52) and tURE3]
(53) require HsplO4, interference with its production can cure
either prion. Guanidine at millimolar concentrations can also cure
either tPSI+] (54) or [URE3] (12, 55) by a mechanism that appears
to involve inactivation of Hsp-104 (56, 574. tURE3] is also cured by
overexpression of parts of Ure2p, particularly when they are fused
to GFP (32~. This curing by the "hair of the dog" method has a
potentially broad application, and is known to be effective in curing
tissue culture cells of PrP (58~. We proposed that the Ure2p
fragments or fusion proteins join the growing filaments, but do not
themselves provide a growing point, thus poisoning the linear
amyloid crystal, but there are other possibilities.
PNAS | December Jo, 2002 | vol. 99 | supp~. 4 | 16389
Table 5. Ability of URE2 homologs to induce or cure [URE3] in S. cerevisiae
URE2 gene Plasmid Induction, USA+/106 cells Plasmid Curing, USA+/USA-
Ve~or pH317 22 pH316 39/1
S. cerevisiae pH739 7,700 pH740 40/0
5. bayanus pH661 52 pH679 40/0
S. paradoxes pH660 6,300 pH678 40/0
C. glabrata pH659 <1 pH677 0/40
A. gossypii pH656 1 pH674 0/40
C. kefyr-1 pH713 2 pH711 0/40
C. kefyr-2 pH714 2 pH712 0/40
C. albicans pH563 6 pH672 0/40
C. maltose pH657 <0.1 pH675 0/40
C. Iipol~ica pH658 22 pH676 40/0
Strain YHE711 (MA T`x ura2 leu2::hisG) was transformed with 2,u plasmids carrying URE2 homologs under
control of the GAL1 promoter. Individual transformants were grown to saturation in leucine dropout medium
containing 2% galactose and 1% rafinose and plated in 10-fold dilutions onto USA plates to assay [URE3]
induction. For curing, centromeric plasmids were transformed into YHE64. Transformants were confirmed to still
carry [URE3], then grown to single colonies on YPAGal2%Raf2% to overexpress the Ure2p homolog. Leu+
colonies were grown as patches three times on dextrose, then tested for USA phenotype.
Here, we defined the parts of the C-terminal and N-terminal
domains of Ure2p necessary for their curing activities. The borders
of the part of Ure2C needed for curing tURE3], are approximately
residues 111 and 347. The domain needed for complementation is
larger, extending from approximately residue 86 to residue 354.
Interaction of Ure2p with Gln3p requires at least Ure2p residues
151-330 (22), and dimer formation has been demonstrated for
Ure2p residues 90-354, but not for Ure2p residues 111-354 (33~.
The C-terminal domain of Ure2p may cure tURE3] by forming
heterodimers with the full-length Ure2p. This interaction might
compete with its incorporation into the filaments. tURE3] prion
stability is not affected by the nitrogen source (29), so it is unlikely
that interactions with Gln3p or other factors involved in nitrogen
regulation explain this curing phenomenon. Mkslp is necessary for
tURE3] prion generation, not for propagation, indicating that
possible interactions with this protein are not likely to be involved.
By using the yeast two hybrid method, evidence for an interaction
of Ure2p residues 1-96 with Ure2p residues 152-354 has been
obtained (59~. However, our results show that a larger segment of
Ure2C is needed for the curing, arguing against explaining the
curing by this interaction of N terminus and C terminus competing
for N terminus-N terminus interactions.
We find that the part of Ure2N (as a fusion with GFP) needed
for curing of tURE3] is N5 to N47, a relatively short region. In this
case, the crystal poisoning mechanism, in which "impurities" pre-
vent crystal growth, is particularly attractive. A slightly smaller
region, amino acid residues N11 to N44, is necessary for interfering
with Ure2p activity on overexpression. All of the constructs able to
interfere with Ure2p appear to be aggregated as judged by the
nonhomogeneous distribution of GFP fluorescence. It is possible
that the overexpressed Ure2-GFP fusion protein forms aggregates
that sequester the full-length Ure2p, but that these aggregates are
not self propagating, or at least do not initiate a self-propagating
aggregation of the full-length Ure2p. Negative complementation of
Sup35p by a fragment of its prion domain has also been observed,
but whether or not this is associated with aggregation is not yet
known (60~.
Whatever the mechanism of inhibition, the N11 to N44 region
probably interacts with full-length Ure2p, though we do not yet have
direct evidence for this interaction. This region corresponds quite
closely with the part of the N-terminal domain that is conserved
among a group of Ure2p homologs (Fig. 1~. The conservation of
this region, despite wide divergence of sequence in the N-terminal
part, suggests that this region is important for some function. In
addition to the two-hybrid data (59), functional data suggests that
Ure2N and Ure2C interact. Deletion of Ure2 residues 1-65 weak-
16390 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 162349599
ens the ability of Ure2C to carry out its function in nitrogen
regulation (29~. Likewise, deletions of all or parts of Ure2C
dramatically increase the frequency with which Ure2p changes to
the prion form (26), suggesting that the C-terminal domain stabi-
lizes the N-terminal prion domain, perhaps by an interaction.
Ure2 Homologs. In surveying clinical isolates, brewing strains, and
baking strains of S. cerevisiae from a variety of sources, we find that
the URE2 sequence is well conserved, and the few amino acid
changes observed are in the N-terminal prion domain. Similar
results were obtained by Jensen et al. (61) studying the Sup35
protein. Examination of URE2 genes from a series of yeasts and the
fungus A. gossypii shows that the C-terminal part of Ure2p is highly
conserved. Although Ure2p is homologous to the ~ group of GSTs
(18, 62), that similarity is only about 30~o, whereas these proteins
are 80-90% identical to each other in their C-terminal domains.
For example, the "cap region" constitutes a loop with an c~ helix (36)
of the S. cerevisiae Ure2p that is missing in most GSTs, but is present
in all of the Ure2p homologs studied here as well as in that of
Klayreromyces lactis (GenBank accession no. AAK;51642~. More-
over, most of the yeast and fungal homologs fully complement the
S. cerevisiae ure27\ mutant, indicating that the nitrogen regulation
function is conserved.
Unlike other ~ class GSTs, the S. cerevisiae Ure2p has Ala-122
and His-187 instead of the consensus Ser and Tyr, respectively. In
the sequences obtained here, the Ala for Ser substitution is main-
tained, but the C. Iipolitica, C. maltosa, and C. albicans Ure2
proteins contain the consensus Tyr-187, and the consensus Gly-136
is replaced with Asp in C. ke~r, K lactis, C. maltosa, and C. albicans
and with Glu in C. Iipolitica. Thus, all of the Ure2p homologs
diverge at critical residues from the GST consensus sequence.
All of the Ure2 homologs studied here have an asparagine/
glutamine-rich N-terminal extension not found in the enzymatically
active GSTs or in homologs from Schizosaccharomyces pombe or
Neurospora crassa (ref. 63 and www-genome.wi.mit.edu). Interest-
ingly, precisely those homologs with an N-terminal extension have
the "cap region" insert in the C-terminal domain. Are they func-
tionally related? The functional significance of the N-terminal
extension of Ure2p remains a mystery. It is unlikely that prion
formation helps cells regulate nitrogen catabolism, because it
differs from the normal regulation mainly in lacking flexibility.
Although Ure2C can regulate nitrogen catabolism without the
prion domain, this regulation is less efficient than that carried out
by the full-length Ure2p (26, 29~. This helper activity of the N
terminus may be sufficient to explain its retention in evolution.
A domain that is only present in yeasts closely related to S.
Edskes ancl Wickner
cerevisiae and the filamentous fungusA. gossypii (also closely related
to S. cerevisiae) is the region between S10 and 135. The conservation
of this region, despite the wide sequence divergence of the remain-
der of the N termini, suggests the presence of some functional
constraint. There is no similarly conserved portion of the Sup35p
prion domain (37-39~.
The curing of tURE3] by homologs of Ure2p is striking in that
all complementing Ure2s can cure except for the Ure2s of Saccha-
romyces species, which can participate in the LURES] process. The
results suggest that the C-terminal domain is doing the curing in
these cases. However, it is not the complementationper se masking
the tURE3] phenotype, because cells are assayed for tURE3] when
the expression of the homolog is repressed on glucose.
Species Barrier for [URE3]. Our efforts to transmit tURE3] from S.
cerevisiae Ure2p to Ure2p of other fungi was only successful for the
Ure2s from other Saccharomyces species. These were the most
closely related in N-terminal sequence, and would thus be expected
to have the lowest barrier to transmission. The fact that transmission
of LURES] was not observed to the C. glabrata or C. keeper Ure2
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