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INTRODUCTION |
Keen interest has been shown during the past several years to a
phenomenon of protein misfolding and aggregation (inside the cells),
particularly due to studies of various amyloidosis and prion diseases
(1-6). Even though it became more or less clear that conformational
changes of proteins are required for the propagation of the diseases
(1-6), it is not yet known how and where these conformational changes
take place in vivo. There are some significant differences
between prion diseases and amyloid diseases, such as transmissibility
of prion diseases, but it is clear that conversion of a soluble form of
a protein into insoluble aggregate is a key mechanism involved in all
the cases (1-6). Such aggregates reveal high resistance to proteases,
thus escaping different common degradation pathways, e.g.
proteosomal complexes (7, 8). These aggregates form deposits (1-6)
that could lead to cytotoxicity.
In contrast to mammalian prions, which in their aggregated form
significantly damage the cells (leading finally to cell death), the
so-called yeast prion-like proteins (Sup35p and Ure2p, when thought to
be aggregated) do not damage yeast cells but do, however, change their
phenotypes (9, 10). This change in yeast cell phenotypes could lead to
an evolutionary advantage under certain conditions, e.g.
giving rise to new proteins due to a readthrough of a stop codon (in
case of the aggregation of Sup35p, known to be a translation
termination factor (see Refs. 9 and 11)), or allow the uptake of both
poor and rich nitrogen sources as happens in case of the Ure2p
aggregation (Ure2p is a transcriptional factor, regulator of nitrogen
metabolism in yeast (12-13)).
It is now widely accepted that formation of insoluble aggregates is a
result of a shift in equilibrium between native soluble conformer of a
prion protein and aggregation-competent molecules (6). Although reasons
for such a shift are rather obscure, the basis for partitioning between
different conformers seems to be provided (at least in case of
mammalian prion proteins) by their specific structural properties (4,
14, 15). Indeed, recent structural studies demonstrated that mammalian
prion proteins possess a large unstructured NH2-terminal
part (14, 15). Since synthetic peptides reproducing various regions in
this domain are capable of polymerizing into amyloid fibrils (for
review see Ref. 16), this part of the molecule has been suggested to
govern PrP aggregation. The most widely accepted model for amyloid
formation hypothesizes that primary conformational changes affect the
NH2-terminal domain of PrP and then propagate to the rest
of the molecule with an efficiency depending on the local structural
properties of different PrP isoforms (4, 6).
In order to understand better the features that can provide the basis
for possible structural plasticity of the yeast prion-like protein
Ure2, we have attempted its purification and characterization (after
overexpression in Escherichia coli cells). Our data
demonstrate that recombinant Ure2p is a soluble monomeric protein that
can self-associate into dimers, tetramers, as well as insoluble high molecular weight oligomers. These high molecular weight oligomers are
fibrillar structures that appear, when examined in the electron microscope, to be very similar to the fibers that are observed in the
case of PrP in its scrapie prion filaments form or that form on Sup35
self-assembly. Ure2p oligomerization is a cooperative process that is
concentration-dependent. Finally, Ure2p fibers bind Congo
Red as do amyloid fibers. We also bring evidence in this work for the
existence of at least two structural domains in Ure2p molecules and
show that only slight conformational changes accompany Ure2p
aggregation. These changes affect essentially the COOH-terminal part of
the molecule.
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MATERIALS AND METHODS |
Reagents--
EGTA, SDS, 1,4-dithiothreitol
(DTT),1 and subtilisin
(Carlsberg P5380) were from Sigma.
Trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone
came from Worthington. Proteinase K was purchased from Stratagene.
[35S]L-Methionine and autoradiography
products came from Amersham Pharmacia Biotech. Acrylamide and all other
electrophoresis reagents were from Bio-Rad. All other chemicals were
analytical grade from Prolabo and Merck.
Construction of Ure2p Expression Vectors in E. coli--
Ure2p
expression constructs were designed as described previously (17).
Briefly, the open reading frame of the URE2 gene was
amplified with the primer
5'-CGCGGATCCAATAACAACGGCAACCAAG-3' and
5'-CCGCTGCAGCTAATCATTATTTTGGCTACC-3' and subcloned
into BamHI-PstI-restricted pUHE21-2 vector after
restriction with the same enzymes (underlined sequences are
BamHI and PstI restriction sites, respectively). The rare codons AGA encoding arginine residues at positions 253 and 254 were changed into CGT codons. These silent mutations were achieved by site-directed mutagenesis using two additional
oligonucleotide primers,
5'-CGGATGAGGTTCGTCGTGTTTACGGTGTAG-3' and
5'-CTACACCGTAAACACGACGAACCTCATCCG-3'. The amplified
cDNA was subcloned into BamHI-PstI-restricted
pUHE21-2 after its digestion with the same enzymes generating the
construct pUHE-URE2* (underlined sequences are the two CGT codons
replacing the AGA codons).
Preparation of 35S-Labeled Ure2p--
A construct
designed to express Ure2p under the control of T7 polymerase was
prepared by subcloning the XhoI-PstI fragment that contains the URE2* cDNA from pUHE-URE2* into pBluescript II
SK+ (Stratagene, Inc.) restricted with the same enzymes.
The resulting plasmid pBluescript II SK+-URE2* was added to
a TNT T7-coupled reticulocyte lysate translation system (Promega). The
reaction was performed according to the manufacturer's recommendations
in the presence of [35S]methionine. Translation products
were added to E. coli lysate where Ure2p was expressed in
order to track Ure2p through the purification process either by
SDS-PAGE followed by autoradiography or scintillation counting using a
Packard CA2000 scintillation spectrometer.
Two additional constructs were designed to express the
NH2-terminal as well as the COOH-terminal domains of Ure2p
under the control of a T7 promoter. First, construction of pBluescript
II SK
-
C-URE2* that encodes the Ure2p 1-94
NH2-terminal fragment: pBluescript II SK
-URE2
was restricted with PmlI and Bsu36I and treated
with T4 DNA polymerase, and the blunt ends were religated. A stop codon was thus generated. Second, construction of pBluescript II
SK+-
N-URE2* that encodes the Ure2p 94-354 COOH-terminal
fragment: pBluescript II SK+-URE2* was linearized with
XmnI, amplified with the primers
5'-TATCGATAAGCTTGATATCGAATTCAACAACGAGGAGTTTCGGATATG-3' and 5'-CCGCTGCAGCTAATCATTATTTTGGCTACC-3', and
subcloned into EcoRI-PstI-restricted pBluescript II SK+ vector (underlined sequences are
EcoRI and PstI restriction sites, respectively).
This procedure introduces a Shine-Dalgarno sequence upstream of the ATG
encoding Met residue at position 94.
Expression and Purification of Recombinant Ure2p--
The
expression construct pUHE-URE2* was transformed into the BMH71.18
E. coli strain, grown on LB medium to an
A550 of 0.8-1.0, induced with 0.5 mM isopropyl-1-thio-
-D-galactopyranoside,
and harvested 2.5 h later. Recombinant Ure2p was found to be in the soluble fraction following lysis in 10 mM Tris, pH 7.5, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and
centrifugation at 25,000 × g. Tracer
35S-labeled Ure2p was added to the centrifuged lysate after
adjusting the buffer conditions to 20 mM Tris, pH 7.5, 20 mM KCl, 1 mM EGTA, 1 mM
MgCl2, 1 mM DTT (buffer A). The lysate was then
filtered through a 0.45-µm Millipore filter and applied to a 50-ml
(2.2 × 15 cm) UNO Q column (Bio-Rad) equilibrated in buffer A. The column was washed with 150 ml of equilibration buffer and developed with a 400-ml linear gradient of 20-500 mM KCl. Fractions
emerging at salt concentrations between 150 and 185 mM KCl
were found to contain Ure2p. Fractions containing Ure2p were pooled,
KCl concentration adjusted to 1 M, and applied to a 25-ml
phenyl-Sepharose high performance column (Amersham Pharmacia Biotech)
equilibrated in 20 mM Tris, pH 7.5, 1 M KCl, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT. The column was washed with 50 ml of equilibration
buffer and developed with a 200-ml linear gradient beginning with this
buffer and ending with the same buffer containing no KCl. Fractions
emerging from the column in the absence of KCl contained Ure2p. They
were pooled and adjusted to 20 mM potassium phosphate, pH
7.2, 1 mM MgCl2, 1 mM DTT by
passage through a Sephadex G-25 column (Amersham Pharmacia Biotech) and
applied to a 20-ml hydroxyapatite column (Pentax, American
International Chemical). Ure2p did not bind to this column and was
eluted during the wash phase, whereas contaminating proteins bind to
the matrix. Of the material emerging from this column, 95%
corresponded to Ure2p, as judged from running this material on a 10%
SDS-polyacrylamide gel. Ure2p-containing fractions were pooled then
frozen in aliquots and stored at
70 °C. The typical yield was 20 mg of Ure2p per liter of culture.
For analytical ultracentrifugation experiments, electron microscopy
observations, and proteolytic digestions, soluble Ure2p was separated
from aggregated proteins by gel filtration through a Superose 6 HR
10/30 column (Amersham Pharmacia Biotech) equilibrated in 50 mM Tris, pH 7.5, 1 mM EGTA, 1 mM
MgCl2, 1 mM DTT and eluted with the same buffer.
Sedimentation Velocity and Molecular Mass
Determination--
Sedimentation velocity experiments were carried out
by the use of a Beckman Optima XLA ultracentrifuge equipped with an AN 60Ti four-hole rotor and cells with two-channel 12-mm path length centerpieces. Measurements were made at 60,000 rpm and at 15 °C. Data were analyzed to provide the apparent distributions of
sedimentation coefficients by means of the program Svedberg (18) and
DC/DT (19). Equilibrium sedimentation was performed in the same
instrument, but at 4 °C. Sample volumes of 100 µl were centrifuged
at 15,000 rpm. Radial scans of absorbance at 278 nm were taken at 4-h
intervals in order to monitor the attainment of equilibrium.
Equilibrium was reached after 20 h of centrifugation. The data
were analyzed to yield weight-average molecular weights by the use of
the programs XLAEQ and EQASSOC supplied by Beckman. The partial
specific volume was calculated from the amino acid composition to be
0.7215 cm3·g
1 using the SEDNTERP software
(John Philo), and the solvent density was 1.01 g/cm3. The
degrees of hydration of the totally unfolded protein were estimated
based on the amino acid composition by the method of Kuntz (20)
according to Laue et al. (21). The degree of hydration used
for all calculations, 0.3707 g of H2O/g of protein, was the result of correcting the calculated degree of hydration by a factor 0.7 (22).
The exact molecular weight of full-length Ure2p as well as Ure2p
fragments was determined by matrix-assisted laser desorption time of
flight mass analysis (Bruker, Bremen, Germany) using
-cyano-4-hydroxycinnamic acid as matrix. Peptide sequence data for
full-length Ure2p as well as Ure2p fragments were obtained by automated
Edman degradation using a sequencer (model 470A or 477A; Applied
Biosystems, Inc., Foster City, CA) equipped with an on-line
phenylthiohydantoin amino acid analysis system (model 120A; Applied
Biosystems, Inc.).
Electron Microscopy--
Samples of soluble or aggregated Ure2p
were negatively stained on carbon-coated grids (200 mesh) with 1%
uranyl acetate and examined in a Philips EM 410 electron microscope.
Fluorescence and Light Scattering Measurements--
The
intrinsic fluorescence of Ure2p was recorded at 20 °C in a
Aminco-Bowman series 2 spectrofluorometer in 10 × 2 mm quartz cuvettes (Hellma) containing 200 µl of Ure2p solution. The excitation monochromator was set at 290 nm, and the emission was recorded between
300 and 400 nm. Ure2p conformational changes were monitored by the
increase of tyrosine fluorescence (excitation 290 nm, emission 333 nm).
Ure2p aggregation was monitored at 20 °C by light scattering.
The increase in the intensity of light scattered at 90° from the
incident beam was measured at 340 nm in a Aminco-Bowman series 2 spectrofluorometer.
Proteolytic Digestions--
Soluble and aggregated Ure2p (0.8 mg/ml) in 50 mM Tris, pH 7.5, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT were treated at
37 °C by either proteinase K (2.4 µg/ml), subtilisin (1.5 µg/ml), or trypsin (1.5 µg/ml). Aliquots were removed at different
intervals following addition of the protease and transferred into
Eppendorf tubes maintained at 90 °C containing sample buffer (50 mM Tris-HCl, pH 6.8, 4% SDS, 2%
-mercaptoethanol, 12%
glycerol 0.01% Serva Blue G and 0.01% bromphenol blue) in order to
arrest immediately the cleavage reaction. After incubation of each tube
for 10 min at 90 °C, the samples were processed to monitor the time
course of Ure2p cleavage by Tricine/SDS-polyacrylamide gel
electrophoresis (23).
The 35S-labeled NH2-terminal and COOH-terminal
domains of Ure2p were generated as described previously (24).
Proteinase K or trypsin (10 µg/ml) was added to the translation
reaction, and the time course of proteolysis was followed as described
above. The reaction products were visualized on the gel using a
PhosphorImager (Molecular Dynamics).
Additional Methods--
Protein concentrations were determined
by either the Lowry et al. (25) or the Bradford (26)
methods. Alternatively, Ure2p concentration was determined
spectrophotometrically (HP 8453 diode array spectrophotometer,
Hewlett-Packard) using an extinction coefficient of 26,000 M
1·cm
1 and a molecular mass
of 40.4 kDa. Congo Red binding was performed as described (32).
Standard SDS-polyacrylamide gel electrophoresis was performed in 10%
gels following the method described by Laemmli (27).
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RESULTS |
Recombinant Ure2p Is a Soluble Protein--
Large amounts of
proteins are required to achieve structural and functional studies.
Since yeast expression systems are well known not to be as efficient as
that of E. coli, we developed an approach to overexpress the
yeast prion-like protein Ure2p in E. coli. To overcome the
problems we noted due to the differences in codon usage upon expression
of wild type Ure2p in E. coli, we substituted two AGA codons
in URE2 gene, that are rare in E. coli, by synonymous CGT
codons (17). The resulting construct was efficiently expressed in
E. coli host cells (Fig.
1A, lane 1). Recombinant
overexpressed Ure2p was purified to homogeneity through three
successive chromatographic dimensions as described under "Materials
and Methods" (Fig. 1A, lanes 3-5). The purified protein
was found to have a molecular mass of 40 kDa upon analysis by SDS-PAGE
(Fig. 1A) and migrated with an apparent mass of 130 kDa upon
gel filtration (Fig. 1B). The molecular mass of this polypeptide (40,415 Da) was determined by mass spectrometry (Fig. 1C). It is consistent with that of the calculated mass of
full-length Ure2p (40,441 Da).

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Fig. 1.
Purification and characterization of
recombinant Ure2p. A, expression in E. coli
and purification of Ure2p. Analysis on a 10% SDS-polyacrylamide gel of
protein samples taken at different steps of Ure2p purification.
Lane 1, soluble fraction of E. coli expressing
recombinant Ure2p. Lanes 2 and 3, protein
fractions containing Ure2p emerging from the UNO Q and the
phenyl-Sepharose columns, respectively. Lane 4, recombinant
Ure2p emerging from the hydroxyapatite ion exchange chromatography
column. Lane 5, pure recombinant Ure2p emerging from the
size exclusion column. Molecular mass markers (in kDa) are shown on the
left. B, size exclusion chromatography analysis
of pure recombinant Ure2p. Elution profile of pure recombinant Ure2p
from a Superose 6 (HR 10/30) column. Arrowheads show the
location of molecular size markers (thyroglobulin, 670 kDa;
immunoglobulin G, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa; and
vitamin B12, 1.35 kDa) run under identical conditions on the same
column. C, mass spectrum of purified recombinant Ure2p
corresponding to the material shown in lane 4 of
A.
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Two species with apparent molecular masses of 38 and 23 kDa were also
present in several purified Ure2p preparations (Fig. 1A).
The molecular masses of these two additional polypeptides were measured
by mass spectrometry and found to be 38,372 and 22,079 Da (Fig.
1C). The 40-, 38-, and 23-kDa polypeptides were separated by
SDS-PAGE and subjected to automated Edman degradation. The polypeptide
that has a molecular mass of 40 kDa corresponds to authentic
full-length Ure2p. Amino acid sequencing of the two other peptides gave
the following sequences, VNI-NRNSN and SHVEYSRI. The polypeptide that
has a molecular mass of 38 kDa corresponds to Ure2p devoid of its 20 first amino acid residues, whereas that of 23 kDa could correspond to
an internal initiation of translation occurring at Met-94, resulting in
a truncated Ure2p molecule.
Quaternary Structure of Ure2p--
We first estimated the size of
recombinant Ure2p by size exclusion chromatography using a Superose 6 column. Ure2p elutes from this column as a single peak between the
molecular weight markers
-globulin (158 kDa) and ovalbumin (44 kDa)
and has an apparent molecular mass of 130 kDa incompatible with the
expected molecular mass of monomeric Ure2p (40.4 kDa) (Fig.
1B). Since the measured molecular mass does not fit with
that of a dimer of Ure2p nor with that of a tetramer, we envisaged the
possibility that Ure2p would not behave as a globular protein. Indeed,
a number of proteins have been found to behave on sizing columns with
apparent molecular masses very different from that derived from their
primary sequences (28-30). These polypeptides are found to be either
rod-shaped or to possess unstructured domains.
To investigate whether the elution behavior of Ure2p from a size
exclusion column is due to its oligomerization or to its behavior as a
non-globular protein, we determined the molecular mass of the protein
by equilibrium sedimentation. The weight-average molecular mass of pure
Ure2p (0.4 mg/ml) in aqueous solution measured by equilibrium
sedimentation was found to be 75,000 (Fig.
2A), inconsistent with its
behavior as a monomeric protein. In order to obtain additional insight
into the oligomeric state of Ure2p, at increasing Ure2p concentrations,
we carried out sedimentation velocity experiments and analyzed them to
yield the apparent distribution of sedimentation coefficients,
g*(s). Fig. 2B shows typical
sedimentation boundaries at series of equally spaced times and at
increasing Ure2p concentrations. Raw data (symbols in Fig.
2C) were fitted by nonlinear least squares procedures (Fig.
2C, solid line) as described by Philo (18). At
low Ure2p concentrations (
0.4 mg/ml), the data fit very well to a
two-component system involving a 2.8 S and a 4.3 S species with
proportions of about 19 and 80%, respectively at a Ure2p concentration
of 0.4 mg/ml. Using the relation
(s1/s2)3 = (M1/M2)2 and
bovine serum albumin as a reference (22), one can obtain apparent
molecular masses of about 40 and 80 kDa consistent with the behavior of
Ure2p as a mixture of monomeric and dimeric molecules. The frictional
ratio values (f/f0) suggest that Ure2p is
asymmetrical. At higher Ure2p concentrations (0.5 mg/ml), the monomeric
species of Ure2p disappeared, and the data fit well to a single
component system involving a 4.3 S species. At even higher Ure2p
concentrations (
0.6 mg/ml), the data fit very well to a two-component
system involving a 4.3 S and a 6.5 S species. The heaviest species
has a sedimentation coefficient consistent with the behavior of a tetramer of Ure2p. The above sedimentation velocity data as well as the
calculated hydrodynamic parameters of Ure2p are summarized in Table
I.

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Fig. 2.
Oligomeric state of Ure2p. A,
measurement of the molecular weight of Ure2p by equilibrium
ultracentrifugation. Equilibrium sedimentation was performed as
described under "Materials and Methods." The data ( ) obtained at
4 °C and 40,000 rpm for Ure2p (0.4 mg/ml) were fitted using a
monomer model (solid line). The top panel shows
the deviation of the data from the fitted curve. The best fit to the
data points was obtained for a molecular weight of 77,000. B, typical sedimentation velocity data for Ure2p (0.36 mg/ml). Images of the boundary taken 12 min apart. The fitted data
curves are represented by a solid line. The steep boundary
corresponds to the peak in C. C, distributions of
sedimentation velocity of Ure2p (0.36 mg/ml). The arrow
indicates the position of the maximum of g·(s).
The solid lines represent the best fit obtained to the data
points ( ) using a monomer-dimer model. Rotor speed is 60,000 rpm and
temperature is 15 °C.
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Table I
Species of Ure2p formed at increasing protein concentrations and their
hydrodynamic parameters
Properties and relative abundance of Ure2p oligomers formed at
increasing protein concentrations calculated from sedimentation
profiles similar to that displayed in Fig. 2B. The
conformational parameters were calculated as described under
"Materials and Methods," using the molecular mass and the partial
specific volume values determined from the amino acid composition of
Ure2p. Numerical values of n-mer of each species were
obtained by dividing experimental molecular mass of each species by the
monomeric Ure2p theoretical mass.
S20,w0 is the sedimentation
coefficient; f and f0 are the friction
coefficients, and RS is the Stokes radius.
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We conclude from these data that soluble Ure2p exists in a monomeric,
dimeric, and tetrameric form in solution. The fact that Ure2p
oligomerization is concentration-dependent suggests that these forms are in equilibrium.
Ure2p Self-associates into High Molecular Weight Oligomers in
Vitro--
Ure2p assembly into insoluble high molecular weight
oligomers was achieved by two means. The first consisted of inducing
self-association by bringing the pH of the solution from 7.5 to 6.5. The second consisted of incubating Ure2p for prolonged periods at
either 4 or 28 °C. Ure2p aggregation was followed
spectrophotometrically or by measurement of light scattering.
Aggregation of Ure2p was instantaneous upon adjustment of the pH to 6.5 (Fig. 3A). In contrast, Ure2p
autoassembly at pH 7.5 was very slow upon incubation of the solution at
4 °C (Fig. 3B) or 28 °C (Fig. 3C). Ure2p
assembly into insoluble high molecular weight oligomers is a
cooperative process and follows a simple sigmoidal curve. Indeed, three
phases can be distinguished in the assembly process as follows: a lag phase where nucleation occur followed by an elongation phase where assembly accelerates preceding the onset of a plateau. The lag phase is
shortened significantly upon addition of Ure2p seeds (not shown)
further demonstrating the cooperative character of Ure2p
autoassembly.

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Fig. 3.
Assembly of Ure2p into high molecular weight
oligomers. A, kinetic of Ure2p assembly monitored by
light scattering. The aggregation of Ure2p in 20 mM
potassium phosphate buffer, pH 7.5, 1 mM MgCl2,
1 mM DTT, 1 mM EGTA at 20 °C was induced at
the time indicated by the arrow by adjustment of the pH of
the solution to pH 6.5 by addition of HCl. B and
C, time course of Ure2p autoconversion from a soluble form
to an insoluble form, in 20 mM potassium phosphate buffer,
pH 7.5, 1 mM MgCl2, 1 mM DTT, 1 mM EGTA, followed by measurement of the turbidity of the
solution at 4 °C (B) and 28 °C (C) in the
absence ( ) or the presence of 100 mM ( ) and 4 M ( ) GdnHCl.
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In all cases, the amount of Ure2p that assembles into high molecular
weight oligomers and that sediments upon centrifugation at 4000 × g represents 95% of the initial concentration of Ure2p in
its soluble form.
Electron microscopy of negatively stained samples of soluble and
aggregated Ure2p revealed that the soluble form of Ure2p contains
structured circular particles with an outer diameter of about 12 nm
(Fig. 4A). In contrast, the
insoluble form of Ure2p obtained upon incubation of the protein for
70 h at 4 °C consists of fibrils that are 15-20 nm wide and
their length varies between 0.5 and 10 µm (Fig. 4B). These
fibers are similar to the fibers that are observed in the case of PrP
in its scrapie prion filaments form (31) or that form upon Sup35
self-assembly (32). Ure2p fibers are often associated laterally and in
some cases twisted together (Fig. 4B, inset).
Finally, the aggregated form of Ure2p obtained upon adjustment of the
pH from 7.5 to 6.5 consisted of filamentous structures that are up to
0.5 µm long and devoid of any regular shape (not shown).

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Fig. 4.
Characterization of Ure2p assembly.
Electron micrographs of negatively stained Ure2p oligomers are shown.
A, soluble Ure2p emerging from the Superose 6 column. The
only organized structures seen are arrowhead-shaped objects
organized in ring-shaped structures, enlarged in the
inset in A, that could correspond to Ure2p
octamers. B, fibers obtained upon autoassembly of the
soluble form of Ure2p. Ring-shaped structures (white
arrow in the inset in B) similar to the ones
observed in A are present in Ure2p fiber preparations. In
some cases two fibers are associated laterally and twisted together
(black arrow in the inset in B)
Bar = 100 and 20 nm in the inset in
A. Time course of Ure2p fiber formation is monitored by
Congo Red binding. C, assembly of the soluble form of Ure2p
at 30 ( ), 10 ( ), and 3 ( ) µM in 20 mM KPO4, 150 mM KCl. D,
time course of Ure2p assembly ( ) upon dilution (1,000-fold) of
fibers assembled from a solution of Ure2p (10 µM) into a
solution containing the soluble form of Ure2p (10 µM).
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Time Course of Ure2p Fiber Formation--
A distinctive property
of amyloid fibers is their capacity to bind the dye Congo Red (33). The
spectral shift that accompanies Congo Red binding to amyloid proteins
(34) was used to document the time course of Ure2p assembly following
the incubation of the soluble form of the protein at 28 °C. A lag
time (hours) preceded Ure2p (10 µM) autoassembly into
fibers (Fig. 4C). The lag time is shortened at higher Ure2p
concentrations (30 µM) and is augmented at lower Ure2p
concentrations (3 µM). This finding strongly suggests that nucleation is a critical step in Ure2p autoassembly into amyloid fibers.
To test this idea further, preformed fibers, obtained upon assembly of
Ure2p (10 µM), were diluted 1000-fold into a solution of
Ure2p (10 µM) in its soluble form, and the time course of
Ure2p assembly was monitored by Congo Red binding. Ure2p assembly under these conditions proceeded without a lag time (Fig. 4D). We
conclude from these data that the autoassembly of the soluble form of
Ure2p into fibers is a nucleated process that is
concentration-dependent.
Ure2p Is a Two-domain Protein--
A number of secondary structure
determination algorithms predict Ure2p to be a two-domain protein. The
NH2-terminal domain appears as very poorly structured
unlike the COOH-terminal domain. Indeed, the COILS program (35)
predicts the NH2-terminal part of Ure2p to have a high
probability of being in coiled coil conformation. These calculations
are displayed in Fig. 5A.
Interestingly, both parts of the gene encoding Ure2p also clearly
reveal differences in codon usage (Fig. 5B). Specific
features of synonymous codon distribution along mRNA were suggested
and shown in certain cases to reflect domain organization of proteins
(36, 37). To determine whether this is indeed the case, Ure2p
NH2-terminal and COOH-terminal domains (amino acid residues
1-94 and 94-354, respectively) were expressed as
35S-labeled polypeptides in rabbit reticulocyte lysate and
subjected to proteinase K or to trypsin treatment. The reaction
products were analyzed by SDS-PAGE and visualized using a
PhosphorImager. The data are presented in Fig.
6. The COOH-terminal domain of Ure2p
which has a molecular mass of 29 kDa (band lettered A) is rapidly
cleaved upon proteinase K treatment (Fig. 6A) into three peptides (14.5, 5, and 3 kDa, labeled B, C, and
D, respectively). The 14.5-kDa peptide is then degraded into
a peptide that has a molecular mass of 10 kDa (band E) and
that resists to protease cleavage for up to 60 min, whereas the 5.5-kDa
peptide generates a polypeptide that has a molecular mass of 3 kDa. The
product that has a molecular mass of 7 kDa resists proteinase K
treatment for up to 60 min. Treatment of the COOH-terminal domain of
Ure2p by trypsin (Fig. 6B) leads to similar observations.
The full-length COOH-terminal domain resists to protease cleavage for
up to 60 min and generates very slowly degradation products that have
molecular masses of 25 and 7 kDa (solid and open
arrowheads, respectively) which resist trypsin treatment for up to
120 min (not shown). In contrast, both proteinase K and trypsin
treatment of the NH2-terminal domain of Ure2p result in a
very quick and total degradation of this domain (0.25 and 1.5 min,
respectively).

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Fig. 5.
Structure predictions for Ure2p.
A, predictions for coiled-coil structures using COIL
algorithm (35). B, prediction for Ure2p domain organization
derived from codon frequency occurrence profile (36).
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Fig. 6.
Differential resistance to protease treatment
of Ure2p NH2- and COOH-terminal domains. The time
courses of Ure2p NH2- and COOH-terminal parts cleavage by
proteinase K (A) or trypsin (B) were monitored by
SDS-electrophoresis followed by autoradiography.
35S-Labeled Ure2p NH2- and COOH-terminal parts
were obtained by in vitro translation in the presence of
[35S]methionine. Aliquots were removed at the times shown
on the top of each slot, and proteolysis was terminated by
heat denaturation after addition of SDS-containing sample buffer. The
reaction products were analyzed on Tricine-SDS gels and
autoradiographed. Quantitation of the amount of polypeptides that have
a molecular mass higher than 3 kDa generated following proteinase K
(top) or trypsin (bottom) treatments of Ure2p
NH2- ( ) and COOH-terminal ( ) parts are displayed on
the left side of each panel.
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We conclude from these data that the COOH-terminal domain of Ure2p, in
agreement with secondary structure predictions, is much more structured
and therefore compact and resistant to limited proteolysis treatments
than the extended NH2-terminal domain of the protein.
The Global Conformation of Ure2p Is Not Affected by
Self-assembly--
To determine whether Ure2p aggregation is
accompanied by a conformational change of the protein, the soluble and
aggregated forms of Ure2p were subjected to limited proteolysis. Three
different proteases were used as follows: proteinase K, subtilisin, and trypsin. The digestion profiles of the soluble (left) and
insoluble (right) forms of Ure2p generated upon proteinase K
(Fig. 7A), subtilisin (Fig. 7B), and trypsin
(Fig. 7C) treatments are shown in Fig.
7.

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Fig. 7.
Differential resistance to protease treatment
of the soluble and insoluble forms of Ure2p. The soluble and
insoluble forms of pure Ure2p were subjected to proteinase K
(A), subtilisin (B), or trypsin (C)
treatments, and the time courses of the different digestions were
monitored by SDS-electrophoresis followed by Coomassie staining. Time
points are shown at the top of each gel. In the slot labeled
00, Ure2p prior to addition of the proteases is shown.
Time 0 corresponds to an aliquot taken immediately after
addition of the various proteases to Ure2p solutions. Quantitation of
the amount of full-length Ure2p remaining after addition at time 0 of
the different proteases to the soluble ( ) or insoluble ( ) forms
of Ure2p are shown on the left side of each panel.
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The soluble form of Ure2p is rapidly degraded by proteinase K (less
than 1 min) into one major product that has a molecular mass of 30 kDa
and four other products that have molecular masses comprised between 28 and 22 kDa. Only the products of molecular masses lower than 26 kDa
persists 15 min after the onset of proteinase K treatment and evolves
toward a final stable product that has a molecular mass of 22 kDa.
Proteinase K treatment of the insoluble form of Ure2p generates a
number of polypeptides that are similar to the ones generated upon
treatment of the soluble form of Ure2p by the same protease. The final
product of proteinase K treatment is also the same. However, and in
contrast with the profile obtained with the soluble form of Ure2p,
full-length Ure2p in its insoluble form appears much more resistant to
proteinase K treatment than the soluble form (5 min compared with
30 s).
Subtilisin degrades in less than 30 s the soluble form of Ure2p
into two products that have molecular masses of 22 and 20 kDa. These
polypeptide chains are then degraded into two lower molecular mass
products (14.5 and 7 kDa). The insoluble form of Ure2p resists
subtilisin cleavage for nearly 1 min. Intermediate products that have
molecular masses of 33 and 30 kDa that barely exist when the soluble
form of Ure2p is subjected to subtilisin treatment persist in the
solution for up to 5 min. These intermediates then disappear in favor
of two polypeptides that have a molecular mass of 22 and 20 kDa.
Finally, trypsin treatment of both the soluble and insoluble forms of
Ure2p generates similar digestion profiles. The major degradation
product is a polypeptide that has a molecular mass of 29 kDa. However,
the insoluble form of Ure2p appear to be, once again, more resistant to
proteolysis than the soluble form of the protein.
We conclude from the comparison of the digestion profiles of the
soluble and insoluble forms of Ure2p by proteinase K, subtilisin, or
trypsin that the products that are generated are identical and that the
soluble form of Ure2p is systematically less resistant to protease
treatments than the insoluble form of the protein. Because the
digestion patterns for the soluble and insoluble forms of Ure2p are
strictly identical, our data further indicate that no major
conformational change affects Ure2p upon its aggregation.
The Structure of Ure2p COOH-terminal Domain Changes Slightly upon
Self-assembly--
Subtle conformational changes occurring during
self-assembly of Ure2p may not affect the cleavage sites of the
proteases used above. Such conformational changes could be documented
by measurement of the intrinsic fluorescence of the protein in the case
where aromatic residues are evenly distributed all along the
polypeptide chain. Alternatively, such conformational changes could be
detected by measurement of the ellipticity of the protein.
Fig. 8A provides, by
measurement of intrinsic fluorescence, an independent indicator of the
conformational differences between the soluble and insoluble forms of
Ure2p. Given that all tyrosine as well as tryptophan residues are
located within the COOH-terminal domain of the protein, these data
allow us to investigate whether the conformation of Ure2p COOH-terminal
domain changes during the self-assembly process. The intensity of the
fluorescence is 50% smaller when Ure2p aggregation into high molecular
weight oligomers is induced by bringing the pH from 7.5 to 6.5 (Fig. 8,
A and B). Furthermore, a slight shift in the
wavelength of the emission maximum is observed (Fig. 8A).
These changes are consistent with a lesser exposure of tyrosine and
tryptophan residues to the aqueous milieu due to self-assembly of Ure2p
into high molecular weight oligomers. Indeed, Ure2p pH-induced
aggregates scatter the light significantly (Fig. 8C).

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Fig. 8.
Ure2p conformation changes upon its
aggregation. Fluorescence excitation (A) and emission
(B) spectra of Ure2p. A, the aromatic amino acid
residues of Ure2p (0.37 mg/ml) at pH 7.5 (dotted line) and
6.5 (solid line) were excited from 250 to 310 nm, and the
fluorescence was recorded at 336 nm. B, tyrosine and
tryptophan residues of Ure2p at pH 7.5 (dotted line) and 6.5 (solid line) were excited at 290 nm, and their emission
spectra were recorded from 300 to 400 nm. Light path is 0.1 cm.
C, variation of the amount of insoluble Ure2p as a function
of the pH quantitated by measurement of light scattering at 340 nm
following adjustment of the pH of the solution to the desired value by
addition of increasing amounts of HCl. D, CD spectra of
soluble Ure2p in 20 mM potassium phosphate buffer, pH 7.5 (solid line), and in 60% TFE (dashed line). The
data are plotted as mean residue weight ellipticity
( mrw) versus wavelength.
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We conclude from these observations that Ure2p aggregation is
accompanied by a slight conformational change of its COOH-terminal domain. This conformational change is due both to a quenching effect
due to the aggregation of Ure2p molecules as well as to a change in the
exposure of tryptophan and tyrosine residues to water molecules.
To determine the secondary structure content of Ure2p, we recorded the
circular dichroism spectrum of Ure2p in aqueous solution, in the low UV
range (Fig. 8D, solid line). The calculated
-helical content of Ure2p is 28%. The CD spectrum of Ure2p in the
presence of 60% TFE, a solvent known to stabilize
-helices, was
also recorded (Fig. 8D, dashed line). The
-helical
content of Ure2p in 60% TFE was found to increase by 8%.
We conclude from these observations that the
-helical content of
Ure2p changes to some extent in the presence of a solvent known to
stabilize
-helices. Given that the COOH-terminal domain of the
protein appears to be compact and structured, the changes in the
-helical content must be due to a stabilization of the NH2-terminal domain of the protein into
-helices. Thus
Ure2p NH2-terminal domain appears not only to be less
structured than its COOH-terminal domain but also more flexible than
the latter.
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DISCUSSION |
Yeast prions Sup35 and Ure2p do not kill the cells that harbor
them and are not hazardous nor pathogenic for humans. They present,
therefore, a useful model system to study the molecular mechanisms of
mammalian prions advent and spread. Our capacity to express recombinant
yeast prion Ure2p as a soluble protein allowed us to characterize some
of its properties. The work presented here provides new insights into
the structure of this protein and the mechanism of its assembly into
various quaternary structures.
Structure of Ure2p--
Soluble Ure2p was found to emerge from a
sizing column with an apparent molecular mass of 130,000 incompatible
with that expected for a monomeric Ure2p molecule that would have a
globular shape. Sedimentation velocity measurements reveal that
recombinant Ure2p forms oligomers in a
concentration-dependent manner. Indeed, soluble Ure2p
preparations are heterogeneous mixtures of monomeric, dimeric, and
tetrameric Ure2p molecules as well as higher order oligomers. Examination of soluble Ure2p preparations by electron microscopy reveals globular but heterogeneous particles. The most striking structures we observed were ring-shaped particles with an outer diameter of 10 nm composed of four arrowheads pointed toward the center
of the particles. The finding that Ure2p oligomerization is
concentration-dependent indicates that the monomeric,
dimeric, and tetrameric forms of Ure2p as well as the higher molecular weight oligomers are in equilibrium. Over 95% of the soluble form of
Ure2p was found to assemble into insoluble high molecular weight oligomers either upon incubation of the protein for prolonged periods
at 4 or 28 °C or adjustment of the pH of the solution from 7.5 to
6.5. In the latter case fibrillar structures highly heterogeneous in
size and shape were obtained while fibrils that are 15-20 nm wide
varying in length between 0.5 and 10 µm were obtained upon incubation
of the soluble form of Ure2p at either 4 or 28 °C. These fibers
appear very similar in the electron microscope to PrP
scrapie-associated filaments and to the fibers observed upon Sup35
autoassembly and bind Congo Red which is a characteristic of amyloid fibers.
The kinetics of Ure2p assembly are sigmoidal indicating a cooperative
process. Furthermore, the lag phase preceding Ure2p assembly depends on
the concentration of the protein and disappears upon addition of
preformed Ure2p fibers to the soluble form of the proteins which
indicates that Ure2p autoassembly is a nucleated process. Finally, the
critical concentration for Ure2p assembly appears to be lower than 0.5 µM since the proportion of protein assembled into fibers
represents 95% of the input soluble Ure2p (10 µM). Taken
together, the properties of Ure2p in vitro can account for
the propagation of Ure2p polymers in a manner similar to what occurs in
the case of Sup 35 and PrP.
Conformational Changes of Ure2p--
Several genetic studies
suggest that Ure2p is a two-domain protein that were recently shown by
means of the two-hybrid system to interact with each other (38).
Indeed, the NH2-terminal third of the molecule appears
essential for the occurrence of [URE3] phenotype, whereas the
catalytic activity of the protein appears to be located in the
COOH-terminal two-thirds of the molecule (10, 39). Secondary structure
calculation algorithms predict that most of the
NH2-terminal third of the protein is unstructured (i.e. essentially constituted by coils, turns, and sheets),
whereas the
-helical structures are mainly located in the
COOH-terminal two-thirds of the protein. Thus Ure2p is predicted to be
at least a two-domain protein. These predictions are supported by the
data obtained upon treatment of the COOH- and NH2-terminal
domains of the protein, expressed independently, with different
proteases. Ure2p NH2-terminal domain showed a much higher
sensitivity to protease treatments than the COOH-terminal domain of the
protein. Indeed, none of the peptides generated upon protease treatment of the NH2-terminal domain resisted longer than 1 min,
whereas the products of Ure2p COOH-terminal domain cleavage were stable for over 1 h. These studies revealed two fragments (22 and 14.5 kDa) highly resistant to proteinase K as well as two others (29 and 7 kDa) highly resistant to trypsin. These peptides were tentatively identified as a breakdown products of Ure2p COOH-terminal domain since
they are generated upon treatment of Ure2p 94-354 fragment by the same
proteases. The largest peptide (29 kDa) would correspond to Ure2p
COOH-terminal domain devoid of its 8-11 NH2-terminal amino
acid residues or to the same domain devoid of its 10 COOH-terminal amino acid residues. Confirmation of the assignment will ultimately require the isolation and sequencing of the 29-kDa fragment. The same
approach will lead to assignment of the 22-, 14.5-, and 7-kDa fragments.
A major conformational change occurring upon Ure2p aggregation would
result in differences in the digestion profiles of the two forms of the
protein. This is not what we observed. Indeed, treatment of full-length
Ure2p in its soluble or aggregated forms by proteinase K or trypsin
yielded peptides that are identical to that generated upon treatment of
Ure2p COOH-terminal domain by the same proteases. However, the kinetics
of cleavage were significantly slower for the insoluble form of Ure2p.
Such results are what one expects in the case of a reduced
accessibility of Ure2p (the substrate) to the protease. Furthermore,
the peptides generated upon either proteinase K or trypsin treatments
of full-length Ure2p were that generated upon treatment of the
COOH-terminal domain of Ure2p by the same proteases. This strongly
suggests that the NH2-terminal domain is rapidly degraded
whether or not the COOH-terminal domain is present. The rapid and total
degradation of Ure2p NH2-terminal domain would be a
consequence of its lack of structure and compactness. Finally, our
findings also indicate that the COOH-terminal domain adopts the same
conformation in the presence or absence of the NH2-terminal
domain, since the same protease cleavage sites are exposed whether the
NH2-terminal domain of the protein is present or not.
Proteolytic treatment of proteins is a powerful tool to probe
conformational changes that may affect the structure of the substrate
polypeptides. Nevertheless, a number of tiny changes may well be
overlooked. In order to detect such small changes, intrinsic
fluorescence as well as circular dichroism measurements were carried
out. Given that all tryptophan and tyrosine residues are located within
Ure2p COOH-terminal domain, intrinsic fluorescence measurements allowed
us to follow conformational changes affecting this domain of the
protein. The data presented in this work clearly demonstrate that the
COOH-terminal domain of Ure2p undergoes a conformational change during
self-assembly process that results in a 50% decrease in exposure of
tyrosine and tryptophan residues to water concomitant with a shift in
the wavelength of the emission maximum. Circular dichroism data suggest
that a number of amino acid residues in Ure2p are in
-helical
structures. The amount of
-helices increases when the protein is in
the presence of TFE, a solvent known to stabilize
-helical
structure. This could be due to the transition of stretches of amino
acid residues in Ure2p NH2-terminal domain from random
coils to
-helical structures. Alternatively, the increase of the
content of
-helices could correspond to amino acid residues located
within the COOH-terminal moiety of the protein that adopts a structure
predominantly
-helical upon TFE addition. Consequently, methods
allowing the specific labeling of the Ure2p NH2-terminal
domain with extrinsic fluorophores will have to be designed in order to
access the effect of the solvent as well as autoassembly on the
flexibility of this domain.
Present genetic, biochemical, and structural data (3-5, 9, 10, 39)
allow us to make a brief comparison of some properties of mammalian
prion protein PrP and yeast prions, Ure2p and Sup35p. In all cases
proteins seem to consist of at least two major parts. Indeed, although
the NH2-terminal part (bearing unusual amino acids repeats,
rich in Gly in the case of PrP (4, 6) and in Asn and Gln in the case of
Ure2p and Sup35 (10)) seems to be in all the cases unstructured and
flexible and at the same time absolutely required for the propagation
of prion conditions (through an extensive aggregation of proteins), the
COOH-terminal part appears to be compactly folded. We show here that
Ure2p like Sup35 and PrPc can undergo extensive aggregation into highly
ordered fibers. Our data demonstrate that preformed Ure2p fibers
incorporate the soluble form of the protein and propagate Ure2p
assembly in a manner similar to what is observed in the case of Sup35
(32, 40) and support a "protein only" seeded polymerization model for Ure2p.
Conversion of the cellular form of the prion protein (PrPc) to the
scrapie isoform (PrPSc) (leading further to its aggregation) is thought
to be driven by an
-helical to
-sheet conformational transition
(4, 5). The poorly structured NH2-terminal part of the
prion protein (14, 15) seems to be ultimately crucial in such
interconversion, providing the plasticity required for a
conformational change. This characteristic is common among PrPc, Sup35,
as well as Ure2p. Nevertheless, such conformational changes are not
necessarily restricted to the NH2-terminal part of these
proteins. Indeed, the majority of the mutations affecting the
efficiency of propagation of prion diseases is associated with the
COOH-terminal part of the protein (4, 5). Our finding that the
conformation of the COOH-terminal part of Ure2p is affected during
aggregation is in favor of a mechanism of assembly similar among all prions.