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Originally published In Press as doi:10.1074/jbc.M306004200 on August 12, 2003

J. Biol. Chem., Vol. 278, Issue 44, 43717-43727, October 31, 2003
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Architecture of Ure2p Prion Filaments

THE N-TERMINAL DOMAINS FORM A CENTRAL CORE FIBER*

Ulrich Baxa{ddagger}§, Kimberly L. Taylor§, Joseph S. Wall||, Martha N. Simon||, Naiqian Cheng{ddagger}, Reed B. Wickner§, and Alasdair C. Steven{ddagger}**

From the Laboratories of {ddagger}Structural Biology, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, and §Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and the ||Department of Biology, Brookhaven National Laboratories, Upton, New York 11973

Received for publication, June 6, 2003 , and in revised form, August 6, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues ~1–70. Fusion protein filaments with diameters of 14–25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ure2p is a cytoplasmic homodimeric protein (2 x 40 kDa) (1, 2) involved in regulation of nitrogen catabolism in Saccharomyces cerevisiae (3, 4). An inactive aggregated prion form of Ure2p has been shown to provide the molecular explanation (5–7) for the nonchromosomal genetic element [URE3] (8). The term "prion" (infectious protein) was first introduced for the mammalian PrP protein (9), but is now used more generally (5). Upon prion conversion, Ure2p is inactivated, apparently by entering an aggregated filamentous state (6, 7, 10). The filaments partition between daughter cells when an infected yeast cell divides, explaining the non-Mendelian genetics; filaments are introduced to a cell lacking [URE3] by cytoplasmic mixing when it fuses with a [URE3] cell, thus explaining the infectivity. Knowledge of the filament structure is essential for gaining further understanding of prionogenesis in this system.

Ure2p consists of a C-terminal functional domain and an N-terminal prion domain (6), a pattern repeated in another yeast protein, Sup35p, which can become the [PSI] prion (11). The Ure2p functional domain binds to the transcription factor Gln3p, preventing its migration to the nucleus (12–14). This interaction is prevented in the prion state, probably by steric blocking of the binding site of Ure2p for Gln3p (15). Residues 90–354 suffice to regulate Gln3p activity (16), and residues 100–354 are conserved among other yeasts (17). This domain has a similar fold to glutathione S-transferase (GST)1 (18, 19), as predicted from sequence similarity (4). The prion domain is necessary for prion-based inactivation of Ure2p and induces the de novo appearance of [URE3] (6, 16, 20). This domain has an unusual amino acid composition, consisting of ~45% Asn and Gln and ~20% Ser and Thr. The Asn-rich tract extends to residue 89, but residues 1–65 suffice for prion-forming ability (although 1–80 are more efficient). In native Ure2p, the N-terminal region is highly protease-sensitive and thought to be unstable or even unfolded (2, 21).

Ure2p forms amyloid filaments in vitro (1), and several findings indicate that these filaments are similar to the aggregated prion form found in vivo: (i) The N-terminal domain is necessary and sufficient for prion behavior and aggregation in vivo (6, 7, 20) and for filament formation in vitro (1). (ii) Filaments of Ure2p could be detected in [URE3] cells in vivo but not in [ure-o] cells (10). These filaments are similar in thickness (~25 nm) to those assembled in vitro. (iii) The proteinase K digestion pattern of Ure2p in extracts of [URE3] cells is very similar to that of in vitro formed filaments (1, 6).

Although considerable effort has been expended on examining Ure2p filaments, a consensus has not been reached as to their overall architecture and conformation (i.e. presence of amyloid2). We have proposed that the prion domains stack to form a core fiber of amyloid (1, 10, 22), which is surrounded by the C-terminal domains. Backbones of polymerized prion domains have also been envisaged for Sup35p filaments (23) and HET-s filaments (24, 25). On the other hand, an alternative model has recently been proposed whereby Ure2p molecules are connected to each other by N-terminal to C-terminal interactions, and the filaments do not contain amyloid (26, 27).

In this study, we have investigated the overall architecture of in vitro assembled Ure2p filaments and fusion protein filaments by a combination of biophysical and biochemical analyses. After controlled proteolytic digestion, the reaction products were analyzed by Western blotting, mass spectrometry, and conventional negative staining EM. We also visualized intact filaments by a high resolution negative staining technique in the scanning transmission electron microscope (STEM) (28) and by cryo-electron microscopy of vitrified specimens, and we measured their values of mass per unit length from dark-field STEM images of freeze-dried filaments (28–30). The results of these experiments substantiate our amyloid core model of the filament structure and show that the prion domain residues, ~1–70 form this core. They further indicate residues ~71–90, although part of the prion domain by amino acid composition and prion-inducing ability are not in the central core but link it to C-terminal appendages.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning Procedures, Protein Expression and Purification, and Filament Assembly
URE2 was cloned into a pFLAG vector (Kodak) by PCR using the primers 5'-ggaactcatatgcatcaccatcaccatcacatgtatccacgtgggaatatgatgaa-taacaacggc-3' and 5'-ggaactgtcgacgaattctgtggttggggtaac-3' and the NdeI and SalI restriction sites. To improve expression levels in Escherichia coli, the AGA codons 253 and 254 for arginine were replaced with CGT using site-directed PCR mutagenesis with the primers 5'-cggatgaggttcgtcgtgtttacggtgtag-3' and 5'-ctacaccgtaaacacgacgaacctcatccg-3' as described (31), yielding plasmid pKT41-1. The N-terminal primer encodes a His6 tag and a thrombin cleavage site, and therefore pKT41-1 codes for a protein with the sequence: MH6MYPRGNUre2p1–354. Overexpression was performed using E. coli BL21 in LB medium containing 0.1 mg/ml ampicillin at 37 °C for 4 h after induction with 1 mM isopropyl {beta}-D-thiogalactosidase at an A550 of ~1.0. After harvesting, the cells were resuspended in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, containing protease inhibitors (Complete EDTA-free, Roche Applied Science), and lysed by high pressure. Insoluble material was removed by centrifugation (40,000 x g, 1 h). The expressed protein was recovered in one step, using a nickel-nitrilotriacetic acid Superflow column (Qiagen). The protein was bound to the column in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, washed extensively with 50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0, and eluted with 50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0. This protein was either used directly in experiments (His6-Ure2p) or digested with thrombin (Roche Applied Science) at room temperature for 20 h to remove the His6 tag. The sequences of Ure2p1–65, Ure2p1–80, and Ure2p1–89 were cloned in the pFLAG vector (Kodak) using PCR and the NdeI and SalI restriction sites, producing plasmids pKT53, pKT54, and pKT55, respectively. For all, the N-terminal primer included a His6 tag, however, without the thrombin cleavage site. The sequences of the proteins were MH6-Ure2p1–65, MH6-Ure2p1–80, and MH6-Ure2p1–89. Synthesis and purification of Ure2p1–65 was as described previously (1). Ure2p1–65-barnase, Ure2p1–65-CA, and Ure2p1–65-GST were prepared as described (15). Plasmid pH469 coding for Ure2p1–80-SDDDDKGGRGFP-H6 was kindly supplied by H. Edskes. Ure2p1–65-GFP was cloned by digestion and religation of pH469 with NotI, exploiting the two NotI sites of pH469 after position 65 of Ure2p and in front of GFP, leading to pUB7. Expression from pUB7 yielded Ure2p1–65-GFP-H6. Expression and purification was performed as described for Ure2p1–80-GFP (15).

To keep numbering of amino acids in the different constructs clear, all proteins and fusion constructs were numbered with 1 being the first Met of Ure2p. Additional sequences at the N terminus (His6 tag and thrombin site) for some constructs were assigned negative numbers.

All proteins were frozen in liquid nitrogen shortly after purification and stored at -80 °C to prevent filament formation. Filaments were assembled by incubating protein solutions (at 1–5 mg/ml) at 4 °C, usually in 20 mM Tris/HCl, 200 mM NaCl, pH 8.0. For Ure2p1–65-barnase the buffer was 20 mM Tris/HCl, 200 mM KCl, pH 7.5. For some fusion proteins, filament formation was accelerated by continual agitation of the solution. Filaments were purified by centrifugation (25,000 x g, 30 min) and washed at least two times with buffer before use.

Antibody Production and Purification
Rabbit antiserum Ure2-#2 has been described previously (1, 5). Although raised against Ure2p, it is very specific for the N-terminal domain (approximately residues 1–80) (Supplementary Fig. S2). Rabbit antiserum Ure2–3C specific for the C-terminal domain of Ure2p (Ure2p64–354) was affinity-purified using recombinant Ure2p (10). To refine the specificity to Ure2p90–354, antibodies recognizing the region 64–89 were removed by passage over a 1.0-ml nickel-nitrilotriacetic acid Superflow column (Qiagen) previously loaded with His6-Ure2p1–89 in 6 M GdnHCl (to prevent filament formation) and then changed back to 20 mM Tris/HCl, 200 mM NaCl, pH 8.0.

Proteinase K Assays
0.5 ml of a protein solution at 4–8 mg/ml in 20 mM Tris/HCl, 200 mM NaCl, pH 8.0, was incubated with proteinase K (Promega) (0.1 mg/ml, 30 units/mg) for 16–20 h at 37 °C. The insoluble material was collected by centrifugation at 25,000 x g for 30 min at 4 °C and washed by resuspension in 20 mM Tris/HCl, 200 mM NaCl, pH 8.0, followed by centrifugation as above, three times. The pellet was then dissolved in either 6 M GdnHCl for mass spectrometry or in 10 M urea, 2% SDS with 5 min boiling for SDS-PAGE.

For less rigorous digestion, the same protein sample was digested with 10 µg/ml proteinase K and incubated at 37 °C. Small aliquots were taken at various times and analyzed directly on SDS-PAGE gels.

SDS-PAGE and Western Blotting
Samples were dissolved in 10 M urea, 2% SDS, and the urea concentration was kept as high as possible throughout the subsequent procedures. SDS-PAGE was performed on 10–20% gradient gels (ICN Biomedicals) with Tris-Tricine buffer. Gels were stained with Colloidal Coomassie (ICN Biomedicals) or used in Western blots to transfer proteins to a polyvinylidene difluoride membrane (Millipore). The membrane was treated with 0.2 M NaOH for 30 min before being treated with the standard procedure. This step proved to be necessary for reproducible staining of the proteinase K digests on Western blots (32).

Liquid Chromatography-Mass Spectrometry
LC-MS was performed on an HP1100 LC-MSD system (Agilent Technologies, Palo Alto, CA), consisting of a binary pump, a degasser, an autosampler, an absorbance detector, an HP1100 MSD, and a Chem-Station for data acquisition and processing. Mass spectra were acquired in positive-ion mode, scanning from 300 to 1700 m/z every 4 s. A Zorbax SB-C3 reversed phase column (150 mm x 2.1 mm) equipped with a guard column was used for chromatographic separation. The flow rate was 0.2 ml/min. Separation was done with a gradient of 5% acetic acid in water to acetonitrile at 40 °C. Proteins and peptides were detected using absorption at 280 nm and MS signal in the positive mode. However, peptides from the N-terminal domain of Ure2p did not show a signal at 280 nm, because they contain neither tryptophan nor tyrosine. Apomyoglobin was used as a calibration standard.

Electron Microscopy
CTEM—For negative staining, samples were adsorbed onto freshly glow-discharged carbon-coated grids, rinsed with water, and stained with 1% uranyl acetate. Micrographs were recorded on a Zeiss EM 902 (Leica, Deerfield, IL). For cryo-EM, drops were adsorbed to holey carbon films, blotted, vitrified, and imaged on a CM200-FEG microscope (FEI, Mahwah, NJ) (33).

STEM—Dark-field micrographs of freeze-dried specimen were recorded at 1.0 or 2.0 nm/pixel at the Brookhaven STEM resource. Mass measurements were performed by interactive analysis of the micrographs using PIC programs (34). Tobacco mosaic virus particles served as a mass standard (131.4 kDa/nm) (35). Some samples were stained with 2% methylamine vanadate (Nanoprobes, Stony Brook, NY).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Filament Formation Requires a Covalently Attached Prion Domain—Ure2p, Ure2p1–89, and the fusion proteins Ure2p1–65-barnase, Ure2p1–65-carbonic anhydrase (CA), Ure2p1–65-glutathione S-transferase (GST), Ure2p1–65-green fluorescent protein (GFP), and Ure2p1–80-GFP were expressed in E. coli and purified. Under the same conditions of incubation, all constructs formed filaments as detected by negative staining EM (Fig. 1). The filaments were either single (Type A) or paired (Type B) (15). The width of single filaments increased with subunit molecular mass from ~14 nm for Ure2p1–65-barnase (19.6 kDa) to ~25 nm for Ure2p (41.8 kDa) (Fig. 1, bottom right panel). In no case did the C-terminal moiety, i.e. the appended protein without the Ure2p prion domain, form filaments under these conditions.



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  		FIG. 1.
Electron micrographs of negatively stained filaments of Ure2p constructs, before and after digestion with proteinase K. Filaments were digested with 0.1 mg/ml proteinase K at 37 °C for 16 h, and the insoluble fraction was collected, washed, and stained with uranyl acetate. Each panel compares undigested (left) and digested (right) filaments. Arrows mark single (non-bundled) 4-nm filaments. Bar = 100 nm. At the bottom right, filament diameters are plotted against subunit masses. Open circles are from digested filaments, and filled circles are from undigested filaments. The colors denote different constructs: red, Ure2p; black, Ure2p1–89; light green, Ure2p1–80-GFP; blue, Ure2p1–65-barnase; orange, Ure2p1–65-GFP; yellow, Ure2p1–80-CA; and green, Ure2p1–65-GST.

 

It has been shown that the prion domain Ure2p1–65 promotes the polymerization of full-length Ure2p to form cofilaments (1). Accordingly, we tested whether it has the same effect on the C-terminal domain alone. This experiment was performed with several prion domain constructs (Ure2p1–65, Ure2p1–80, and Ure2p1–89) mixed with C-terminal domain constructs Ure2p81–354 or Ure2p65–354, or with Ure2p as a positive control. By SDS-PAGE, Ure2p was detected in the insoluble fraction with each prion domain construct, and filaments were observed by negative staining. However, neither Ure2p65–354 nor Ure2p81–354 was detected in the insoluble fraction for any prion domain construct. These data show that a covalently attached prion domain is needed for the protein to enter filaments.

Filaments of Ure2p and Fusion Constructs All Have Protease-resistant Core Fibers—All filament preparations were subjected to extensive digestion with proteinase K (100 µg/ml at 37 °C for 16 h). In each case, the reaction left sedimentable material consisting of much thinner filaments (Fig. 1). These filaments had the same diameter (4 nm) and tendency to aggregate into bundles (Fig. 1), whether they were derived from Ure2p or any of the fusion protein filaments; in these properties, they resemble polymers of Ure2p1–65 (1) and Ure2p1–89 (Fig. 1). Similarly, thin filaments were observed in earlier work after proteinase K digestion of cofilaments of Ure2p and Ure2p1–65 (1), with the qualification that those digestions were less rigorous and some of the resulting filaments had patches of greater width between 4-nm segments.3 Congo Red staining of the digested material produced apple-green birefringence, indicating that they are amyloid (Supplementary Fig. S1). Again, this is a property that they share with Ure2p1–65 filaments (1, 32).

The Protease-resistant Core Fibers Consist of N-terminal Peptides—To determine which fragments of Ure2p make up the thin filaments, this material was analyzed by SDS-PAGE (Fig. 2). With Coomassie Blue staining, all constructs yielded a fuzzy band at ~10 kDa (Fig. 2). The specific affinity of these bands for the dye is about 2-fold lower than that of intact Ure2p, as determined by quantitative gel analysis. All of these bands reacted positively with a polyclonal antiserum that specifically recognizes Ure2p1–80; conversely, an antibody specific for Ure2p90–354 did not detect this material (Fig. 2).



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  		FIG. 2.
SDS-PAGE and Western blot analysis of rigorously digested Ure2p filaments. Either without (-) or after (+) digestion with proteinase K (100 µg/ml, 37 °C, 16 h), filaments were collected, solubilized with 10 M urea (a necessary step for the protein to enter the gel reproducibly), and analyzed by SDS-PAGE. Proteins were detected with Coomassie Blue (Coom) or Western blotting with antibodies specific for Ure2p1–89 (N-AB), Ure2p90–354 (C-AB), or GFP (GFP-AB). The numbers on the vertical axis denote the masses (kDa) of marker proteins. The bottom panel shows fluorescence and gel analysis of Ure2p1–80-GFP filaments without (-) and after (+) digestion. In each case, the soluble (sup) and insoluble (pel) fractions were separated by centrifugation (40,000 x g, 30 min). Mass spectrometry of the soluble fraction after digestion showed that the proteinase K-resistant part of GFP consists of GFP2–231, GFP3–231, and GFP2–232 (~70, 15, and 15%, respectively).

 

In contrast, digestions of soluble Ure2p with proteinase K for 16 h produced very little insoluble material, and no filaments were detected by EM. Moreover, no band was visible on Coomassie-stained gels or Western blots using either the C- or N-terminal antibody (data not shown).

If the proteinase K digestion was less rigorous (10 µg/ml at 37 °C for up to 1 h) and soluble material was not separated from insoluble material, then Ure2p filaments yielded similar digestion patterns to those obtained from soluble Ure2p or C-terminal domain (Ure2p65–354) (Fig. 3). The main bands revealed by Coomassie staining correspond to fragments of the C-terminal domain, as identified by Western blots (Fig. 3). The N-terminal fragments are inconspicuous, because fragments of the C-terminal domain run in the same region of the gel (7–10 kDa) and stain more strongly with Coomassie Blue.



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  		FIG. 3.
SDS-PAGE and Western blot analysis of less rigorously digested Ure2p filaments. Samples from a proteinase K digest (10 µg/ml, 37 °C) were stopped after the times (min) indicated above the lanes and analyzed by SDS-PAGE with Coomassie Blue staining (Coomassie) or on Western blots with antibodies specific for Ure2p1–89 (N-term AB) or Ure2p90–354 (C-term AB). The numbers on the vertical axis denote the masses (kDa) of marker proteins. Mass spectrometry of the same samples led to the assignment of the digestion pattern to the Ure2p fragments indicated on the left side of the upper panel.

 

We further characterized these earlier stages of proteinase K digestion of Ure2p filaments by mass spectrometry and gel analysis (Fig. 3). The first cut is between the N-terminal and C-terminal domains around residue 95 and produces a C-terminal fragment of ~30 kDa. This fragment is not stable, however, and a second cut follows around residue 285 in the so-called cap region, a flexible loop that is unique to Ure2p among GST-like proteins (18), producing relatively stable fragments of ~20 and ~8 kDa (Fig. 3). These fragments are subject to fraying at the ends as detected by mass spectrometry and by slight shifts to lower masses on SDS gels. Fraying is observed from residue 95 up to 120 and in the cap region from residue 285 down to 272 and up to 294. Further cleavage then takes place at sites that appear to be protected initially but become exposed as a result of prior cuts and fraying: cutting around residue 150 generates another relatively stable fragment at ~14 kDa and cutting around residue 230 generates a fragment at ~9 kDa.

When fusion protein filaments were subjected to similar, less rigorous, proteinase K digestion (10 µg/ml at 37 °C for up to 1 h) without separation of insoluble material, each construct gave a characteristic pattern on SDS gels with Coomassie staining, indistinguishable from that obtained from unpolymerized protein (data not shown). Again, because of the weak staining of Ure2p N-domain, these patterns were dominated by fragments of the C-terminal moieties, in this case the appended proteins. GST and CA were digested slightly more slowly than Ure2p but were completely degraded after 16 h. However, GFP and barnase were rather resistant to proteinase K. Most of the GFP was still native and functional but detached from the filaments and found in the soluble fraction (Fig. 2, bottom panel). Some barnase was also found in native and active form in the post-digestion supernatant. These observations indicate that proteinase K simply shaves off GFP and barnase from the core fibers, cutting only in the region between the prion domain and GFP or barnase. In fact, consistent with this scenario, a fast cut was observed between the prion domain and the proteins fused to it for each species of fusion protein filament.

Analysis of Residual Core Fibers by Mass Spectrometry—We identified the peptide fragments that make up the 4-nm filaments by liquid chromatography-mass spectrometry (LC-MS). To solubilize this material, 6 M GdnHCl was used. A typical LC-MS run for a Ure2p filament digest is shown in Fig. 4. The number of peptides identified ranged from 10 (Ure2p1–65-CA and Ure2p1–89) to 42 (Ure2p1–80-GFP) (Supplementary Table SI). The five most abundant peptides are listed in Table I for each filament species. In contrast, no peptides were detected in the "insoluble" fraction after soluble Ure2p was digested in the same way. Proteinase K is a rather unspecific enzyme, which tends to complicate identification of peptides generated by it. Nevertheless, a consistent solution for these data, with similar peptides in corresponding peaks of the LC elution profiles, could be obtained only by assigning all of the peptides to the N-terminal domain of Ure2p (Fig. 4). Furthermore, for Ure2p filaments, the possibility of major contributions from the C-terminal domain was excluded by the Western blots (see above). For Ure2p1–80-GFP and Ure2p1–65-GFP, the possibility that GFP might contribute to this material was excluded by the fact that this protein resisted digestion throughout the experiment (Fig. 2, bottom panel). A similar albeit less compelling argument applies to Ure2p1–65-barnase.



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  		FIG. 4.
Liquid-chromatography mass-spectrometry of proteinase K-digested Ure2p filaments. The bottom panel shows the HPLC profile of a run with digested Ure2p as measured in the positive ion mass signal. Absorbance at 280 nm did not produce a signal, because N-terminal peptides derived from Ure2p lack Trp and Tyr. The mass spectra correspond to the indicated peaks of the HPLC chromatogram. For the second spectrum, a fit is shown. Mass/charge peaks corresponding to the same peptide are shown in the same color, and the number of positive charges is indicated, e.g. A:5 (red) indicates the peak corresponding to peptide A (5744.5 Da) with five positive charges.

 

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  		TABLE I
Most abundant peptides identified by mass spectrometry after rigorous proteinase K digest of filaments of Ure2p constructs and reisolation of thin filaments Supplementary Table SI lists all detected peptides.

 

Similar experiments carried out with trypsin, a more specific protease, showed complete cleavage at all predicted sites in soluble Ure2p but substantial protection of sites in the N-terminal domain in Ure2p filaments (Table II). Some insoluble C-terminal peptides were produced by trypsin digestion and transiently on proteinase K digestion of soluble or filamentous Ure2p, but these aggregates were not filamentous.


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  		TABLE II
N-terminal trypsin fragments from filaments of Ure2p and soluble Ure2p

 

Filaments of All Prion Domain-containing Constructs Have the Same Number of Subunits per Unit Length—Determinations of mass per unit length impose a strong constraint on possible packing schemes in protein filaments and illuminate relationships between filaments of different kinds. We measured this parameter directly on individual filaments by darkfield STEM imaging of unstained filaments of Ure2p and the various fusion proteins (Fig. 5). As with negative staining (Fig. 1), both Type A (single) and Type B (double) filaments were readily distinguished in the STEM micrographs (Fig. 5). Type B filaments are obtained mainly with constructs that have small C-terminal domains (Ure2p1–65-barnase) or none (Ure2p1–65 and Ure2p1–89). With larger C-terminal domains (Ure2p1–80-GFP and Ure2p), Type B filaments are infrequent. Ure2p1–65-GST is an exception to this trend; we observed few Type B filaments for this construct, although its C-terminal moiety is smaller than those of Ure2p1–65-CA and Ure2p1–65-GFP, both of which produced significant amounts of Type B filaments (Fig. 5).



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  		FIG. 5.
STEM mass analysis of filaments of Ure2p constructs. The upper panel shows dark-field micrographs. Type A (single) and Type B (paired) filaments are indicated with A and B. Bar = 100 nm. The lower panels show the mass per unit length measurements for all constructs. Ordinates represent numbers of measurements. The curves represent Gaussian distributions fitted to the data. In some cases, more than one peak was observed, corresponding to Type A and Type B filaments, respectively, except for Ure2p1–65 whose two peaks correspond to Type B filaments and paired Type B filaments (four Type A filaments). Color coding of different constructs are as in Fig. 1, plus gray, Ure2p1–65. At the bottom right, the average mass per unit length for each construct is plotted against subunit mass, Type A (full circles) and Type B (open circles). The linear relationship indicates that all filaments have the same number of subunits per unit length.

 

Ure2p1–65 filaments have a strong tendency to bundle (Ref. 1; confirmed in this study), and mass measurements for this construct were restricted to well separated filaments. Two peaks are apparent in the resulting distribution (Fig. 5), whose means are related by a factor of two. Because filaments in the lower density peak had a typical Type B appearance, we conclude that the higher density peak represents paired Type B filaments (four Type A filaments). Essentially no single Type A filaments were present in this preparation.

Mass per unit length data for Type A filaments are plotted against the masses of the component subunits in Fig. 5 (bottom right). Because Type B filaments consist of two laterally associated Type A filaments, we halved their mass per unit length values for inclusion in this plot (open symbols). The data show a linear relationship; all constructs, including Ure2p, have 2.2 ± 0.1 subunits per nm of filament (or one subunit per 0.45 ± 0.03 nm), as calculated from the slope of the regression line (R2 = 0.92). The finding, that Ure2p and Ure2p1–65-GST, which are dimers in solution, have the same number of subunits per unit length in filaments as the monomeric proteins Ure2p1–80-GFP, Ure2p1–65-GFP, Ure2p1–65-barnase, and Ure2p1–65-CA, agrees with the report by Bousset et al. (26) that they convert to monomers on entering the filamentous state.

High Resolution Negative Staining and Cryo-EM of Filaments Reveal a Core Fiber Surrounded by Globular Domains— STEM observation of vanadate-stained specimens has been found to give highly informative images of some proteins (e.g. Refs. 36 and 37). We used this technique to visualize filaments of Ure2p and some fusion proteins (Fig. 6). These images reveal two main features: 1) In specimens that showed good stain penetration, a thin core fiber was clearly visible (arrows in Fig. 6). Its diameter is 3.9 ± 0.5 nm (n = 100). 2) Individual globules are well resolved along the filament surfaces. They have diameters of 4.9 ± 0.7 nm (n = 111) for Ure2p, and similar values were obtained from the fusion protein filaments except Ure2p1–65-barnase, whose globules are appreciably smaller at 2.3 ± 0.5 nm (n = 58). These figures tally well with the average diameters of the Ure2p95–354 monomer (~5 nm) and the various appendages, GFP, CA, and GST (3–5 nm) and barnase (2–3 nm), as estimated from their crystal structures.



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  		FIG. 6.
Filaments visualized by dark-field STEM of vanadate-stained specimens (upper panels: bar = 50 nm) and cryo-EM of vitrified specimens (lower panels; bar = 150 nm). Left, filaments of Ure2p; right, filaments of Ure2p1–80-GFP. Asterisks indicate tobacco mosaic virus reference particles. Arrows in both panels indicate regions where the core fiber is clearly visualized.

 

Cryo-electron microscopy of frozen-hydrated specimens gives optimal preservation of native structure (38), and, because the contrast arises from the density difference between protein and buffer, stain infiltration is not a problem. Cryo-micrographs of Ure2p and Ure2p1–80-GFP filaments, shown in Fig. 6, also reveal the "thin core filament" motif seen in the vanadate-stained specimens. In both sets of observations, the core filament diameter matches those of the thin filaments by proteinase K digestion (Fig. 1) or assembled from Ure2p1–65 (1). Visualization of this structure in intact filaments implies that the 4-nm filaments seen after proteinase K digestion are residual protease-resistant entities.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Models of Ure2p Filaments—Two models have been proposed for the basic architecture of Ure2p filaments. According to the "amyloid backbone" model (1, 10), they have an amyloid core of polymerized N-terminal prion domains, surrounded by the C-terminal functional domains. The latter are in native or near-native conformation (15). This model was initially based on the observed ability of the prion domain to form amyloid filaments in the absence of the C-terminal domain, and the relative accessibility of C-terminal domains in filaments to proteases and antibodies. In contrast, Bousset et al. (26, 27) have proposed that filament architecture is based on interactions between the C-terminal domain and the N-terminal domain and that filaments have no content of amyloid. For brevity, we refer to this as the C-N model. This proposal was based on their inability to detect changes in secondary structure or proteinase K-resistance upon filament formation (26, 39) or alterations in filament diameter upon treating them with proteinase K (26). In this study, we have used EM and mass spectrometry to investigate Ure2p filaments and filaments modified in various ways. The results illuminate filament structure while discriminating decisively between the two hypotheses.

Implications of Fusion Protein Filament Formation—The observation that fusions of the Ure2p N-terminal domain with four different proteins all form filaments (15) is a specific prediction of the "amyloid backbone" hypothesis, although the failure of any given fusion protein to form filaments would not rule it out, because steric blocking or solubility considerations might intervene. According to the C-N hypothesis, for such filaments to form, there would have to be some motif present on all four fused appendages that emulates the putative interaction surface on the C-terminal domain of Ure2p in its ability to interact with the N-terminal domain to form filaments, or all five proteins should fortuitously have patches with the same property. Given the diversity of their amino acid sequences and folds, we see this proposition as being extremely unlikely.

Susceptibility to Proteolysis in the Soluble and Filamentous States—Similarity of the breakdown patterns of soluble Ure2p and filamentous Ure2p exposed to proteinase K, as seen on Coomassie Blue-stained gels, was interpreted as evidence that the protein conformation is unchanged (39). However, we show here that the prominent bands in question derive exclusively from the C-terminal domain (Fig. 3), which is not expected to undergo a conformational change in either model (15, 26). Accordingly, we focused our attention on fragments containing the N-terminal domain as identified by specific antibody. We found that, when filaments are digested, the N-terminal domain contributes a diffuse band at 7–10 kDa that survives protracted proteolysis (Figs. 2 and 3). This band is not obtained from soluble Ure2p, but very similar bands are produced from fusion protein filaments. It is noteworthy that the N-terminal domain is the most proteolytically sensitive portion of soluble Ure2p and the least sensitive portion of filamentous Ure2p, providing strong evidence that this domain undergoes a change in conformation between the two states.

The Universal 4-nm Residual Filament—Filaments of Ure2p and of the various fusion proteins were digested with proteinase K and examined by EM. Although the original filaments ranged in diameter from 14 to 25 nm, depending on the construct, the residual filaments were ~4 nm across in each case. Moreover, the residual filaments are locally straight and smooth-sided and have a strong tendency to bundle, regardless of source. In all these properties they resemble amyloid filaments of polymerized Ure2p1–65 (1). These observations imply that the residual filaments are composed of some element that is common to all. This requirement is readily met in the backbone hypothesis in which that element is the N-terminal domain. In contrast, it is no more apparent from the C-N hypothesis why these filaments should be trimmed to the same diameter than that they should be assembled in the first place.

We observe widths of 20–25 nm for Ure2p filaments by negative staining TEM, vanadate staining STEM, and cryo-TEM of vitrified specimens (Figs. 1 and 6, see also (32)). These figures are consistent with the observed dimensions of the components; i.e. a 4-nm core, plus globular domains of 4- to 5-nm diameter connected by a linker of 2–4 nm. In fact, the cryo-EM data give the diameter of Ure2p filaments as ~20 nm, and the slightly larger figures obtained by negative staining suggest a small degree of spreading when the filaments are dried.

In another report (26), which used atomic force microscopy, no thinning of Ure2p filaments was observed following proteinase K treatment. In their experiments, filament widths, measured as the spacing between the half-height points on either side, were reported to be 40–60 nm. Their height was given as 7–10 nm. We suspect that low spatial resolution in the x-y plane of atomic force microscopy as used in this study exaggerated the filament widths and failed to discriminate between intact filaments, partially digested filaments, and bundles of core filaments.

Composition and Properties of the 4-nm Residual Filaments—According to mass spectrometry, the insoluble material produced by proteinase K digestion of filaments was in each case composed of fragments of the N-terminal domain. This outcome is predicted by the "amyloid core" hypothesis but not the C-N hypothesis.

The amount of material recovered in this fraction was near-quantitative. For example, with Ure2p filaments, ~15% of the initial protein mass was recovered. If the median fragment mass is the same as that of the major product (residues 14–65), this corresponds to ~100% of the initially 25-nm filaments being reduced to 4-nm filaments.

4-nm Residual Filaments Are Not Reassembly Products—It is not unknown for protein fragments produced by proteolysis to aggregate or even crystallize. However, strong indications that the 4-nm filaments are not reassembly products are served by three sets of observations on intact filaments, i.e. the vanadate-stained images, the cryo-EM data, and the STEM measurements of mass per unit length. The former two sets of images explicitly revealed, within intact filaments, core fibers with the same diameter of 4 nm as residual (post-digestion) filaments. These observations of an internal backbone confirm a central tenet of the "amyloid backbone" model. In contrast, the C-N model (26) makes no mention of internal filaments.

According to the STEM measurements of mass per unit length (Fig. 5), a given length of filament contains the same number of subunits in each case. Again, this is the outcome predicted by the backbone model, given the same or similar stacking of the N-terminal domains in each case. The C-N model, on the other hand, offers no explanation for these data. The repeat length per subunit along the filament is also germane. The observed spacing of 0.45 nm correlates with an important repeat distance in amyloid structures, the spacing between two {beta}-strands.

High Resolution Negative Staining—STEM micrographs of negatively stained specimens have been found to be very informative for some proteins (e.g. Refs. 28 and 40), and we have found vanadate to be a valuable stain for this method (36). Contrast is enhanced by the STEM dark-field mode, and image quality is further improved by stabilizing the specimen at low temperature (here, -196 °C). Moreover, at focus, the STEM contrast transfer function (41) gives near-uniform transmittal of all frequencies, whereas conventional transmission electron microscopy (CTEM) bright-field images must be defocussed to generate sufficient contrast at low and moderate frequencies, which introduces complications with phase reversals and amplification of noise at higher frequencies. Such images have relatively high resolution and a signal-to-noise ratio that can only be achieved in CTEM by image averaging, which is not readily applicable to poorly ordered filaments

The Core Fibers Are Surrounded by Globular Domains—In conventional TEM images of prion filaments stained with uranyl acetate, filament diameter increases with the size of the appendage (Fig. 1). In vanadate-stained STEM micrographs (Fig. 6, top), the peripheral material is seen to consist of globules whose diameters match those expected for appendage monomers, explaining the observed variations in filament diameter. These images also detect an internal core fiber whose diameter (4 nm) matches those of residual filaments left after treating filaments of all kinds with proteinase K. The "core fiber plus globular appendages" motif is also apparent in cryomicrographs (Fig. 6, bottom), although their lower signal-to-noise ratio makes it difficult to make precise measurements of globule diameters.

Direct observation of globules of the appropriate size for the folded proteins supports prior evidence from retained functional properties that the appended moieties have their native conformations (15, 26). The "backbone" model incorporates these features. The C-N model also anticipates globules of the appropriate size for folded monomers but not a core fiber.

The Core Fiber Is an Amyloid Filament with High {beta}-Sheet Content—We found that, in addition to their high resistance to proteolysis, residual filaments show apple-green birefringence upon Congo Red staining. Both properties are hallmarks of amyloid. Moreover, they contain much the same polypeptides as Ure2p1–65 filaments and are morphologically indistinguishable from them. The latter filaments have been shown by two independent spectroscopic techniques, Raman (1) and Fourier transform infrared spectroscopy (32), to have high contents (~60%) of {beta}-structure. Pending similar measurements on core filaments, current data strongly suggest that they share this property and accordingly are amyloid by this criterion as well.

Secondary Structures of Soluble and Filamentous Ure2p— Conformational changes have been a major feature of models of prionogenesis (e.g. Refs. 42–44), and there has been considerable interest in comparing the structures of prion proteins in their soluble and filamentous states. Although there are crystal or NMR structures for several such proteins or fragments thereof in their soluble versions, there are no high resolution structures for filaments. Consequently, such comparisons have tended to rely on spectroscopic estimates of secondary structure.

In this context, the amyloid backbone model anticipates a conformational change in the N-terminal domain of Ure2p when filaments are formed and little or no change in the C-terminal part. The C-N model envisages no change whatever (26). Whereas spectroscopic determinations of secondary structure have been unambiguous for Ure2p1–65, there has been considerable variability for other related specimens (1, 26, 27, 32). Ure2p94–354 provides a useful reference point, because it has been solved by crystallography (19, 45). Its structure is mainly {alpha}-helical (47%) with little {beta}-sheet content (5–6%). Fourier transform infrared spectroscopy determinations yielded 33% {alpha}-helix in crystals (27) and 63% in the soluble state (26), in both cases imputing >20% of {beta}-sheet. Our Raman spectroscopic measurements correctly measured the {alpha}-helical content (48–50%) of C-terminal domain, but its {beta}-sheet estimate (16–21%) was too high,4 and we have encountered similarly large discrepancies between Amide I- and Amide III-based determinations (1). Thus it appears that there are quite large experimental uncertainties in the spectroscopic measurements to date.

On the other hand, the secondary structure change predicted by the amyloid backbone model is quite small and depends on (i) the fraction of the N-terminal domain (residues 1–90) that changes its conformation; (ii) its conformation in the soluble protein. If, as an extreme case, Ure2p1–65 were to have 60% {beta}-sheet content in filaments and zero in the soluble state, this would correspond to an 11% change overall. On the other hand, this domain may have a substantial content of {beta}-sheet in unassembled Ure2p but nevertheless be acutely sensitive to proteolysis, in which case the expected change in secondary structure upon filament formation would be much lower.

Architecture of Ure2p Filaments—We conclude that, in Ure2p filaments, the prion domains form a central core in amyloid conformation surrounded by the C-terminal domains in largely native conformation. The central part of the prion domain, from about residue 14 to 65 or 70 exhibits the highest degree of protease resistance. The segment from 71 to 94 does not appear to be integral part of the amyloid backbone: its amino acid composition resembles that of the "inner core," but it may simply serve as a linker to attached domains. Two further observations point in this direction: 1) When Ure2p1–89 filaments are treated with proteinase K, the portion distal to residue 65 or so is removed (Table I), suggesting that it occupies an exposed peripheral location; 2) to date, our electron micrographs do not yield rich helical diffraction patterns, implying that the C-terminal domains are not particularly well ordered. The so-called M domain in Sup35p (46) might provide a similar function for the [PSI+] yeast prion system but the extent of the protease-resistant part of Sup35p amyloid is not yet known.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Figs. S1 and S2 and Table SI. Back

Present address: Nabi Biopharmaceuticals, Rockville, MD 20852. Back

** To whom correspondence should be addressed: Bldg. 50, Rm. 1517, 50 South Dr., MSC 8025, Bethesda, MD 20892-8025. Tel.: 301-496-0132; Fax: 301-480-1191; E-mail: Alasdair_Steven{at}nih.gov.

1 The abbreviations used are: GST, glutathione S-transferase; CTEM, conventional transmission electron microscopy; STEM, scanning transmission electron microscopy; GFP, green fluorescent protein; CA, carbonic anhydrase; GdnHCl, guanidine hydrochloride; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; LC-MS, liquid chromatography-mass spectrometry; EM, electron microscopy. Back

2 Amyloid is defined as a filamentous, protease-resistant, conformational state of a protein that is rich in {beta}-structure and binds the dye Congo Red, producing apple-green birefringence. The term "amyloid filament" tends to be used not only for filaments consisting entirely of amyloid but also for any filament that has a significant amyloid content. Back

3 K. L. Taylor, N. Cheng, R. B. Wickner, and A. C. Steven, unpublished results. Back

4 R. Williams, K. L. Taylor, R. B. Wickner, and A. C. Steven, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Lewis Pannell, David Anderson, and Sonja Hess for expert performance of the LC-MS; Dr. Vladislav Speransky for performing preliminary STEM mass measurements; and Dr. Herman Edskes for plasmid pH469 and Todd Cassese for helpful suggestions.



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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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