Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p

] prion-forming proteins are, respectively, the translational termination factor Sup35 and the yet poorly characterized Rnq1 protein that is rich in glutamines and asparagines. The prion domain of Rnq1 (RnqPD) polymerizes more readily in vitro than the full-length protein. As is typical for amyloidogenic proteins, the reaction begins with a lag phase, followed by exponential growth. Seeding with pre-formed aggregates significantly shortens the lag. A generic antibody against pre-amyloid oligomer inhibits the unseeded but not the selfseeded reaction. As revealed by electron microscopy, RnqPD polymerizes predominantly into spherical species that eventually agglomerate. We observed infrequent fiber-like structures in samples taken at 4 h of polymerization, but in overnight samples SDS treatment was required to reveal fibers among agglomerates. Polymerization reactions in which RnqPD and the prion domain of Sup35 (Sup35NM) cross-seed each other proceeded with a shortened lag that only depends weakly on the protein concentration. Cross-seeded Sup35NM fibers appear to sprout from globular RnqPD aggregates as seen by electron microscopy. RnqPD spherical aggregates appear to associate with and, later occlude, Sup35NM seed fibers. Our kinetic and morphological analyses suggest that, upon cross-seeding, the aggregate provides the surface on which oligomers of the heterologous protein nucleate their subsequent amyloid formation.Prions are currently viewed as proteinaceous genetic elements because of their potential to encode, propagate, and discretely modify phenotypes produced by an infectious alternative conformational state of the prion protein (1). In this state, proteins form amyloid aggregates characterized by a rigid structure that is stabilized by inter ␤-sheet bonds (2). These aggregates can convert molecules of the prion-forming protein from the normal conformation into the prion form. Propagation of the prion state in vivo is based upon fragmentation of already formed aggregates that results in multiplication of prion seeds. In mammals, the prion PrP Sc is the causative agent of a series of neurodegenerative disorders, e.g. CreutzfeldtJakob disease in humans, bovine spongiform encephalopathy, scrapie of sheep, etc. (for review see Ref.3).Several prions have been characterized in the yeast Saccharomyces cerevisiae (for review see Refs. 4 and 5), and a growing body of evidence suggests that there are interactions between them (for review see Ref. 6). These interactions range from the ability of one prion to facilitate the appearance of another to incompatibility between some (7-9).Here we focus on two yeast prions, [PSI ϩ ] and [PIN ϩ ], as a model for prion-prion interactions. [PSI ϩ ] is composed of the alternatively folded translational termination factor Sup35 (10, 11) and is characterized by an increased level of readthrough of stop codons (nonsense suppression) because of the sequestration of Sup35 into prion aggregates (12). A Sup35 fragment spanning amino acids 1-253 (Sup35NM) 3 forms fibers in vitr...

Section: Discussionmentioning

confidence: 87%

Visualization of Aggregation of the Rnq1 Prion Domain and Cross-seeding Interactions with Sup35NM

Vitrenko¹,

Gracheva²,

Richmond

et al. 2007

“…R17F, R17L, and R17C and its truncated variants were purified immediately before use; buffer exchange was performed rapidly using a desalting column (HiTrap desalting, GE Healthcare), and the proteins were then used directly for experiments because of their tendency to form fibrils extremely rapidly, even during the processes of freezing and thawing. Other proteins were prepared in an identical manner and used immediately or were dialyzed at 4°C, flash-frozen, and stored at Ϫ80°C in Buffer A; the latter leads to slightly faster fibril formation presumably because of the presence of small quantities of preformed seeds (15,39). Therefore, for direct comparison of mutants (i.e.…”

Section: Methodsmentioning

confidence: 99%

Disulfide Bond Formation Significantly Accelerates the Assembly of Ure2p Fibrils because of the Proximity of a Potential Amyloid Stretch

Fei

Perrett

2009

Aggregation of the Ure2 protein is at the origin of the [URE3]prion trait in the yeast Saccharomyces cerevisiae. The N-terminal region of Ure2p is necessary and sufficient to induce the [URE3] phenotype in vivo and to polymerize into amyloid-like fibrils in vitro. However, as the N-terminal region is poorly ordered in the native state, making it difficult to detect structural changes in this region by spectroscopic methods, detailed information about the fibril assembly process is therefore lacking. Short fibril-forming peptide regions (4 -7 residues) have been identified in a number of prion and other amyloid-related proteins, but such short regions have not yet been identified in Ure2p. In this study, we identify a unique cysteine mutant (R17C) that can greatly accelerate the fibril assembly kinetics of Ure2p under oxidizing conditions. We found that the segment QVNI, corresponding to residues 18 -21 in Ure2p, plays a critical role in the fast assembly properties of R17C, suggesting that this segment represents a potential amyloid-forming region. A series of peptides containing the QVNI segment were found to form fibrils in vitro. Furthermore, the peptide fibrils could seed fibril formation for wild-type Ure2p. Preceding the QVNI segment with a cysteine or a hydrophobic residue, instead of a charged residue, caused the rate of assembly into fibrils to increase greatly for both peptides and full-length Ure2p. Our results indicate that the potential amyloid stretch and its preceding residue can modulate the fibril assembly of Ure2p to control the initiation of prion formation.The [URE3] phenotype of Saccharomyces cerevisiae arises because of conversion of the Ure2 protein to an aggregated propagatable prion state (1, 2). Ure2p contains two regions: a poorly structured N-terminal region and a compactly folded C-terminal region (3, 4). The N-terminal region is rich in Asn and Gln residues, is highly flexible, and is without any detectable ordered secondary structure (4 -6). This region is necessary and sufficient for prion behavior in vivo (2) and amyloid-forming capacity in vitro (5, 7), so it is referred to as the prion domain (PrD). 2 The C-terminal region has a fold similar to the glutathione S-transferase superfamily (8, 9) and possesses glutathionedependent peroxidase activity (10). Upon fibril formation, the N-terminal region undergoes a significant conformational change from an unfolded to a thermally resistant conformation (11), whereas the glutathione S-transferase-like C-terminal domain retains its enzymatic activity, suggesting that little conformational change occurs (10, 12). Ure2p fibrils show various morphologies, including variations in thickness and the presence or absence of a periodic twist (13-16). The overall structure of the fibrils imaged by cryoelectron microscopy suggests that the intact fibrils contain a 4-nm amyloid filament backbone surrounded by C-terminal globular domains (17).It is widely accepted that disulfide bonds play a critical role in maintaining protein stability (18 -21) an...

“…(4,6). Using AFM, a variety of globular and fibrillar species have been observed during the time course of fibril formation (8,45,46). AFM was used to monitor the morphology of aggregates generated by Ure2 in both the presence and absence of Ydj1.…”

Section: In Vivo Effects Of Co-chaperone Overexpression On [Ure3]mentioning

confidence: 99%

Hsp40 Interacts Directly with the Native State of the Yeast Prion Protein Ure2 and Inhibits Formation of Amyloid-like Fibrils

Lian

Zhao

Zhang

et al. 2007

Ure2 is the protein determinant of the [URE3] prion phenotype inHere we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.The epigenetic factor [URE3] in the yeast Saccharomyces cerevisiae represents the prion form of the protein Ure2 (1). Like the mammalian prion, the heritable [URE3] phenotype is conveyed by a structural change in its protein determinant to an aggregated form (1, 2). Ure2 is a 354-amino acid homodimeric protein consisting of a relatively flexible and protease-sensitive N-terminal region (ϳ90 amino acids) and a globular C-terminal region (3-5). The N-terminal region is required for its prion properties in vivo (2) and to form amyloid-like filaments in vitro (6 -8). However, deletion of the N-terminal region has no detectable effect on the stability or folding of the protein in vitro (9). The C-terminal region, for which the crystal structure has been determined in both apo (5, 10) and glutathione-bound (11) forms, shows structural similarity to glutathione transferases (GSTs), 3 and is necessary and sufficient for its regulatory function in vivo: Ure2 interacts with the transcription factor Gln3, allowing control of nitrogen catabolite repression and blocking the uptake of poor nitrogen sources in the presence of a good nitrogen source (12, 13). In addition, Ure2 possesses glutathione-dependent peroxidase (GPx) activity, which is maintained upon formation of fibrillar aggregates, indicating that the C-terminal globular domains of Ure2 retain their native structure within the fibrils (14).Ydj1 from S. cerevisiae is a molecular chaperone of the type I Hsp40 family and is involved in multiple functions, including import of proteins into mitochondria, secretion of mating pheromones, and regulation of the activity of the cytoplasmic Hsp70s (15-17). Ydj1 can bind to nonnative polypeptides and pair with Hsp70 Ssa proteins to prevent aggregation and facilitate refolding of denatured p...