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• W2020140985 abstract "Article9 February 2006free access Molecular chaperones and the assembly of the prion Sup35p, an in vitro study Joanna Krzewska Corresponding Author Joanna Krzewska Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette Cedex, France Search for more papers by this author Ronald Melki Corresponding Author Ronald Melki Search for more papers by this author Joanna Krzewska Corresponding Author Joanna Krzewska Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette Cedex, France Search for more papers by this author Ronald Melki Corresponding Author Ronald Melki Search for more papers by this author Author Information Joanna Krzewska 1 and Ronald Melki 1Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette Cedex, France *Corresponding authors: Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette Cedex, France. Tel.: +33 169 823 503; Fax: +33 169 823 129; E-mails: [email protected] or E-mail: [email protected] The EMBO Journal (2006)25:822-833https://doi.org/10.1038/sj.emboj.7600985 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The protein Sup35 from Saccharomyces cerevisiae possesses prion properties. In vivo, a high molecular weight form of Sup35p is associated to the [PSI+] factor. The continued propagation of [PSI+] is highly dependent on the expression levels of molecular chaperones from the Hsp100, 70 and 40 families; however, so far, their role in this process is unclear. We have developed a reproducible in vitro system to study the effects of molecular chaperones on the assembly of full-length Sup35p. We show that Hsp104p greatly stimulates the assembly of Sup35p into fibrils, whereas Ydj1p has inhibitory effect. Hsp82p, Ssa1p and Sis1p, individually, do not affect assembly. In contrast, Ssa1p together with either of its Hsp40 cochaperones blocks Sup35p polymerization. Furthermore, Ssa1p and Ydj1p or Sis1p can counteract the stimulatory activity of Hsp104p, by forming complexes with Sup35p oligomers, in an ATP-dependent manner. Our observations reveal the functional differences between Hsp104p and the Hsp70–40 systems in the assembly of Sup35p into fibrils and bring new insight into the mechanism by which molecular chaperones influence the propagation of [PSI+]. Introduction The epigenetic factor [PSI+] in the yeast Saccharomyces cerevisiae (Cox, 1965) is due to the prion properties of the protein Sup35 (Wickner, 1994). In the prion state, insoluble aggregates of Sup35p (or eRF3, eukaryotic translation termination factor) give rise to [PSI+] cells with altered translation termination, as manifested by an increased tendency of ribosomes to read through nonsense ochre stop codons (Cox et al, 1988). ‘Weak’ and ‘strong’ [PSI+] variants have been described that possess different mitotic stabilities and stop codon suppression capacities, possibly as the consequence of different prion structures (Derkatch et al, 1996). In vivo, Sup35p aggregates associated to [PSI+] have increased resistance to proteolysis, while in [psi−] cells, Sup35p is soluble and proteinase K sensitive (Patino et al, 1996; Paushkin et al, 1996). The Sup35p-containing complexes characteristic of the [PSI+] determinant are composed of Sup35p and associated proteins. These complexes vary in size in different [PSI+] variants (Kryndushkin et al, 2003). The full-length Sup35p assembles spontaneously in vitro into protein fibrils with an average diameter of 11–17±1–2 nm that are over 1 μm long. The region critical for assembly into fibrils is the N-terminal domain extending from amino-acid residue 1 to 123 (Sup35pN). This domain alone or together with the middle domain (Sup35pNM) of the protein (amino-acid residues 124–253) forms fibrils, which exhibit the characteristics of amyloids in that they (i) have increased resistance to proteolysis, (ii) bind the dye Congo red and exhibit the 4.7 Å reflection in X-ray fiber diffraction images, and (iii) assemble in a cooperative manner, following a nucleation growth process that can be greatly facilitated with preformed fibrils, thus mimicking in a test tube the invasive propagation of [PSI+] (Glover et al, 1997; King et al, 1997; Serio et al, 2000). The majority of in vitro studies on the assembly of Sup35p have been performed using its prion-determining region (Sup35pNM) as fibrils made of Sup35pNM assembled in vitro propagate [PSI+] when reintroduced in yeast cells (King and Diaz-Avalos, 2004; Tanaka et al, 2004). However, there is no evidence that Sup35p is degraded upon its aggregation in [PSI+] cells. In vivo, the continued propagation of yeast prions is highly dependent on the expression levels of a number of molecular chaperones (Tuite and Cox, 2003). Indeed, members of the Hsp100, Hsp70 and Hsp40 protein families modulate the propagation of [PSI+], but their roles at the molecular level are subject to debate. The effect of Hsp104p on the propagation of [PSI+] has been actively studied, since it was shown that either inactivation or overproduction of the molecular chaperone caused the loss of [PSI+] (Chernoff et al, 1995). Hsp104p has been reported to act both on the soluble and insoluble high molecular weight forms of Sup35p in vivo. When Hsp104p activity is decreased, large aggregates of Sup35p accumulate within the cells and the faithful propagation of [PSI+] is impaired (Wegrzyn et al, 2001), while the overexpression of Hsp104p is accompanied by an increase in the amount of soluble Sup35p (Paushkin et al, 1996). Two alternative models have been proposed to explain the effect of Hsp104p on [PSI+] propagation. As Hsp104p, in coordination with the Hsp70–40 chaperone system, is involved in the solubilization and refolding of aggregated proteins (Parsell et al, 1994; Glover and Lindquist, 1998), it was proposed that Hsp104p fragments prion particles into smaller oligomers, thus allowing their successful propagation to daughter cells upon division (Kushnirov and Ter-Avanesyan, 1998). However, the mode of action of Hsp104p in the disaggregation of heat-aggregated proteins and the potential mechanistic differences with prion aggregate fragmentation are not well characterized. Alternatively, Hsp104p is believed to be primarily required for an inherently unstable protein-folding event, necessary for prion formation (Patino et al, 1996; Serio and Lindquist, 2000). Molecular chaperones belonging to the Hsp70 and Hsp40 families, although not strictly required, have also been shown to influence [PSI+] propagation. Mutations in the ATPase domain of Ssa1p, as well as in residues important for the interaction with Hsp40, impair [PSI+] propagation (Jung et al, 2000; Jones and Masison 2003). Unbalanced levels of overexpressed Ydj1p and Ssa1p were shown to cause prion loss in either ‘weak’ native or chimeric [PSI+] strains (Kushnirov et al, 2000). While elevated levels of Ssa1p counteract [PSI+] loss upon overexpression of Hsp104p (Newnam et al, 1999), other members of the Hsp70 family, Ssb1 and Ssb2, enhance [PSI+] curing under similar conditions and influence the frequency of spontaneous [PSI+] formation in [psi−] cells (Chernoff et al, 1999). Most studies of the role of molecular chaperones in the propagation of [PSI+] have been performed in vivo where the effects of a molecular chaperone can be counterbalanced by another chaperone. The limited in vitro studies that have been carried out employed the Sup35pNM fragment (Inoue et al, 2004; Shorter and Lindquist, 2004). Here we document, for the first time by in vitro techniques, the effect of molecular chaperones from the Hsp40, Hsp70, Hsp90 and Hsp100 families, individually and in concert, on the assembly of the full-length Sup35p. Our observations reveal the functional differences between Hsp104p and the Hsp70–40 systems in the assembly of Sup35p into protein fibrils, provide a rationale for the effect of the different molecular chaperones on prion formation and bring new insight into the mechanism by which molecular chaperones interplay influences the propagation of [PSI+]. Results Full-length Sup35p is a suitable substrate for prion assembly studies Recombinant, renatured Sup35NM fragment is used in the vast majority of studies performed in vitro on the assembly of Sup35p. To make sure that the conclusions derived from our study of the interaction of molecular chaperones with Sup35p would not be subject to limitations due, in particular, to differences in the surfaces of Sup35pNM and full-length Sup35p, we chose to use soluble, full-length protein. For this purpose, we developed a reproducible protocol for the purification of recombinant, full-length Sup35p, using nondenaturing conditions (see Materials and methods). The full-length Sup35p assembles into high molecular weight oligomers (Figure 1), as does its NM fragment (Glover et al, 1997). Three phases can be distinguished in the assembly reaction. A lag phase, where nucleation occurs, followed by an elongation phase preceding a steady state (plateau). The assembly reaction is highly cooperative as the lag phase can be shortened significantly by increasing the Sup35p concentration (Figure 1A). The nucleation phase is the limiting step in the assembly reaction since it is abolished by the addition of preformed Sup35p fibrils (Figure 1B). Fibrils obtained under our experimental conditions are long (several μm), 25 nm wide, rigid and twisted, and have a high tendency to bundle (Figure 1C). All the experiments presented in this study were performed at a protein concentration of 4–6 μM. Under these conditions, the lag phase extends over 6–8 h and a steady state is reached within 35 h. Figure 1.Assembly of the full-length Sup35p into protein fibrils. (A) Dependence of the lag time preceding assembly on the concentration of Sup35p. Soluble Sup35p, 4 μM (•) and 6 μM (▪) was incubated without shaking at 10°C in the assembly buffer. Aliquots were removed at regular time intervals and assayed using the thioflavin T binding assay described in the Materials and methods section. (B) The lag time preceding assembly is the rate-limiting reaction in the assembly process. Assembly of soluble Sup35p (6 μM) in the absence (•) or the presence (▴) of preformed Sup35p fibrils (1 μM). AU, arbitrary units. (C) Negative-stained electron micrographs of fibrils made from the full-length Sup35p. Bar, 0.3 μm. Download figure Download PowerPoint Effects of individual yeast molecular chaperones on the assembly of Sup35p into high molecular weight oligomers The effects of Hsp104p (Hsp100), Hsp82p (Hsp90), Ssa1p (Hsp70), Ydj1p and Sis1p (Hsp40) on Sup35p assembly were monitored using thioflavin T binding. Molecular chaperones were tested both in sub- and equimolar monomer concentrations (Figure 2). Hsp82p, Ssa1p and Sis1p in sub- or equimolar concentrations have no effect on Sup35p assembly (Figure 2A and B, respectively). In contrast, Ydj1p and Hsp104p strongly influenced the reaction (Figure 2C). While Ydj1p has a strong inhibitory effect, Hsp104p promotes Sup35p assembly. Indeed, the thioflavin T fluorescence intensities at steady state in the presence of Ydj1p and Sup35p at a ratio of 1:10 and 1:1 are, respectively, two- and four-fold lower than in its absence (Figure 2C). This effect is not due to a generic Hsp40 molecular chaperone activity, as it is not observed with Sis1p (Figure 2A), another member of the Hsp40 family. In the presence of an equimolar concentration of Hsp104p, the thioflavin T fluorescence in the elongation phase and at steady state is 2.5-fold higher than in its absence and the lag phase is reduced by a factor of 2 (Figure 2C). Figure 2.Assembly of Sup35p in the presence of individual molecular chaperones. (A, B) Time courses of Sup35p (4 μM) assembly at 10°C in the absence of molecular chaperones (•) and in the presence of submolar, 0.4 μM (open symbols), and equimolar (filled symbols) concentrations of Ssa1p (□, ▪), Sis1p (▵, ▴) and Hsp82p (⋄, ♦). (C) Time courses of Sup35p (4 μM) assembly in the absence (•) and in the presence of submolar 0.4 μM (▾, ♦) and equimolar (▴, ▪) concentrations of Ydj1p and Hsp104p, respectively. All assembly reactions were monitored by thioflavin T binding. AU, arbitrary units. Download figure Download PowerPoint Functional interplay of molecular chaperones during the assembly of Sup35p To further characterize the influence of molecular chaperones on Sup35p assembly, the following combinations of members from the Hsp40, Hsp70 and Hsp100 families were used: Hsp40/Hsp70, Hsp40/Hsp100, Hsp70/Hsp100 and Hsp40/Hsp70/Hsp100 (Figure 3A–D). Remarkably, in the presence of Ssa1p and Ydj1p or Sis1p (Hsp70/Hsp40 combinations), Sup35p assembly was totally inhibited (Figure 3A). Although we have shown that Ydj1p by itself possesses a significant inhibitory activity, neither Sis1p nor Ssa1p alone display an appreciable ability to inhibit assembly (Figure 2B and C). Thus, Ssa1p acquires an inhibitory capacity only in the presence of its Hsp40 cochaperones. We next studied whether Hsp40 or Hsp70 chaperones influence the stimulatory activity of Hsp104p. When Hsp104p is incubated with Sis1p (Figure 3B), the Sup35p assembly reaction is stimulated to the same extent as in the presence of Hsp104p alone (Figure 2C). In contrast, the assembly reaction of Sup35p in the presence of Hsp104p and Ydj1p (Figure 3B) resembles that obtained in the presence of Ydj1p alone (Figure 2C). This suggests that Ydj1p counteracts the stimulatory activity of Hsp104p, whereas Sis1p has no such effect. Also, the assembly reaction of Sup35p in the presence of Hsp104p and Ssa1p is similar to that in the presence of Hsp104p alone, thus indicating that Ssa1p has a marginal effect on the stimulatory effect of Hsp104p (Figure 3C). Figure 3.Functional interplay of molecular chaperones during the assembly of Sup35p. (A–D) Time courses of Sup35p (4 μM) assembly at 10°C in the absence (•) and (A) the presence of equimolar amounts of Ydj1p/Ssa1p (▴) and Sis1p/Ssa1p (□), (B) Ydj1p/Hsp104p (▴) and Sis1p/Hsp104p (♦), (C) Ssa1p/Hsp104p (▴) and (D) Ydj1p/Ssa1p/Hsp104p (▴), Sis1p/Ssa1p/Hsp104p (♦). Assembly was monitored by thioflavin T binding. AU, arbitrary units. Download figure Download PowerPoint Finally, having demonstrated a total inhibition of Sup35p assembly by the Hsp70–40 system, we examined whether these chaperones might counteract the stimulatory activity of Hsp104p. In a manner similar to what is observed in reactions where Hsp40 family members are associated to Hsp70 in the absence of Hsp104 (Figure 3A), the assembly of Sup35p into protein fibrils is abolished in reactions containing the following combinations Ydj1p/Ssa1p/Hsp104p or Sis1p/Ssa1p/Hsp104p (Figure 3D). This indicates that when combined, Hsp70–40 complexes over-ride the effect of Hsp104p. The capacity of Ssa1p to inhibit Sup35p fibril formation is greatly stimulated by its cochaperones Sis1p and Ydj1p The results presented above suggest that Sis1p and Ydj1p enhance the ability of Ssa1p to inhibit Sup35p assembly. To further quantify this effect, Sup35p (4 μM) was assembled in the presence of a constant amount of Ssa1p (4 μM) and increasing concentrations of Ydj1p or Sis1p, and the amount of fibrillar Sup35p at steady state determined using the thioflavin T binding assay. We demonstrate that the addition of submolar amounts of Ydj1p (Figure 4A) or Sis1p (Figure 4B) to Ssa1p causes a dramatic inhibition of Sup35p assembly (50% inhibition is observed in the presence of 0.11 or 0.14 μM Ydj1p and Sis1p, respectively). This inhibition does not correspond to an additive effect of individual molecular chaperones. Sis1p alone has no effect (Figure 4B) and a 10-fold higher concentration of Ydj1p is required to reach comparable assembly inhibition levels (50% inhibition is observed in the presence of 1 μM Ydj1p; Figure 4A). Similar inhibition levels are observed upon lowering the concentration of Ssa1p to 0.5 μM and Sis1p and Ydj1p, respectively, to 0.05 and 0.1 μM (not shown), indicating that Ssa1p acts on Sup35p assembly in a substoichiometric manner. Figure 4.Sis1p and Ydj1p stimulate the Sup35p sequestering capacity of Ssa1p. Proportion of fibrillar Sup35p (4 μM) at steady state in the presence of Ssa1p (4 μM) and increasing concentrations (0–8 μM) of (A) Ydj1p (▪) and (B) Sis1p (▪). The thioflavin T fluorescence intensity values at steady state were averaged over three measurements taken at 30, 40 and 50 h following the onset of assembly and expressed as a fraction of the fluorescence of fibrillar Sup35p (4 μM) in the presence of Ssa1p alone. Control reactions where the effect of increasing concentrations of Ydj1p (• in A) and Sis1p (• in B) on the amount of fibrillar Sup35p in the absence of Ssa1p are also presented. Download figure Download PowerPoint Quantitative analysis of the interplay between Hsp104p and the Hsp70–40 systems on Sup35p assembly To explore the functional interplay between Hsp104p and Hsp70–40 family members on the assembly of Sup35p into fibrils, we measured the fraction of assembled Sup35p (4 μM) at steady state in the presence of a constant, nonlimiting concentration of Hsp104p (6 μM) and increasing concentrations of Ssa1p and Ydj1p or Sis1p, at an Hsp70:40 ratio of 5:1. This ratio is commonly used in in vitro studies of the disaggregating and folding properties of the Hsp70–40 system (Goloubinoff et al, 1999; Mogk et al, 1999; Krzewska et al, 2001). The fraction of polymerized Sup35p decreases from 100% (arbitrary value) in the presence of Hsp104p alone to about 75% in the presence of a 12-fold excess of Hsp104p over Ssa1p and either Hsp40 cochaperone (Table I). Increased inhibition is observed at lower Hsp104p/Hsp70–40 ratios, and over 85% inhibition takes place using an Hsp104p/Hsp70–40 ratio of 1.5, with equimolar concentrations of Hsp70 and Hsp40 (Table I). Table 1. Quantitative analysis of the effect of the interplay between Hsp104p and Hsp70–40 systems on Sup35p assembly Concentration of Hsp104p, Hsp70, Hsp40 (μM) Ratio of Hsp104p/Hsp70–40 Fraction of assembled Sup35p in the presence of Ssa1p and Ydj1p Fraction of assembled Sup35p in the presence of Ssa1p and Sis1p 6, 0, 0 ∞ 100 100 6, 4, 0.8 1.5 41 52 6, 2, 0.4 3 50 59 6, 1, 0.2 6 57 68 6, 0.5, 0.1 12 74 77 6, 4, 4 — 10 15 The fraction of assembled Sup35p (4 μM) at steady state in the presence of a constant, nonlimiting concentration of Hsp104p (6 μM) and increasing concentrations of Ssa1p and Ydj1p or Sis1p at an Hsp70/40 ratio of 5:1 are listed. A control measurement at an Hsp70/40 ratio of 1:1 in the presence of Hsp104p (6 μM) is also presented (bottom line). The thioflavin T fluorescence intensity values at steady state were averaged over three measurements made at 30, 40 and 50 h following the onset of assembly and expressed as a fraction of the fluorescence of fibrillar Sup35p (4 μM) in the presence of Hsp104p (6 μM). The results were obtained from two independent measurements. The standard deviation is ±5%. Ydj1p maintains Sup35p in a soluble state and counteracts the ability of Hsp104p to promote fibril growth The interaction of Sup35p oligomeric species that form at different stages of assembly with Ydj1p and Hsp104p, separately or together, was further characterized in a quantitative manner using a sedimentation assay. The resulting pellets and supernatants were subjected to SDS–PAGE analysis (Figure 5A). As expected, during the early stage of assembly, in the absence or presence of molecular chaperones, 100% of Sup35p (as determined from densitometric analysis using the software NIH Image) is in the supernatant fractions (Figure 5A). At steady state, in the absence of molecular chaperones, over 85% of Sup35p is in the pellet fraction (Figure 5A). In contrast, Sup35p remains in the supernatants of samples containing Ydj1p alone (Figure 5B) or Ydj1p together with Hsp104p (Figure 5E), whereas 70% of Sup35p is in the pellet fraction in the presence of Hsp104p alone (Figure 5C). These observations are consistent with our kinetic measurements and indicate that Ydj1p maintains Sup35p in a soluble form, thus counteracting Hsp104p activity. Figure 5.Effect of Ydj1p and Hsp104p on Sup35p assembly. (A–E and H) Sedimentation analysis of soluble and fibrillar Sup35p (4 μM) (A) in the absence and (B) the presence of Ydj1p (4 μM), (C) Hsp104p (6 μM), (D) Hsp104pTRAP (6 μM) and (E) Ydj1p and Hsp104p (4 and 6 μM, respectively), in the presence of ATP, GTP and an ATP-regenerating system. Aliquots from each sample were subjected to ultracentrifugation at the time indicated. The resulting supernatants (S) and pellets (P) were analyzed by SDS–PAGE. (F, G) Negative-stained electron micrographs of fibrils made from full-length Sup35p (4 μM) in the presence (F) and the absence (G) of Hsp104 (12 μM). Bar, 0.5 μm. Length distribution analysis of 400 fibrils made in the presence and 200 fibrils made in the absence of Hsp104p are presented below the electron micrographs. (H) SDS–PAGE analysis of supernatants (S) and pellets (P) of insoluble fractions of Sup35p fibrils from the second half of the elongation phase in the presence of Hsp104p and AlF4− (S3 and P3), Hsp104p, AlF4− and Ssa1p (S4 and P4), Hsp104p, AlF4− and Ydj1p (S5 and P5) and Hsp104p, AlF4−, Ssa1p and Ydj1p (S6 and P6). Control reactions where Hsp104p alone (S1 and P1) or in the presence of Sup35p fibrils and the absence of AlF4− (S2 and P2) were subjected to the same sedimentation conditions are shown. Download figure Download PowerPoint Shorter Sup35p fibrils are generated in the presence of Hsp104p, yet no disassembling activity of fibrillar Sup35p is observed Although Sup35p assembly is stimulated by Hsp104p, we never detect the latter chaperone upon sedimentation of the fibrils, which implies that the interaction, if any, is short-lived or weak. It is possible that the binding capacity of Hsp104p to Sup35p fibrils is lost with time as ATP hydrolysis and nucleotide exchange on Hsp104p following interaction leads to the dissociation of Hsp104p from the fibrils. Thus, we carried out our sedimentation assay using elongating fibrils and either wild-type Hsp104p blocked in its ADP-Pi transition state using the phosphate structural analog, aluminum fluoride (AlF4−), or an Hsp104p mutant, termed Hsp104pTRAP, that binds ATP but is unable to hydrolyze it, because two conserved Glu residues in the Walker B motif of both nucleotide-binding domains have been replaced with Gln (E285Q/E687Q) (Bösl et al, 2005). The ATPase activity of wild-type Hsp104p is markedly reduced (5.5-fold) in the presence of AlF4−, consistent with the binding of AlF4− to Hsp104p in its transition state (Supplementary Figure 1). Nevertheless, the latter form of Hsp104p is not found bound to Sup35p fibrils (Figure 5H). Furthermore, the addition of Ssa1p, Ydj1p or both chaperones to Sup35p fibrils assembled in the presence of Hsp104p and AlF4− has no additional effect, as all chaperones are found in the supernatant (Figure 5H). Similarly, we were not able to reveal a stable interaction between Hsp104p and Sup35p using the variant Hsp104pTRAP (Figure 5D). Finally, we were unable to detect a stable, physical interaction between soluble, full-length Sup35p and Hsp104p by other classical methods, for example, crosslinking with glutaraldehyde (Supplementary Figure 2) and gel filtration chromatography with both wild-type Hsp104p and the Hsp104TRAP mutant (data not shown). As examined by electron microscopy, Sup35p fibrils at steady state obtained in the absence and presence of Hsp104p appear morphologically indistinguishable at the resolution used. Strikingly however, in the presence of Hsp104p, the fibrils are significantly shorter than those assembled in its absence (Figure 5F and G, upper panels). This is confirmed by a length distribution analysis (Figure 5F and G, lower panels). In addition, while Sup35p fibrils tend to stack laterally into large bundles, very few form in the presence of Hsp104p (not shown), presumably due to shorter fibrillar length. Finally, the length of Sup35p fibrils preassembled in the absence of Hsp104p does not change upon further incubation for up to 12 h with Hsp104p (8 μM), as illustrated by the electron micrograph (Supplementary Figure 3). Also, the incubation of Sup35p fibrils, assembled in the absence or presence of Hsp104p for up to 12 h with Hsp104p, Ssa1p and Ydj1p, or Ssa1p and Ydj1p does not lead to fibril depolymerization or solubilization, as the amount of insoluble Sup35p, estimated by our sedimentation assay remains constant (Supplementary Figure 4). Altogether, this ensemble of data seems to indicate that the full-length Sup35p fibrils that can be sedimented are not a target for Hsp104p activity. Hsp104 neither changes the length of fibrils nor solubilizes them, even with the assistance of Ssa1p and Ydj1p. Hsp104p acts on Sup35p oligomers that form at the early stages of assembly To better understand how Hsp104p affects the time course of Sup35p assembly, Hsp104p was added at various times following the onset of the assembly reaction. When Hsp104p is added at time zero, or during the lag phase, this phase shortens and the thioflavin T fluorescence level increases two-fold at steady state (Figure 6); a similar increase is observed upon addition of Hsp104p during the elongation phase (time 20 h). Last, a slow and limited increase in fluorescence is observed when Hsp104p is added at steady state to mature fibrils (time 40 h). However, the fluorescence intensity never reaches the level measured when Hsp104p is added at the earlier stages of assembly. We conclude from these observations that Hsp104p acts on Sup35p oligomers that form at the early stages of assembly. Figure 6.Hsp104p promotes the formation of assembly competent Sup35p oligomeric species. Effect of Hsp104p (6 μM) addition on the nucleation (▪), the elongation (▴) and the steady state (♦) phases of Sup35p (4 μM) assembly. The arrows indicate the time where Hsp104p was added to the assembly reaction, time 0, 20 and 40 h. The assembly of Sup35p in the absence of Hsp104p is shown as a control (•). Assembly was carried out at 10°C and monitored using thioflavin T binding. AU, arbitrary units. Download figure Download PowerPoint Fibrils assembled in the presence of Hsp104p show increased seeding activity To further characterize the stimulatory effect of Hsp104p on Sup35p assembly, we compared the seeding activities of increasing amounts of Sup35p fibrils preassembled in the absence of Hsp104p to those of a constant amount of fibrils assembled in the presence of increasing amounts of the molecular chaperone. If Hsp104p promotes Sup35p nucleation, the number of seeds generated in the presence of high Hsp104p concentrations would be higher and therefore have an increased seeding capacity as compared to that obtained in the presence of low Hsp104p concentrations or in its absence, at a constant Sup35p concentration. We find that the apparent rate of Sup35p assembly is accelerated by the addition of increasing amounts of preformed Sup35p fibrils (Figure 7A). As expected, this rate varies linearly with the amount of added seeds in the range 0.5–2 μM (Figure 7B). Importantly, fibrils preassembled in the presence of increasing amounts of Hsp104p (6–18 μM) also show a linear increase in seeding activity, yet significantly higher (Figure 7C), suggesting that more seeds are actually present in the latter samples. When fibrils preassembled in the presence of increasing amounts of Hsp104p are used as seeds, the final concentration of Hsp104p in the elongation reaction is 1.25–3.6 μM. To check whether Hsp104p affects the elongation reaction, the seeding capacities of fibrils assembled without Hsp104p supplemented or not with 3.6 μM of the chaperone were compared. Similar elongation rates are observed in the two reactions (data not shown), thus indicating that the increased nucleation activity of fibrils assembled in the presence of Hsp104p is not due to Hsp104p on its own but to the fibrils. Figure 7.Seeding properties of Sup35p fibrils assembled in the presence and absence of Hsp104p. (A) The elongation rates of increasing concentration of fibrillar Sup35p (0.5, ▪; 1, • and 2 μM, ♦), and that of 1 μM fibrillar Sup35p made in the presence of 6 (○) and 18 μM (⋄) Hsp104p were measured in the presence of 5 μM Sup35p. Control reactions show the assembly of Sup35p in the absence of added seeds (□) and upon addition of 3.6 μM of Hsp104p (▴). The elongation reactions were carried out at 10°C in assembly buffer and were monitored using thioflavin T binding. AU, arbitrary units. (B) Linear dependence between the rate of elongation and the amount of fibrils preformed in the absence of Hsp104p, at a constant soluble Sup35p concentration (5 μM). (C) Linear dependence between the seeding activity of Sup35p fibrils (1 μM) and the amount of Hsp104p present in the reaction where they were assembled. Panels B and C are based on the experiment described in panel A. Download figure Download PowerPoint Comparison of the elongation rates of seeds made in the presence and the absence of Hsp104p allowed us to estimate the increase in the number of elongation sites due to Hsp104p activity. We show that the apparent assembly rate of 1 μM seeds assembled in the presence of Hsp104p (6–18 μM) is two- to three-fold greater than that of 1 μM seeds assembled in the absence of Hsp104p (Figure 7C). This rate is even greater than that of seeds (2 μM) made in the absence of the" @default.
• W2020140985 created "2016-06-24" @default.
• W2020140985 creator A5008122548 @default.
• W2020140985 creator A5084373973 @default.
• W2020140985 date "2006-02-09" @default.
• W2020140985 modified "2023-10-16" @default.
• W2020140985 title "Molecular chaperones and the assembly of the prion Sup35p, an in vitro study" @default.
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