Molecular Chaperones and the Assembly of the Prion Ure2p in Vitro
2008; Elsevier BV; Volume: 283; Issue: 23 Linguagem: Inglês
10.1074/jbc.m800728200
ISSN1083-351X
AutoresJimmy Savistchenko, Joanna Krzewska, Nicolas Fay, Ronald Melki,
Tópico(s)Neurological diseases and metabolism
ResumoThe protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3]is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled full-length Ure2p and Ure2p-(94–354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3]. The protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3]is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled full-length Ure2p and Ure2p-(94–354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3]. The [URE3] trait, discovered in the early 70s (1Lacroute F. J. Bacteriol. 1971; 106: 519-522Crossref PubMed Google Scholar), is an inheritable prion factor in the yeast Saccharomyces cerevisiae (2Wickner R.B. Science. 1994; 264: 566-569Crossref PubMed Scopus (1073) Google Scholar). In a manner similar to what is observed for the vertebrate prion PrP, the [URE3] prion state is associated with a change in the solubility of the protein Ure2 (3Masison D.C. Wickner R.B. Science. 1995; 270: 93-95Crossref PubMed Scopus (329) Google Scholar).Similarly to other proteins with prion properties from S. cerevisiae, Ure2p is a two-domain protein. The physical boundary between the two domains of this 354-amino acid polypeptide is amino acid residue 94 (4Thual C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The asparagine-, glutamine-, serine-, and threonine-rich N-terminal domain of Ure2p (62% of amino acid residues) is crucial for prion propagation and flexible (4Thual C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 5Perrett S. Freeman S.J. Butler P.J. Fersht A.R. J. Mol. Biol. 1999; 290: 331-345Crossref PubMed Scopus (79) Google Scholar, 6Thual C. Bousset L. Komar A.A. Walter S. Buchner J. Cullin C. Melki R. Biochemistry. 2001; 40: 1764-1773Crossref PubMed Scopus (75) Google Scholar), whereas its C-terminal domain is compactly folded and mainly α-helical (7Bousset L. Belrhali H. Janin J. Melki R. Morera S. Structure. 2001; 9: 39-46Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 8Umland T.C. Taylor K.L. Rhee S. Wickner R.B. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1459-1464Crossref PubMed Scopus (95) Google Scholar). The latter domain binds glutathione (9Bousset L. Belrhali H. Melki R. Morera S. Biochemistry. 2001; 40: 13564-13573Crossref PubMed Scopus (60) Google Scholar), has glutathione peroxidase activity (10Bai M. Zhou J.M. Perrett S. J. Biol. Chem. 2004; 279: 50025-50030Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and is sufficient to regulate nitrogen metabolism in bakers' yeast cells (11Mitchell A.P. Magasanik B. Mol. Cell. Biol. 1984; 4: 2758-2766Crossref PubMed Scopus (97) Google Scholar, 12Courchesne W.E. Magasanik B. J. Bacteriol. 1988; 170: 708-713Crossref PubMed Scopus (129) Google Scholar, 13Coschigano P.M. Magasanik B. Mol. Cell. Biol. 1991; 11: 822-832Crossref PubMed Scopus (222) Google Scholar, 14Cooper T.G. FEMS Microbiol. Rev. 2002; 26: 223-238Crossref PubMed Google Scholar).In vitro, at neutral pH, soluble Ure2p spontaneously forms long, twisted fibrils (4Thual C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 15Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1343Crossref PubMed Scopus (262) Google Scholar, 16Jiang Y. Li H. Zhu L. Zhou J.M. Perrett S. J. Biol. Chem. 2004; 279: 3361-3369Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 17Ranson N. Stromer T. Bousset L. Melki R. Serpell L.C. Protein Sci. 2006; 15: 2481-2487Crossref PubMed Scopus (17) Google Scholar). It is widely believed that similar fibrils form in vivo and are at the origin of the [URE3] trait (18Speransky V.V. Taylor K.L. Edskes H.K. Wickner R.B. Steven A.C. J. Cell Biol. 2001; 153: 1327-1336Crossref PubMed Scopus (74) Google Scholar).In vivo, the continued propagation of yeast prions is highly dependent on the expression levels of a number of molecular chaperones. Indeed, members of the Hsp100, Hsp70, and Hsp40 protein families modulate the propagation of the [PSI+], [PIN+], and [URE3] traits. As for [PSI+] propagation (19Chernoff Y.O. Lindquist S.L. Ono B. Inge-Vechmotov S.G. Liebman S.W. Science. 1995; 268: 880-884Crossref PubMed Scopus (913) Google Scholar), Hsp104p is required for maintenance of [URE3] (20Moriyama H. Edskes H.K. Wickner R.B. Mol. Cell. Biol. 2000; 20: 8916-8922Crossref PubMed Scopus (238) Google Scholar). The over-expression of the Hsp70 protein Ssa1 cures [URE3] (21Schwimmer C. Masison D.C. Mol. Cell. Biol. 2002; 22: 3590-3598Crossref PubMed Scopus (157) Google Scholar). A mutation in the peptide binding domain of another Hsp70 family member, Ssa2p, abolishes [URE3] propagation (22Roberts B.T. Moriyama H. Wickner R.B. Yeast. 2004; 21: 107-117Crossref PubMed Scopus (58) Google Scholar). In addition, [URE3] propagation is affected by mutations in the ATP binding domain of both Ssa1p and Ssa2p (23Loovers H.M. Guinan E. Jones G.W. Genetics. 2007; 175: 621-630Crossref PubMed Scopus (34) Google Scholar). Finally, Ydj1p overexpression leads to the loss of [URE3] (20Moriyama H. Edskes H.K. Wickner R.B. Mol. Cell. Biol. 2000; 20: 8916-8922Crossref PubMed Scopus (238) Google Scholar). It is worth noting that in vivo, the effect of a molecular chaperone can be counterbalanced by another chaperone. Furthermore, the concentrations of molecular chaperones and Ure2p cannot be determined with accuracy. It is, therefore, very difficult to access the exact role of each molecular chaperone in [URE3] propagation. It is for this reason that a number of in vitro studies have been carried out since 2006 to document the exact role of each molecular chaperone on yeast prion assembly (24Krzewska J. Melki R. EMBO J. 2006; 25: 822-833Crossref PubMed Scopus (89) Google Scholar, 25Shorter J. Lindquist S. Mol. Cell. 2006; 23: 425-438Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 26Lian H.Y. Zhang H. Zhang Z.R. Loovers H.M. Jones G.W. Rowling P.J. Itzhaki L.S. Zhou J.M. Perrett S. J. Biol. Chem. 2007; 282: 11931-11940Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar).Here we report the effect of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families (Table 1) and the chaperonin CCT/Tric 3The abbreviations used are: CCT, cytosolic chaperonin containing TCP1; Tric, TCP1 ring complex; IAEDANS, 5-[2-(iodoacetamido)ethylamino]naphthalene-1-sulfonic acid. 3The abbreviations used are: CCT, cytosolic chaperonin containing TCP1; Tric, TCP1 ring complex; IAEDANS, 5-[2-(iodoacetamido)ethylamino]naphthalene-1-sulfonic acid. individually and in concert on the assembly of full-length Ure2p. Although Hsp82p, Ssa1p, Ydj1p, and Sis1p inhibit to different extents Ure2p assembly, Hsp104p promotes and the chaperonin CCT/Tric does not affect assembly. The nature of the high molecular weight Ure2p species that form in the presence of the different molecular chaperones is described. The nucleotide dependence of the interaction between full-length Ure2p and the compactly folded C-terminal domain of the protein (Ure2p-(94–354)) and Ssa1p alone or in the presence of its co-chaperone Ydj1p and Hsp104p is particularly documented. Our results indicate that Ure2p-molecular chaperone complex formation is nucleotide- and domain-dependent.TABLE 1Identity of the molecular chaperones used throughout this studyMolecular chaperone familyHsp40Hsp70Hsp60Hsp90Hsp100Member usedSis1p, Ydj1pSsa1pCCT/TricHsp82pHsp104p Open table in a new tab MATERIALS AND METHODSExpression and Purification of Ure2p—Recombinant full-length Ure2p, Ure2p-(94–354), and the variants Ure2pC355 and Ure2p-(94–354)C355 were produced as previously described (4Thual C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 6Thual C. Bousset L. Komar A.A. Walter S. Buchner J. Cullin C. Melki R. Biochemistry. 2001; 40: 1764-1773Crossref PubMed Scopus (75) Google Scholar, 27Fay N. Inoue Y. Bousset L. Taguchi H. Melki R. J. Biol. Chem. 2003; 278: 30199-30205Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Protein concentrations were determined spectrophotometrically (HP 8453 diode array spectrophotometer, Hewlett-Packard) using an extinction coefficient of 0.67 mg·cm–2 at 280 nm and molecular masses of 40.2 and 30 kDa for full-length Ure2p and Ure2p-(94–354), respectively. The proteins were stored at –80 °C in buffer A (75 mm KCl, 20 mm Tris, pH 7.5, 1 mm EGTA, 1 mm dithiothreitol) at a concentration of 2.8 mg/ml.Expression and Purification of Other Proteins—Sup35p, Hsp104p, Ssa1p, Ydj1p, and Sis1p were overexpressed either in Escherichia coli or S. cerevisiae and purified and stored as described (24Krzewska J. Melki R. EMBO J. 2006; 25: 822-833Crossref PubMed Scopus (89) Google Scholar). CCT was isolated from rabbit reticulocyte lysate (28Melki R. Batelier G. Soulie S. Williams Jr., R.C. Biochemistry. 1997; 36: 5817-7826Crossref PubMed Scopus (78) Google Scholar), and purified Hsp82p (29Prodromou C. Piper P.W. Pearl L.H. Proteins Struct. Funct. Genet. 1996; 25: 517-522PubMed Google Scholar) was generously provided by Dr. C. Combeau (Sanofi-Aventis Pharma, Vitry, France). All monomeric protein concentrations were measured using their absorbance at 280 nm and their calculated extinction coefficients.Assembly of Ure2p into Fibrils—Soluble full-length wild-type Ure2p was assembled at 8 °C in buffer A supplemented with 2 mm ATP unless specified. The assembly reaction was monitored using thioflavin T binding (30McParland V.J. Kad N.M. Kalverda A.P. Brown A. Kirwin-Jones P. Hunter M.G. Sunde M. Radford S.E. Biochemistry. 2000; 39: 8735-8746Crossref PubMed Scopus (306) Google Scholar) using a Quantamaster QM 2000-4 spectrofluorimeter (Photon Technology International, Inc.). Ure2p fibrils were also examined after negative staining with 1% uranyl acetate on carbon-coated grids (200 mesh) in a Philips EM 410 electron microscope (Philips Inc., The Netherlands).Fluorescent Labeling of Ure2p—Soluble Ure2pC355 and Ure2p-(94–354)C355 (25 μm) were dialyzed in buffer A without dithiothreitol for 2 h at 4 °C. The proteins were then labeled by adding 250 μm IAEDANS (Molecular Probes). The proteins were incubated on ice for 15 min. The unbound dye was removed by a desalting step using Sephadex G25 column (GE Healthcare) equilibrated in buffer A after quenching the reaction with 250 μm dithiothreitol. The labeled proteins were separated into aliquots and stored at –80 °C. Before use, the protein samples were spun for 10 min at 12,500 rpm and 4 °C to eliminate any aggregated material from the solutions. Their concentration was determined spectrophotometrically before dilution in the presence of unlabeled Ure2p and Ure2p-(94–354).Fluorescence Polarization Measurements—Complex formation between Ure2p or Ure2p-(94–354) and the different molecular chaperones was analyzed using fluorescence polarization. Labeled Ure2p or Ure2p-(94–354) and the different molecular chaperones dialyzed in buffer A at various concentrations were mixed in the presence of ATP or ADP and 5 mm MgCl2, and the time course of increase in fluorescence polarization upon binding of labeled Ure2p and Ure2p-(94–354) to the added molecular chaperone was measured at 20 °C in a thermostatted Quantamaster QM 2000-4 spectrofluorimeter (Photon Technology International, Inc.) equipped with autopolarizers. Monochromators were set to 337 nm (excitation) and 475 nm (emission), respectively.Filtration Assay, SDS-PAGE Electrophoresis, and Western Blotting—The time courses of Ure2p assembly into fibrils in the presence of the different molecular chaperones was also followed by a filtration assay allowing the quantification of fibrillar Ure2p. Aliquots (5 μl) were withdrawn from the assembly reactions at different time intervals, diluted into 200 μl of water or SDS 2% incubated for 5 min at 37 °C, and filtered through cellulose acetate membranes (0.20-μm pore size, Millipore Corp., Bedford, MA). After filtration of the sample, 200 μl of water or SDS 2% were filtered in each slot twice. The membrane was then washed with distilled water and processed for Western blotting. The cellulose acetate membranes were incubated with 5% skimmed milk, probed with an antibody directed against full-length Ure2p, and developed with the enzyme-coupled luminescence technique (ECL, GE Healthcare) according to the recommendation of the manufacturer.SDS-polyacrylamide gel electrophoresis were performed in 10–12% polyacrylamide gels (14 × 15 × 0.15 cm) following the standard method described by Laemmli (31Laemmli U.K. Nature. 1970; 27: 680-685Crossref Scopus (206024) Google Scholar). The gels were stained with Coomassie Blue, destained, imaged using a CCD camera (Sony, Inc.), and further analyzed on a MacIntosh (Apple Computer, Inc., Cupertino, CA) computer using the software NIH Image (developed at the United States National Institutes of Health and available at rsb.info.nih.gov/nih-image).RESULTSEffects of the Different Yeast Molecular Chaperones on the Assembly of Ure2p Using Thioflavin T Fluorescence—The effect of Hsp104p (Hsp100), Hsp82p (Hsp90), Ssa1p (Hsp70), Ydj1p (Hsp40), Sis1p (Hsp40), and CCT/Tric on Ure2p assembly was monitored in the absence or the presence of ATP using thioflavin T binding. Only CCT was found not to have a significant effect on Ure2p assembly at sub- or equimolar monomer concentrations (Fig. 1A). All the other molecular chaperones tested, namely, Hsp104p, Hsp82p, Ssa1p, Ydj1p, and Sis1p influenced the assembly reaction at sub- or equimolar concentrations (Fig. 1, B–F). Although Hsp82p (Fig. 1C), Ssa1p (Fig. 1D), Ydj1p (Fig. 1E), and Sis1p (Fig. 1F) inhibit to various extents Ure2p assembly, Hsp104p (Fig. 1B) promotes assembly. Indeed, the elongation rate increases 2.5-fold without a significant reduction in the lag phase preceding assembly. Moreover, in the presence of an equimolar concentration of Hsp104p, the thioflavin T fluorescence at steady state is 2.5-fold higher in the presence of Hsp104p than in its absence.Role of ATP—Although the assembly of Ure2p is affected by Hsp104p only in the presence of ATP, the opposite effect is observed for Ssa1p. Interestingly, Hsp82p did not affect assembly in an ATP-dependent manner. We, therefore, documented the effect of the N-terminal domain of Hsp82p on Ure2p assembly. The latter domain inhibits Ure2p assembly as efficiently as intact Hsp82p (not shown), thus confirming that Hsp82p interacts with Ure2p in an ATP-independent manner. Finally, as expected, the effects of Ydj1p and Sis1p were not ATP-dependent.Nature of the High Molecular Weight Ure2p Species That Form in the Presence of the Different Molecular Chaperones—To further characterize the high molecular weight species that form in the presence of the different molecular chaperones, aliquots were withdrawn at different time intervals from each assembly reaction and examined by electron microscopy after negative staining. In the absence of any molecular chaperone, Ure2p assembles into twisted fibrils (Fig. 2A). At steady state and in the presence of Hsp104p and ATP at a Ure2p to Hsp104p ratio 1:1 (Fig. 2B), very large aggregates with no obvious structural motifs were observed. In the presence of Hsp82p (Fig. 2C), Ssa1p (Fig. 2D), Ydj1p (Fig. 2E), and Sis1p (Fig. 2F) at a molecular chaperone to Ure2p molar ratio of 1:1, globular high molecular weight species were observed. At lower molecular chaperones to Ure2p molar ratios, for example 0.2:1, mixtures of ordered fibrils, large aggregates, and the globular particles were observed (Fig. 2, G–K). We conclude from these observations that whereas the observed decrease in thioflavin T binding upon the addition of Hsp82p, Ssa1p, Ydj1p, and Sis1p is due to the sequestering of Ure2p in high molecular weight, assembly-incompetent species with decreased ability to bind the dye thioflavin T, the significant increase in thioflavin T binding observed in the presence of Hsp104p is not due to an increased fibrillation but to the formation of high molecular weight species with no obvious structural motif and increased thioflavin T binding capacity.FIGURE 2High molecular weight Ure2p species that form in the presence of molecular chaperones. Negative stained electron micrographs of Ure2p (50 μm) assemblies obtained in the absence of molecular chaperones (A) and in the presence of Hsp104p (B and G), Hsp82p (C and H), Ssa1p (D and I), Ydj1p (E and J), and Sis1p (F and K) at a molecular chaperone to Ure2p molar ratio of 1:1 and 0.2:1, respectively. Bar, 0.2 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To better characterize the high molecular weight oligomeric Ure2p species that form in the presence of Hsp104p, Hsp82p, Ssa1p, Ydj1p, and Sis1p, we used a filter trap assay derived from that first designed for polyglutamine-containing proteins by Wanker et al. (32Wanker E.E. Scherzinger E. Heiser V. Sittler A. Eickhoff H. Lehrach H. Methods Enzymol. 1999; 309: 375-386Crossref PubMed Scopus (197) Google Scholar) that allows distinguishing fibrillar and nonfibrillar Ure2p. Although fibrillar Ure2p is retained on the cellulose acetate filters in the presence of 2% SDS, none of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones used is retained on the filters under the same experimental conditions, thus indicating that unlike fibrillar Ure2p, the high molecular weight Ure2p-molecular chaperone complexes are labile in 2% SDS (Fig. 3A). We then used the filter trap assay to assess whether Ure2p fibrils are labile in the presence of Hsp104p or Ssa1p alone or together with its co-chaperone Ydj1p. Although the SDS-resistant fibrils are unaffected by the addition of Ssa1p alone (Fig. 3B) or together with its co-chaperone (Fig. 3C) in the presence of ATP, Hsp104p appears to convert Ure2p fibrils into molecular species that are no more retained on the filters (Fig. 3D).FIGURE 3Nature of the Ure2p species that form in the presence of molecular chaperones and effect of molecular chaperones on preformed fibrils. A, aliquots (5 μl) of soluble, fibrillar Ure2p (50 μm) and Ure2p oligomers that form upon incubation of Ure2p in the presence of Hsp104p for 90 h were diluted in water or SDS 2% (200 μl). The samples were incubated for 5 min at 37 °C and filtered through cellulose acetate membranes (0.20 μm pore size, Millipore) using a vacuum manifold with wide rectangular wells (GE Health-care). The cellulose acetate membrane was kept in place after the samples (205 μl) were completely filtered, and each slot on the membrane was further washed twice by filtering either 200 μl of water or 2% SDS depending on whether the initial sample was diluted in water or 2% SDS. After these washes, the membrane was removed from the filtering apparatus, washed with distilled water, and immunostained as described under "Material and Methods" with an antibody directed against Ure2p. Fibrillar Ure2p (50 μm) incubated the time indicated in the presence of equimolar concentrations of Ssa1p (B), Ssa1p and Ydj1p (C), and Hsp104p (D) were subjected to the same treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of the Interaction of Ssa1p and Ure2p—A total inhibition of Ure2p assembly is observed at an Ssa1p: Ure2p ratio of 1:4 (Fig. 4A). The comparison of the assembly of Ure2p at different concentrations with that of Ure2p at a constant concentration in the presence of increasing concentrations of Ssa1p is very informative on the mode of action of Ssa1p. The time courses of Ure2p (30 μm) in the presence of Ssa1p, 0.5 and 1 μm, superimpose perfectly to that of Ure2p, 27.5 and 25 μm, respectively. This indicates that five Ure2p molecules are sequestered in an assembly-incompetent state by each molecule of Ssa1p. We previously showed that hexameric Ure2p is the precursor of the fibrillar form of the protein (27Fay N. Inoue Y. Bousset L. Taguchi H. Melki R. J. Biol. Chem. 2003; 278: 30199-30205Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 33Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Crossref PubMed Scopus (129) Google Scholar). Thus, our results strongly suggest that Ssa1p binds to the hexameric form of Ure2p.FIGURE 4Interaction of Ssa1p and Ure2p. A, assembly of Ure2p (filled circle), 30 μm in the presence of increasing amounts of Ssa1p (skewed filled triangle, 0.5; inverted filled triangle, 1μm; filled diamond, 2μm; filled triangle, 4μm; filled square, 8 μm). The assembly reactions of Ure2p (dotted lines and gray square, 27.5 μm; gray triangle, 25 μm) are also shown. B, SDS-PAGE analysis of the supernatant (s) and pellet (p) fractions of the Ure2p assembly reactions shown in panel A subjected to sedimentation at steady state. C, rate of Ure2p fibrils (μm) elongation at a constant Ure2p concentration (30 μm) and increasing Ssa1p concentrations (0.2–8 μm). D, SDS-PAGE analysis of the supernatant and pellet fractions of Ure2p fibrils (30 μm) incubated for 24 h in the presence of Ssa1p (8 μm). The molecular mass markers (in kilodaltons) are shown to the left of B and D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)When aliquots are removed at steady state from the assembly reactions presented in Fig. 4A and are subjected to ultracentrifugation and the supernatant and pellet fractions are analyzed by SDS-PAGE, a fraction of Ure2p that increases with increasing concentrations of added Ssa1p is found in the supernatant fractions instead of being in the pellet (Fig. 4B). This further demonstrates that Ssa1p sequesters Ure2p in an assembly-incompetent soluble form.The affinity of Ssa1p for Ure2p (Fig. 4C) was derived from measurements of the elongation rates of the fibrils assembled at a constant Ure2p and increasing Ssa1p concentrations (Fig. 4A). It is 0.5 μm.To determine whether Ssa1p interacts with fibrillar Ure2p, Ure2p fibrils made in the absence of Ssa1p were incubated in the presence of Ssa1p for 24 h. The fibrils were pelleted, and the supernatant and pellet fractions were analyzed by SDS-PAGE. The data presented in Fig. 4D clearly indicate that Ssa1p neither binds stably to the fibrils nor disassembles them.Functional Interplay of Molecular Chaperones during the Assembly of Ure2p—Because Ssa1p, Ydj1p, and Sis1p inhibit independently Ure2p assembly, and because size exclusion chromatography is a slow non-equilibrium method where labile complexes may irreversibly dissociate during the time course of analysis, we employed fluorescence polarization to monitor the interaction of Ure2p with Ssa1p alone or together with its co-chaperones from the Hsp40 family and in the presence of Hsp104p.The polarization signal of a fluorophore depends on its rotational correlation time that is related to its size (34Lakowicz, J. R. (ed) (1999) Principles of Fluorescence Spectroscopy, 2nd Ed., pp. 291–346, Kluwer Academic Publishers/Plenum Publishers, New YorkGoogle Scholar). Ure2p should display a smaller fluorescence polarization as compared with that of larger Ure2p-molecular chaperone complexes if such complexes form. To validate the method, we first measured the polarization signal of fluorescently labeled Sup35p (3 μm) in the absence or the presence of equimolar amounts of Ssa1p and Ydj1p (1 μm). We indeed recently showed that Ssa1p sequesters Sup35p in an assembly incompetent state in the presence of its co-chaperone Ydj1p. A stable Sup35p·Ssa1p·Ydj1p complex that resists dissociation when it is subjected to size exclusion chromatography indeed forms. The fluorescence polarization of Sup35p increased very significantly in the presence of Ssa1p and Ydj1p (Fig. 5, inset) indicating that a Sup35p·Ssa1p·Ydj1p complex forms in solution. Similarly to what is observed for Sup35p, the polarization signal of AEDANS-labeled Ure2pC355 (3 μm) significantly increased upon addition of equimolar amounts of Ssa1p alone or in the presence of its co-chaperone Ydj1p as well as upon the addition of Hsp104p (Fig. 5). This indicates that large Ure2p-molecular chaperone complexes form in the presence of molecular chaperones. It is worth noting that the fluorescence polarization of Sup35p and Ure2p alone increases within the time frame of the measurements, consistent with the oligomerization of these proteins into high molecular weight soluble oligomers that can be seen in the electron microscope before or concomitant to the formation of insoluble protein fibrils (4Thual C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 35Krzewska J. Tanaka M. Burston S.G. Melki R. J. Biol. Chem. 2007; 282: 1679-1686Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar).FIGURE 5Binding of AEDANS-labeled Ure2pC355 and Sup35p to Ssa1p and Hsp104p. The binding reaction was monitored by measurement of fluorescence polarization. Ure2p, 3 μm was incubated alone (•) and in the presence of 3 μm Ssa1p (♦), 3 μm Ssa1p and 1 μm Ydj1p (▴), and 3 μm Hsp104p (▾). The change in the fluorescence polarization of AEDANS-labeled Sup35p, 3 μm, alone (•) and in the presence of Ssa1p, 3 μm, and Ydj1p, 1 μm (▴) over time is presented in the inset.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nucleotide Dependence of Ure2p-Molecular Chaperone Interactions and Identification of the Ure2p Region That Interacts with Molecular Chaperones—We then examined whether Ssa1p alone or in the presence of Ydj1p, on the one hand, and Hsp104p, on the other, interact with Ure2p in an ATP-dependent manner. We, therefore, incubated AEDANS-labeled Ure2p (0.3 μm) in the presence of increasing concentrations of Ssa1p, Ydj1p, Ssa1p, and Ydj1p at a Ydj1p:Ssa1p molar ratio of 1:5 and Hsp104p in the presence of 5 mm ATP or ADP. The interaction between Ssa1p and Ure
Referência(s)