Sup35 Md destabilizes [PSI⁺] in vivo and functionally interacts with Hsp104. A, Western blot of copper-induced Md expression in yeast strain OT55. The protein concentrations of extracts were equalized prior to loading. Yeast harboring a plasmid lacking an insert was used as control. B, Md expression was induced by growth in medium containing copper and cultures were diluted daily to maintain growth. At intervals, aliquots of cells were washed to remove residual copper and plated on medium lacking copper. Day 0 and day 9 plates are shown. C, the number of red-pigmented colonies (cured) on all plates was scored as a percentage of the total number of colonies counted. D, the ATPase activity of Hsp104^E285A was measured in the presence of Md, the Hsp104-binding peptide p370, and the nonbinding peptide pSGG (35) and expressed as the fold-increase relative to unstimulated activity. E, fRCMLa binding to Hsp104^Trap in the presence of ATP (defined as maximal binding), ADP (no binding), and in the presence of ATP and 7.5 μm Md.

Md inhibits chaperone-dependent fibrilization of NM in vitro. A, 2 μm NM was diluted either into buffers FB or FIB (see “Experimental Procedures”). Tween 20 (T20; 0.03% v/v) and glycerol (gly; 10% v/v) were added to FIB as indicated. After overnight incubation with gentle agitation the increase in ThT binding was determined relative to the initial fluorescence of each reaction. B, NM was diluted into reactions into FIB with Tween 20 and glycerol (FIB+) containing molecular chaperones and ATP as indicated. After incubation for 15–17 h in undisturbed reactions, reaction products were analyzed by ThT binding (upper panel; n = 3) and SDS-PAGE (lower panel). Proteins in sample buffer were heated either to 99 °C (boiled) or 38 °C to determine SDS resistance. The asterisk in each panel draws attention to the sample with the highest ThT binding and highest SDS resistance indicated by the retention of NM at the interface between the stacking and resolving gels. C, reactions in FIB+ containing huHsp70, Ydj1, ATP, and an ATP-regenerating system were conducted in the presence of wild type Hsp104 (WT), Hsp104^Trap, Hsp104 with substitutions in pore loops in the second (Hsp104^Y662A) or first (Hsp104^Y257A) AAA+ domain, or without Hsp104 (CON). D, reactions with Hsp104, huHsp70, and Ydj1 were incubated with or without 5 mm guandinium HCl (Gdm). E, negative stain TEM analysis of NM under the following conditions: (left to right) FIB+ with no additions; FIB+ with Hsp104, huHsp70, Ydj1, and 5 mm Gdm; FIB+ with chaperones but no ATP; FIB+ with chaperones and ATP; fibers formed spontaneously in FB. F, 3 μm Sup35 NM (25% acrylodan-labeled Sup35Y106C) was incubated in FIB+ with a complete set of chaperones (0.6 μm monomers each), ATP, an ATP regenerating system, and Md. Fluorescence was expressed as a percentage of the maximum fluorescence measured in the control reaction without Md. G, left panel, 3 μm NM was incubated in FB for 16 h with gentle agitation in the presence of the indicated concentration of Md. Right panel, 3 μm NM was incubated in FIB+ without agitation along with 0.1 μm Hsp104 hexamers, 0.3 μm Ydj1 dimers, and 0.6 μm huHsp70 monomers, ATP, and an ATP regenerating system, and Md at the indicated concentrations. In both panels fibrilization was determined by ThT fluorescence and expressed as the fold-increase in fluorescence compared with freshly prepared reactions (n = 3).

The basic region of Md functionally interacts with Hsp104. A, overlapping 13-mer peptides spanning the Sup35 N- and M-domains (amino acids 1–253) were spot-synthesized on cellulose membrane and probed with 35 nm Hsp104^Trap in the presence of 2 mm ATP. Bound protein was transferred to membrane and detected with anti-Hsp104 antibodies. Three binding regions were identified highlighted on the blot in boxes labeled i, ii, and iii with the amino acid sequences of the Hsp104-binding peptides shown below. B, fibrilization of Sup35 NM was carried out as described in the legend to Fig. 2E in the presence of polypeptides corresponding to Sup35 residues 105–163 and 164–253. C, the ATPase activity of Hsp104 was measured in the presence of various concentrations of Md, and the two subsegments used in panel B, and expressed as the fold-increase over activity measured in the absence of proteins. D, fluorescence anisotropy was used to measure fRCMLa binding to Hsp104^Trap in the presence of ATP, ADP, and in the presence of 7.5 μm Md, Md(105–163), and Md(164–253).

The functional interaction of soluble Md peptides with Hsp104. Overlapping 20-mer peptides corresponding the basic region of the Sup35 Md were assayed for Hsp104^E285A ATPase stimulation (right panel) at a concentration of 26 μm and inhibition of ATP-dependent fRCMLa binding to Hsp104^Trap (left panel) at a concentration of 7.5 μm. These concentrations were chosen so that the positive control peptide p370 effect was about 50% of its maximum. The nonbinding peptide pSGG was used as a negative control.

Deletion of amino acids 129–148 diminishes functional interactions with Hsp104. A, the ATPase activity of Hsp104 was measured in the presence of Md and MdΔ129–148 and expressed as the fold-increase over activity measured in the absence of peptide. B, the fibrilization of 3 μm NM in the presence of Hsp104, huHsp70, and Ydj1 (0.6 μm monomers each) was monitored in the presence of increasing concentrations of MdΔ129–148. The enhancement of acrylodan fluorescence was normalized to the maximum value obtained in reactions without addition of peptide. C, the binding of Hsp104^Trap to fluorescently labeled Md and MdΔ129–148 was measured using fluorescence anisotropy in the presence of ATP. D, spontaneous and chaperone-dependent fibrilization of intact NM and NMΔ129–148 were compared. For spontaneous fibrilization, 3 μm of the each protein was rotated overnight and fibrilization was measured by ThT fluorescence and expressed as the fold-increase in fluorescence in samples compared with the starting material. Chaperone-dependent reactions were as described in panel B but ThT fluorescence was used to determine the extent of fibril formation. E, comparison of fibrils produced from NM and NMΔ129–148 seeded with preformed NM fibrils using negative stain TEM (inset) and thermal stability of fibrils. Top panels show Coomassie-stained gels of proteins entering the gel after heating at the indicated temperature in SDS. Lower panel shows quantitation of solubility.

Sup35Δ129–148 displays a propagation defect in vivo. A, schematic of the plasmid shuffle procedure (see text for a description of the procedures). B, after plasmid shuffling, expression of full-length Sup35 and Sup35Δ129–148 was determined by Western blot analysis. C, the suppression phenotypes of two isolates each of [psi⁻] and [PSI⁺] strains were assessed by streaking on ¼ YPD. D, the particle size distributions of SDS-resistant Sup35 in the [PSI⁺] parental strain and the plasmid-shuffled strains were analyzed by SDS-PAGE and Western blotting. E, Hsp104 dependence of [PSI⁺] propagation was examined during continuous culturing of cells in selective media supplemented with 5 mm Gdm. Aliquots were periodically drawn and plated. Red colonies were scored as “cured” and expressed as a percentage of the total number of colonies counted. F, suppression phenotypes in “diploid” [PSI⁺] intermediates compared with single-copy derivatives. G, SDS-PAGE analysis of Sup35 particles in strains shown in panel F. H, curing of [PSI⁺] diploid intermediates after 2 days of growth in 5 mm Gdm.

[PSI⁺] maintained by Sup35Δ129–148 is resistant to curing by Hsp104 overexpression. A, [PSI⁺] cells were cultured overnight in selective medium containing 2% galactose and 0.03% glucose. Galactose-inducible Hsp104 expression was compared by Western blotting to cells grown in glucose. Blots were reprobed with an antibody against yeast alcohol dehydrogenase (YADH) as a loading control. B, cells were washed and plated onto glucose-containing selective medium to suppress further Hsp104 overexpression. C, red colonies were scored as cured and expressed as a percentage of the total number of colonies counted.

[PSI⁺] maintained by Sup35Δ129–148 is not a different strain. [PSI⁺] cells were transformed with a URA3 marked SUP35 plasmid and grown for many generations without selection to induce loss of the LEU2-marked plasmid. A, the SDS-resistant Sup35 particle size distributions of the parental strain, the plasmid-swapped strains, and the restored strains were analyzed by SDS-PAGE and Western blotting. B, the strain that was restored following the maintenance of [PSI⁺] by Sup35Δ129–148 was grown overnight as described in the legend to Fig. 7B.