The effects of amino acid composition of glutamine-rich domains on amyloid formation and fragmentation

Abstract

Fragmentation of amyloid polymers by the chaperone Hsp104 allows them to propagate as prions in yeast. The factors which determine the frequency of fragmentation are unclear, though it is often presumed to depend on the physical strength of prion polymers. Proteins with long polyglutamine stretches represent a tractable model for revealing sequence elements required for polymer fragmentation in yeast, since they form poorly fragmented amyloids. Here we show that interspersion of polyglutamine stretches with various amino acid residues differentially affects the in vivo formation and fragmentation of the respective amyloids. Aromatic residues tyrosine, tryptophan and phenylalanine strongly stimulated polymer fragmentation, leading to the appearance of oligomers as small as dimers. Alanine, methionine, cysteine, serine, threonine and histidine also enhanced fragmentation, while charged residues, proline, glycine and leucine inhibited polymerization. Our data indicate that fragmentation frequency primarily depends on the recognition of fragmentation-promoting residues by Hsp104 and/or its co-chaperones, rather than on the physical stability of polymers. This suggests that differential exposure of such residues to chaperones defines prion variant-specific differences in polymer fragmentation efficiency.

Figure 1. Polymerization of polyQX proteins in the presence of [PIN ⁺].

Lysates of 74-D694/ΔS35 [PIN ⁺] cells producing indicated QX proteins were analyzed by SDD-AGE. (A) Most QX proteins readily form SDS-insoluble polymers of varying size. Staining with anti-S35NM antibodies. (B) Polymerization of QX proteins, which did not polymerize in a [PIN ⁺] background on their own, in the presence of 85Q-GFP polymers and after loss of the plasmid encoding 85Q-GFP. Staining with anti-Sup35C.

Figure 2. Polymerization of polyQX proteins in the absence of [PIN ⁺].

Lysates of 74-D694/ΔS35 ΔRNQ1 cells producing QX proteins of different length were analyzed by SDD-AGE. Cells were grown for 20 generations after obtaining transformants. Staining with anti-S35NM antibodies.

Figure 3. Polyglutamine domain length influences polymer size and not polymer stability.

(A) Lysates of 74-D694/ΔS35 [PIN ⁺] cells producing QX proteins of different length were analyzed by SDD-AGE. Staining with anti-S35NM antibodies. (B) The lysates of 74-D694/ΔS35 [PIN ⁺] cells producing QA71 and QA110 proteins were incubated at different temperatures in the presence of sample buffer containing 2% SDS and analyzed by SDD-AGE. The thermal denaturation curves were obtained by densitometric analysis of the stained blot images.

Figure 4. Small SDS-insoluble polymers of QX proteins.

(A) Lysates of 74-D694/ΔS35 [PIN ⁺] Δprb1 cells producing 76QY, 96QW and 81QF were analyzed by SDS-PAGE without boiling the samples using large pore 5% gel. (B) 74-D694/ΔS35 [PIN ⁺] Δprb1 cells producing 76QY were grown for different periods of time in the presence of GuHCl (3 mM), and then their lysates were analyzed by SDD-AGE (left panel) and SDS-PAGE without boiling the samples (large pore 5% gel) (right panel).

Figure 5. Thermal stability of prion polymers of full-size and truncated Sup35.

(A) The lysates of [PSI ⁺] yeast cells producing full-sized or truncated Sup35 were analyzed by SDD-AGE. (B, C) Lysates were heated in the presence of sample buffer containing 2% SDS at different temperatures and analyzed by SDD-AGE. The thermal denaturation curves were derived from densitometric analysis of the stained blot images.

Figure 6. Staggered and fixed structures of amyloid polymers.

Schematic representation of amyloid polymers with a fixed fold, as proposed for yeast prion polymers, and a staggered fold, as proposed for polyQX amyloids.

Figure 7. Schematic model of the structure of the Sup35 prion domain.

Fragmentation-promoting residues (blue rectangles), such as tyrosine, are hidden inside the amyloid core structure and cannot act as recognition signals for chaperones. Residues in the exposed area (yellow triangles) are available and can thus facilitate fragmentation.

Figure 8. Amino acid content of yeast prion domains.

Percentages represent the proportion of the appropriate amino acids in yeast prion domains. The extent of the prion domains were taken from . Fragmentation-promoting amino acids which are abundant in certain prion domains are marked in bold. Numbers on the left represent the group number we assigned to certain residues according to their effect on amyloid formation and fragmentation.