Mechanism of inactivation on prion conversion of the Saccharomyces cerevisiae Ure2 protein

Abstract

The [URE3] infectious protein (prion) of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The C-terminal domain of Ure2p controls nitrogen catabolism by complexing with the transcription factor, Gln3p, whereas the asparagine-rich N-terminal "prion" domain is responsible for amyloid filament formation (prion conversion). On filament formation, Ure2p is inactivated, reflecting either a structural change in the C-terminal domain or steric blocking of its interaction with Gln3p. We fused the prion domain with four proteins whose activities should not be sterically impeded by aggregation because their substrates are very small: barnase, carbonic anhydrase, glutathione S-transferase, and green fluorescent protein. All formed amyloid filaments in vitro, whose diameters increased with the mass of the appended enzyme. The helical repeat lengths were consistent within a single filament but varied with the construct and between filaments from a single construct. CD data suggest that, in the soluble fusion proteins, the prion domain has no regular secondary structure, whereas earlier data showed that in filaments, it is virtually all beta-sheet. In filaments, the activity of the appended proteins was at most mildly reduced, when substrate diffusion effects were taken into account, indicating that they retained their native structures. These observations suggest that the amyloid content of these filaments is confined to their prion domain-containing backbones and imply that Ure2p is inactivated in [URE3] cells by a steric blocking mechanism.

Figure 1

Purified chimeras of the Ure2p prion domain fused with four other proteins. The fusion proteins were expressed in E. coli, purified, and analyzed by SDS/PAGE on a 10–20% gel with Coomassie blue staining. (Lane 1) Ure2^1–65-barnase; (lane 2) Ure2^1–80-GFP; (lane 3) Ure2^1–65-CA; and (lane 4) Ure2^1–65-GST. M, molecular weight markers. For all fusion proteins, some slight proteolytic trimming was observed. Protracted incubation at 4C° (e.g., for initial filament formation) leads to further proteolytic shortening, but only the largest (full or near-full-length) molecules were incorporated into the amyloid filaments.

Figure 2

Amyloid filaments of proteins containing the Ure2p prion domain visualized by negative-staining EM. The filaments were assembled in vitro under uniform conditions from purified soluble protein and negatively stained with uranyl acetate. Some filaments exhibit well defined axial repeats: on examples, the repeats are marked with black dots. With Ure2^1–80-GFP (Upper Middle and Right), this periodicity gives the filaments a corkscrew appearance whose repeat distance varies from filament to filament. The diameter of unitary (A-type) filaments varies according to the construct (Table 1). Some thicker filaments (B-type) consist of two or more A-type filaments wrapped around a common axis. Ure2^1–65-barnase produces only B-type filaments. [Bar = 150 nm.]

Figure 3

Fluorescence spectra of Ure2^1–80-GFP in amyloid filaments and in soluble form. The small differences between the spectra probably represent slight conformational changes in the GFP fold imposed by its incorporation into filaments, but the generally close resemblance between the spectra indicate that its fold is largely preserved.

Figure 4

CD spectra of Ure2p prion domain fused with barnase and CA. In the top two images, the spectra of the soluble fusion proteins are compared with the spectra of the unfused enzymes. In the third image, spectra of Ure2p and its C-terminal domain Ure2p^66–354 are compared. These spectra are affected little by the presence of the prion domain although this moiety accounts for about 35% of the mass of Ure2^1–65-barnase. In the last image, the difference spectra, corresponding to the prion domain contribution, are shown, including that for the prion domain in soluble Ure2p. None of the spectra indicates a substantial content of secondary structure. The differences among them may reflect small conformational differences between the prion domain in close proximity to the respective folded domains or slight changes associated with the formation of small aggregates before filament formation, which proceeds at different rates for the respective constructs.