Amyloid Nucleation and Hierarchical Assembly of Ure2p Fibrils

Abstract

The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro. This ability depends on the N-terminal prion domain, which contains Asn/Gln repeats, a motif thought to cause human disease by forming stable protein aggregates. The Asn/Gln region of the Ure2p prion domain extends to residue 89, but residues 15-42 represent an island of "normal" random sequence, which is highly conserved in related species and is relatively hydrophobic. We compare the time course of structural changes monitored by thioflavin T (ThT) binding fluorescence and atomic force microscopy for Ure2 and a series of prion domain mutants under a range of conditions. Atomic force microscopy height images at successive time points during a single growth experiment showed the sequential appearance of at least four fibril types that could be readily differentiated by height (5, 8, 12, or 9 nm), morphology (twisted or smooth), and/or time of appearance (early or late in the plateau phase of ThT binding). The Ure2 dimer (h ‫؍‬ 2.6 ؎ 0.5 nm) and granular particles corresponding to higher order oligomers (h ‫؍‬ 4 -12 nm) could also be detected. The mutants 15Ure2 and ⌬15-42Ure2 showed the same time-dependent variation in fibril types but with an increased lag time detected by ThT binding compared with wild-type Ure2. In addition, ⌬15-42Ure2 showed reduced binding to ThT. The results imply a role of the conserved region in both amyloid nucleation and formation of the binding surface recognized by ThT. Further, Ure2 amyloid formation is a multistep process via a series of fibrillar intermediates.

Molecular Chaperones and the Assembly of the Prion Ure2p in Vitro

“…Hsp82p, Ssa1p, Ydj1p, and Sis1p inhibited assembly in a concentration-dependent manner, whereas Hsp104p stimulated the formation of large unstructured Ure2p aggregates that bind the dye thioflavin T. Globular high molecular weight particles were observed at equimolar Ure2p:Hsp82p, Ure2p: Ssa1p, Ure2p:Ydj1p, and Ure2p:Sis1p ratios, whereas a mixture of globular particles, amorphous aggregates, and fibrils was observed at submolar Ure2p-molecular chaperone ratios. We and others (4,16,33) previously reported the existence of a globular species at the early stages of Ure2p assembly into protein fibrils. The presence of these particles at high molecular chaperone concentrations together with the inability of Ure2p to polymerize suggests that Hsp82p, Ssa1p, Ydj1p, and Sis1p interact with these potential precursors of Ure2p fibrils and inhibit assembly into fibrils through this interaction.…”

“…The asparagine-, glutamine-, serine-, and threonine-rich N-terminal domain of Ure2p (62% of amino acid residues) is crucial for prion propagation and flexible (4, 5, 6), whereas its C-terminal domain is compactly folded and mainly ␣-helical (7, 8). The latter domain binds glutathione (9), has glutathione peroxidase activity (10), and is sufficient to regulate nitrogen metabolism in bakers' yeast cells (11)(12)(13)(14).In vitro, at neutral pH, soluble Ure2p spontaneously forms long, twisted fibrils (4,(15)(16)(17). It is widely believed that similar fibrils form in vivo and are at the origin of the [URE3] trait (18).…”

“…In vitro, at neutral pH, soluble Ure2p spontaneously forms long, twisted fibrils (4,(15)(16)(17). It is widely believed that similar fibrils form in vivo and are at the origin of the [URE3] trait (18).…”

The Yeast Prion Protein Ure2 Shows Glutathione Peroxidase Activity in Both Native and Fibrillar Forms

“…The reaction mixture contained 30 M full-length Ure2 in 50 mM sodium phosphate buffer, pH 7.5, containing 0.2 M NaCl. The samples were incubated in parallel at a constant temperature of 37°C with shaking as described previously (35,36). Under these conditions, the increase in fluorescence due to ThT binding correlates directly with the appearance of fibrillar aggregates of Ure2 (36).…”

“…The samples were incubated in parallel at a constant temperature of 37°C with shaking as described previously (35,36). Under these conditions, the increase in fluorescence due to ThT binding correlates directly with the appearance of fibrillar aggregates of Ure2 (36). At each time point, one of the samples was placed on ice.…”