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 Delta 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, Delta 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.
Highlights
The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro
Ure2 Mutants 15Ure2 and ⌬15– 42Ure2 Show an Increase in the Lag Time Detected by thioflavin T (ThT) Binding—ThT binding provides a convenient method to assay the effect of different factors on the kinetics of amyloid formation [9, 33]
We compared the ThT-monitored kinetics of amyloid formation for full-length Ure2 and a series of prion domain (PrD) deletion mutants
Summary
The yeast prion protein Ure 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 lag time can be circumvented by seeding with preformed amyloid-like fibrils [5,6,7] and is protein concentration-dependent [6, 9] This is consistent with a nucleation-dependent mechanism [34, 35], where the lag time reflects the kinetic barrier to association of a sufficient number of molecules to form a thermodynamically stable nucleus or seed.
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