In the present study we show that the prion domain of Ure2p in whole or in parts assemble into fibrils of amyloid nature. In contrast, the full-length prion assembles into helical fibrils. Thus, the intrinsic assembly propensity of Ure2p prion domain into fibrils of amyloid nature appears to be abolished by its covalent association with the functional domain of the protein that leads to the generation of fibrils with high α-helical content. The counteracting activity of Ure2p C-terminal domain strongly suggests the existence of a crosstalk between the two protein moieties. The latter could theoretically lead to the acquisition of a defined structure by the N-terminal domain. Comparison of the CD difference spectra of full-length Ure2p and Ure2pC-terminal domain 
and that of the GST-prion domain fusion and GST reveal however that Ure2p prion domain is poorly structured whether in its natural context or fused to the C-terminal end of GST i.e.
establishing interaction with the rest of the protein or not, respectively.
The finding that Ure2p 42–79 has a higher propensity to assemble into amyloid fibrils than Ure2p 1–42 suggests that the latter polypeptide stretch modulates aggregation in its natural context in a negative manner. This agrees with the finding that amino-acid residues centred around residue 6 interact with those centred around residue 137 in assembly competent Ure2p 
. The vast majority of natively unfolded polypeptides have been shown to form fibrils of amyloid nature. The finding that free Ure2p 42–79 readily forms fibrils of amyloid nature strongly suggests this peptide is natively unfolded. In contrast, the complex FTIR spectra recorded for free Ure2p 1–42 assemblies suggest that the peptide retain elements of secondary structure. Taken together our observations suggest that the N-terminal half of Ure2p prion domain not only interact with the C-terminal moiety of the prion protein but also possess elements of secondary structure.
It is worth noting that GST-Ure2p 1-42 has a higher assembly propensity than free Ure2p 1-42. It is possible that the ensemble of conformational states adopted by free Ure2p 1–42 is wider than that the polypeptide can explore when it is physically attached to the C-terminal end of GST. Thus, one reasonable explanation for this observation is that Ure2p 1–42 in GST-Ure2p 1–42 populates an assembly competent state faster than free Ure2p 1–42 because of its limited freedom and assemble more readily, as measured using thioflavin T binding.
Prions propagate by incorporation of soluble prion proteins into high molecular weight, fibrillar, oligomers. As the formation of stable nuclei following the conformational change that yield assembly competent prions is rate limiting, preformed prion aggregates seed very efficiently the assembly of soluble prions into fibrillar structures. The fibrillar form of full-length Ure2p generated under physiological experimental conditions seeds very efficiently the assembly of intact Ure2p. In contrast, fibrils made of the prion domain of the protein lack the ability to incorporate full-length Ure2p. This indicates that Ure2p N-terminal domain within its natural context does not populate a conformational state that incorporate within the amyloids that are generated when the prion domain is free in solution and out of its physiological context. This further support the model we proposed several years ago where the assembly of the prion Ure2p is driven by the establishment of intermolecular interactions between the N- and C-terminal domains of two consecutive Ure2p dimers and where the functional C-terminal domain of the protein is tightly involved in the fibrillar scaffold. Indeed, if the alternative model where assembly is driven by the stacking of Ure2p N-termini into a systematically H-bonded β-sheet core running along the fibrils to which the C-terminal domain is attached through a flexible region 
was correct, amyloids made of free Ure2p N-terminal domain would seed the assembly of full-length Ure2p.
Fibrils made in vitro
of Ure2p N-terminal domain were shown recently to induce, although to a lesser extent than those made of full-length Ure2p, [URE3
] occurrence when reintroduced in yeast cells 
. The results we report in this study suggest that [URE3
] occurrence might be the consequence of the conversion of soluble cytosolic Ure2p into an insoluble form upon introduction of fibrils made of full-length Ure2p as fibrillar Ure2p seeds very efficiently the assembly of full-length Ure2p. Given that the amyloid fibrils made of the N-terminal domain of the protein i-exhibit no full-length Ure2p seeding capacity, ii- most likely expose to the solvent surface areas that differ from those exposed by full-length Ure2p fibrils, the process by which they induce [URE3
] must differ from that of full-length Ure2p fibrils. The occurrence of [URE3
] has been reported to be highly dependent on the expression levels of a number of molecular chaperones 
. In addition, we have shown that the assembly of Ure2p is finely tuned by molecular chaperones in vitro 
. Thus, it is possible that the induction of the prion trait upon reintroduction of fibrils made of Ure2p N-terminal domain is the consequence of their interaction interaction with molecular chaperones leading to de novo
occurrence of [URE3
]. The latter considerations and the finding that the [URE3
] induction efficiency by full-length Ure2p fibrils is higher than that of fibrils made of the N-terminal domain of the protein 
might thus reflect two independent pathways leading to a similar end point observable.
Further characterization of the affinity of molecular chaperone for fibrillar full-length Ure2p and Ure2p N-terminal domain using binding measurements and proteomic analysis should allow establishing whether the differences in the efficiency of [URE3] induction recorded for the two kinds of fibrils is due to two independent pathways leading to prion trait occurrence. This will undoubtedly contribute to a better understanding of the molecular events leading to [URE3] occurrence and propagation.