Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p

The [URE3] factor of Saccharomyces cerevisiae propagates by a prion-like me chanism and corresponds to the loss of the function of the cellular protein Ure2. The molecular basis of the propagation of this phenotype is unknown. We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C- terminal region is compactly folded. Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils [Thual et al. (1999) J. Biol. Che m. 274, 13666-13674]. To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conf ormation, and capacity to assemble into fibrils, we have further analyzed t he properties of Ure2p N- and C-terminal regions. We show here that Ure2p d imerizes through its C-terminal region. We also show that the N-terminal re gion is essential for directing the assembly of the protein into a particul ar pathway that yields amyloid fibrils. A full-length Ure2p variant that po ssesses an additional tryptophan residue in its N-terminal moiety was gener ated to follow conformational changes affecting this domain. Comparison of the overall conformation, folding, and unfolding properties, and the behavi or upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no a dditional stability to the protein. This study reveals the existence of a s table unfolding intermediate of Ure2p under conditions where the protein as sembles into amyloid fibrils. Our results contradict the intramolecular int eraction between the N- and C-terminal moieties of Ure2p and the single unf olding transitions reported in a number of previous studies.