Structural Characterization of Saccharomyces cerevisiae Prion-like Protein Ure2

Abstract

Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH 2 -terminal region is found poorly structured, whereas its COOHterminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.

The yeast prion protein Ure2: Structure, function and folding

“…In the case of Ure2, to obtain ideal results in this two-way experiment requires a careful balance of pH, temperature, protein concentration and choice of buffer [27]. The conditions found to be 'ideal' for folding experiments (Tris-HCl buffer pH 8.4, 0.2 M NaCl, 25°C and 1 μM protein concentration) unsurprisingly resemble the conditions adopted for Ure2 purification in a variety of labs [26,27,32,109], and coincide with maximal stabilization of the native state of Ure2 with respect to partially-folded intermediates [36]. Nevertheless, under these conditions, the folding kinetics of Ure2 is extremely complex and a variety of folding intermediates are transiently populated during unfolding or refolding [27,35,36].…”

“…Subsequent biophysical analysis of the purified Ure2 protein has shown that the structural properties of the N-terminal and C-terminal regions are also distinct. The N-terminal domain is extremely sensitive to protease digestion [26][27][28]104] and progressive deletion of the N-terminal domain has no effect on the dimerisation, stability or folding kinetics of the protein in vitro [27,33,35,36,105] (see Fig. 6).…”

“…Ure2 is a 354-amino acid protein consisting of a relatively flexible and protease-sensitive N-terminal region, and a globular C-terminal region [26][27][28]. The C-terminal region of Ure2 shows structural similarity to glutathione transferases (GSTs) and is necessary and sufficient for its regulatory function: Ure2 interacts with the transcription factor Gln3 allowing control of nitrogen catabolite repression, blocking the uptake of poor nitrogen sources in the presence of a good nitrogen source [29,30].…”

Assembly of the Yeast Prion Ure2p into Protein Fibrils

“…The assembly reaction follows a nucleationgrowth mechanism with a lag phase that can be eliminated by the addition of minute amounts of preformed fibrils to a solution of native soluble Ure2p (11). The assembly reaction of native full-length Ure2p into fibrils under physiologically relevant conditions is believed to be at the origin of the [URE3] phenotype in the yeast.…”

“…Native soluble Ure2p assembles in vitro into fibrils that exhibit the characteristics of amyloids in that they are about 20 nm wide and more than 1 m long, bind thioflavin T and Congo Red, show yellow-green birefringence in polarized light upon Congo Red binding, and exhibit an increased resistance to proteolysis (11)(12)(13). The assembly reaction follows a nucleationgrowth mechanism with a lag phase that can be eliminated by the addition of minute amounts of preformed fibrils to a solution of native soluble Ure2p (11).…”