The prion domain of yeast Ure2P induces autocatalytic formation of amyloid fibers by a recombinant fusion protein

Abstract

The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion‐like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [ URE3 ] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [ URE3 ] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione‐ S ‐transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST‐UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid‐like characteristics, such as Congo red dye binding and green‐gold birefringence. The fibrils exhibited high β‐sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full‐length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [ URE3 ] state is caused by a self‐propagating inactive form of Ure2p. We also found that the uncleaved GST‐UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, β‐sheet‐rich conformation. The findings on the GST‐UPD fusion protein indicate that the ability of the prion domain to mediate a prion‐like conversion process is not specific for or limited to the Ure2p.