Self-Seeded Fibers Formed by Sup35, the Protein Determinant of [PSI+], a Heritable Prion-like Factor of S. cerevisiae

Abstract

The [PSI+] factor of S. cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid. The molecular basis of its propagation is unknown. We report that purified Sup35 and subdomains that induce [PSI+] elements in vivo form highly ordered fibers in vitro. Fibers bind Congo red and are rich in beta sheet, characteristics of amyloids found in certain human diseases, including the prion diseases. Some fibers have distinct structures and these, once initiated, are self-perpetuating. Preformed fibers greatly accelerate fiber formation by unpolymerized protein. These data support a "protein-only" seeded polymerization model for the inheritance of [PSI+].

The yeast prion protein Ure2: Structure, function and folding

“…The Ure2 system is therefore a useful model not only to investigate the prion concept, but also to understand the properties of Gln/ Asn-repeat proteins and hence the molecular basis of the related diseases. [27,29,58], Sup35 [114,115], Rnq1 [13], Het-s [22] and PrP [23,24,116,117]. The functional regions are as indicated.…”

[PSI+] prion generation in yeast: characterization of the ‘strain’ difference

“…This region may be further subdivided into the middle (M) domain, of unknown function, and the N-terminal (N) domain of 123 amino acids, required for [PSI + ] maintenance (Figure 1). The N domain plays a key role in the [PSI + ] phenomenon, being required for [PSI + ] propagation in vivo and solely responsible for Sup35p prion conversion and oligomerization into amyloid-like ®brils in vitro (Paushkin et al, 1997a, b;Glover et al, 1997;King et al, 1997).…”

Production of a Polymer-Forming Fusion Protein inEscerichia coliStrain BL21

“…have at least three cellular components, [URE3], 3,4) [PSI þ ], [5][6][7] and [PIN þ ], [8][9][10] that persist in the cell in the same manner as mammalian prion; hence they are often referred to as yeast prions. The yeast prions are not only attractive research objects in themselves but also useful model experimental systems for elucidating conformational alteration of mammalian prion and polymerforming proteins in general.…”

“…10,17) It has been found that Sup35, Sup35NM, and Sup35N form polymers in vivo and also in vitro. 7,[18][19][20] Since it is a common practice in these studies to construct a fusion gene in which glutathione S-transferase (GST: EC2.5.1.18) is combined with the peptide of interest and to introduce it into the E. coli expression system, we constructed a plasmid that carried a gene encoding GST-Sup35NM in which Sup35NM was fused to the C terminus of GST. Cells producing this fusion protein grew normally to the stationary phase, but their colony-forming ability in the stationary phase decreased much faster than that of the control cells.…”