Conservation of the prion properties of Ure2p through evolution

Abstract

The yeast inheritable [URE3] element corresponds to a prion form of the nitrogen catabolism regulator Ure2p. We have isolated several orthologous URE2 genes in different yeast species: Saccharomyces paradoxus, S. uvarum, Kluyveromyces lactis, Candida albicans, and Schizosaccharomyces pombe. We show here by in silico analysis that the GST-like functional domain and the prion domain of the Ure2 proteins have diverged separately, the functional domain being more conserved through the evolution. The more extreme situation is found in the two S. pombe genes, in which the prion domain is absent. The functional analysis demonstrates that all the homologous genes except for the two S. pombe genes are able to complement the URE2 gene deletion in a S. cerevisiae strain. We show that in the two most closely related yeast species to S. cerevisiae, i.e., S. paradoxus and S. uvarum, the prion domains of the proteins have retained the capability to induce [URE3] in a S. cerevisiae strain. However, only the S. uvarum full-length Ure2p is able to behave as a prion. We also show that the prion inactivation mechanisms can be cross-transmitted between the S. cerevisiae and S. uvarum prions.

Figure 1.

Multiple alignment of full-length URE2p in nearly related species of S. cerevisiae. The gray box corresponds to the N-terminal prion domain. Within this domain, two hatched boxes delimit the small subregions A and B described in the text. The dark box corresponds to the C-terminal GST-like domain.

Figure 2.

Complementation assays. A strain deleted for URE2 (AF36) was transformed by the different plasmids allowing the overexpression of orthologous URE2. The different plasmids used are indicated on the left. After a 2-d growth on galactose medium, the transformants were streaked on both glucose and galactose medium, supplemented with the indicated nutriments (U, USA; A, adenine; D, histidine; I, tryptophan). The Petri dishes were observed after 4 d at 30°C. The expression of a functional Ure2p protein on galactose makes the strain unable to grow on USA medium.

Figure 3.

Curing effect of the PrDs. The ability of the AB34 [URE3_Sc] strain to grow on USA medium was tested after overexpression of the different PrDs. After selection, overexpression of the Prd was allowed (growth on galactose medium for 48 h) or repressed (growth on glucose medium), and the cells were streaked on Glu + USA. For each construct, the result is shown for one clone with two 10-fold dilutions.

Figure 4.

Analysis of the species barrier. The AF36 strain (deleted for URE2) expressing Sc or heterologous URE2 ORFs under the control of a Gal promoter was crossed with the strain AB34 [URE3]. Diploids were grown on galactose medium for 48 h and tested on Glu + USA and Gal + USA.