Scrambled prion domains form prions and amyloid

Abstract

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The amino-terminal prion domain of Ure2p is necessary and sufficient for prion formation and has a high glutamine (Q) and asparagine (N) content. Such Q/N-rich domains are found in two other yeast prion proteins, Sup35p and Rnq1p, although none of the many other yeast Q/N-rich domain proteins have yet been found to be prions. To examine the role of amino acid sequence composition in prion formation, we used Ure2p as a model system and generated five Ure2p variants in which the order of the amino acids in the prion domain was randomly shuffled while keeping the amino acid composition and C-terminal domain unchanged. Surprisingly, all five formed prions in vivo, with a range of frequencies and stabilities, and the prion domains of all five readily formed amyloid fibers in vitro. Although it is unclear whether other amyloid-forming proteins would be equally resistant to scrambling, this result demonstrates that [URE3] formation is driven primarily by amino acid composition, largely independent of primary sequence.

FIG. 1.

Amino acids sequences of scrambled prion domains. Amino acids 1 to 89 are shown for wild-type Ure2p and for each of the scrambled Ure2p variants. For each of the Ure2p variants, amino acids 90 to 354 are the same as in wild-type Ure2p.

FIG. 2.

Stability and curability of the USA⁺ phenotype. Strains expressing URE2-21, -22, -23, and -25 form stable, curable USA⁺ colonies. USA⁺ colonies from strains expressing the URE2 variants were streaked for single colonies onto YPAD and YPAD with 5 mM guanidine HCl. Single colonies were taken from the YPAD plate (+) and from YPAD plus guanidine (cured) and streaked on ammonia medium with uracil (right) or with USA (left) to test for the ability to utilize USA. The [ure-o] parent was included as a negative control (−).

FIG. 3.

URE2-22 strains form unstable prions. (A) USA⁺ colonies from strains expressing URE2-22 retain the ability to utilize USA when maintained under selective pressure but lose the ability when maintained under nonselective conditions. USA⁺ cells were pregrown on either YPAD or ammonia medium with USA in the place of uracil and then tested for the ability to grow on ammonia medium containing uracil (right) or USA (left). The [ure-o] parent was included as a negative control (−). (B) The USA⁺ phenotype in URE2-22 cells is transmissible by cytoduction when cells are maintained under selective pressure. Cytoductions with donors that were maintained either on ammonia medium containing USA (+) or on YPAD (−) were streaked directly onto ammonia medium containing limiting adenine and with USA in the place of uracil. For cells maintained under selective pressure, both USA⁺ diploids (white) and cytoductants (red) are observed.

FIG. 4.

Scrambled Ure2p prion domains form filaments. Prion domains were purified under denaturing conditions and dialyzed into water overnight at 4°C. Significant precipitate was visible for all constructs except Ure2-25p^1-90. Precipitate was observed for Ure2-25p^1-90 after further 12 h rotation at 4°C. Precipitates were stained with uranyl acetate and visualized by negative-staining electron microscopy. Bar, 100 nm.

FIG. 5.

Full-length scrambled Ure2p variants form fibers with protease resistant cores. Full-length Ure2-21p, -22p, and -25p were purified under native conditions. Significant precipitate was observed after overnight rotation at 4°C. Precipitates were digested with 0.1 mg of proteinase K/ml at 37°C for 16 h, and the insoluble fraction was collected, washed, and stained with uranyl acetate. Electron micrographs compare undigested (left) and digested (right) filaments. Bar, 100 nm.