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. 2018:1779:313-339.
doi: 10.1007/978-1-4939-7816-8_19.

Study of Amyloids Using Yeast

Reed B Wickner  1 , Dmitry Kryndushkin  2   3 , Frank Shewmaker  3 , Ryan McGlinchey  2 , Herman K Edskes  2
Affiliations

Study of Amyloids Using Yeast

Reed B Wickner et al. Methods Mol Biol. 2018.

Abstract

We detail some of the genetic, biochemical, and physical methods useful in studying amyloids in yeast, particularly the yeast prions. These methods include cytoduction (cytoplasmic mixing), infection of cells with prion amyloids, use of green fluorescent protein fusions with amyloid-forming proteins for cytology, protein purification and amyloid formation, and electron microscopy of filaments.

Keywords: Cytoduction; Electron diffraction; Mass per length; Protein transformation.

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Figures

Figure 1.
Figure 1.
Strain 74D-694 [psi-] was transformed with amyloid fibrils formed in vitro from recombinant Sup35 prion protein mixed with pRS425 (carrying LEU2). Transformants were selected on -Leu plates containing 2% of the normal amount of adenine. [PSI+] transformants are large, Ade+ and white, while [psi-] transformants are small, Ade- and red.
Figure 2.
Figure 2.
(A) The application of a 10 ul amyloid suspension to a carbon-coated copper grid. (B) Sup35 prion domain fibers stained with uranyl acetate. (C) Typical amyloid electron diffraction of unaligned fibers (mouse Pmel rpt). (D) electron diffraction of thallous chloride crystals. (E) TMV and Sup35NM fibers viewed in the darkfield mode (tilted beam). (F) histogram representation of Sup35NM mass-per-length measurements (from Chen et al (97)).
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References

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