An efficient protein transformation protocol for introducing prions into yeast.
Although a range of robust techniques exists for transforming organisms with nucleic acids, approaches for introducing proteins into cells are far less developed. Here we describe a facile and highly efficient protein transformation protocol suitable for introducing prion particles, produced in vitro from pure protein or purified from an in vivo source, into yeast. Prion particles composed of amyloid forms of fragments of Sup35p, the protein determinant of the yeast prion state [PSI(+)], lead to dose-dependent de novo induction of [PSI(+)] with efficiencies approaching 100% at high protein concentrations. We also describe a procedure for generating distinct, self-propagating amyloid conformations of a prionogenic Sup35p fragment termed Sup-NM. Remarkably, infection of yeast with different Sup-NM amyloid conformations leads to distinct [PSI(+)] prion strains, establishing that the heritable differences in prion strain differences result directly from self-propagating differences in the conformations of the infectious protein. This protein transformation protocol can be readily adapted to the analysis of other yeast prion states, as well as to test the infectious (prion) nature of protein extracts from less well-characterized epigenetic traits. More generally, the protein transformation procedure makes it possible to bridge in vitro and in vivo studies, thus greatly facilitating efforts to explain the structural and mechanistic basis of prion inheritance.
Tanaka
Motomasa
M
Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California San Francisco, 94143, USA.
Weissman
Jonathan S
JS
D011328:Prions / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D011328:Prions; D029701:Saccharomyces cerevisiae Proteins