A protein transformation protocol for introducing yeast prion particles into yeast.
A range of methods for transforming organisms with nucleic acids has been established. However, techniques for introducing proteins, or particularly protein aggregates, into cells are less developed. Here, we introduce a highly efficient protocol for introducing protein aggregates such as prions into yeast. The protein transformation protocol allows one to infect yeast with amyloid fibers of recombinant fragments (Sup-NM) of Sup35p, the protein determinant of the yeast prion state [PSI+], or in vivo Sup35p prions. Infectivity is dependent on the concentration of Sup-NM fibers and approaches approximately 100% at high Sup-NM concentrations. We also describe a method to create distinct conformations of Sup-NM amyloids. Using the protein transformation protocol, infection of yeast with different Sup-NM amyloid conformations leads to distinct [PSI+] strains. This protein transformation procedure is readily adaptable to other prion proteins and makes it possible to bridge in vitro and in vivo studies and greatly helps to elucidate the principles of prion inheritance.
Tanaka
Motomasa
M
Tanaka Research Unit, RIKEN Brain Science Institute, Hirosawa, Wako, Saitama, Japan.
D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D044366:Transition Temperature
D009097:Multienzyme Complexes; D011328:Prions; C025310:lyticase; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D010447:Peptide Hydrolases