title: Biochemical and functional analysis of the assembly of full-length Sup35p and its prion-forming domain.
abstract: The protein Sup35 has prion properties. Its aggregation is at the origin of the [PSI(+)] trait in Saccharomyces cerevisiae. In vitro, the N-terminal domain of Sup35p alone or with the middle domain assembles into fibrils that exhibit the characteristics of amyloids. The vast majority of in vitro studies on the assembly of Sup35p have been performed using Sup35pNM, as fibrils made of Sup35pNM assembled in vitro propagate [PSI(+)] when reintroduced into yeast cells. Little is known about the assembly of full-length Sup35p and the role of the functional C-terminal domain of the protein. Here we report a systematic comparison of the biochemical and assembly properties of full-length Sup35p and Sup35pNM. We show that the native structure of the C-terminal domain is retained within the fibrils. We determined the size of Sup35p nuclei and the critical concentration for assembly that both differ from that of Sup35pNM. We demonstrate that Sup35pNM co-assembles with the full-length protein and that fibrils made of Sup35p or Sup35pNM seed the assembly of soluble Sup35pNM and Sup35p with different efficiencies. Finally, we show that fibrils made of full-length Sup35p induce with higher efficiency [PSI(+)] appearance as compared with those made of Sup35pNM. Our findings reveal differences and similarities in the assembly of Sup35p and its NM fragment and validate the use of Sup35pNM in studying some aspects of Sup35p aggregation but also underline the importance of using full-length Sup35p in studying prion propagation both in vivo and in vitro.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
authors.forename: Joanna
authors.initials: J
authors.lastname: Krzewska
authors.forename: Motomasa
authors.initials: M
authors.lastname: Tanaka
authors.forename: Steven G
authors.initials: SG
authors.lastname: Burston
authors.forename: Ronald
authors.initials: R
authors.lastname: Melki
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D002845:Chromatography; D002942:Circular Dichroism; D005656:Fungal Proteins / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000378:metabolism; D008854:Microscopy, Electron; D010454:Peptide Termination Factors; D011328:Prions / Q000737:chemistry* / Q000502:physiology; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D013997:Time Factors
chemical_list: D005656:Fungal Proteins; D010454:Peptide Termination Factors; D011328:Prions; D011994:Recombinant Proteins; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D020558:GTP Phosphohydrolases