In vivo evidence for the fibrillar structures of Sup35 prions in yeast cells.
Yeast prion [PSI(+)] is caused by aggregated structures of the Sup35 protein. Although Sup35 forms typical amyloid fibrils in vitro, there is no direct evidence for the fibrillar structures of Sup35 in vivo. We analyzed [PSI(+)] cells in which Sup35 fused with green fluorescent protein (GFP) formed aggregates visible by fluorescence microscopy using thin-section electron microscopy (EM). Rapid-freeze EM combined with an immunogold-labeling technique as well as correlative light EM, which allows high-resolution imaging by EM of the same structure observed by light (fluorescence) microscopy, shows that the aggregates contain bundled fibrillar structures of Sup35-GFP. Additional biochemical and fluorescent correlation spectroscopy results suggest that the Sup35 oligomers diffused in the [PSI(+)] lysates adopt fibril-like shapes. Our findings demonstrate that [PSI(+)] cells contain Sup35 fibrillar structures closely related to those formed in vitro and provide insight into the molecular mechanism by which Sup35 aggregates are assembled and remodeled in [PSI(+)] cells.
Kawai-Noma
Shigeko
S
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan.
Pack
Chan-Gi
CG
Kojidani
Tomoko
T
Asakawa
Haruhiko
H
Hiraoka
Yasushi
Y
Kinjo
Masataka
M
Haraguchi
Tokuko
T
Taguchi
Hideki
H
Hirata
Aiko
A
D000682:Amyloid / Q000648:ultrastructure*; D007150:Immunohistochemistry; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D010454:Peptide Termination Factors / Q000235:genetics / Q000648:ultrastructure*; D011328:Prions / Q000648:ultrastructure*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000648:ultrastructure*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000648:ultrastructure*
D000682:Amyloid; D010454:Peptide Termination Factors; D011328:Prions; D011993:Recombinant Fusion Proteins; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins