Single mother-daughter pair analysis to clarify the diffusion properties of yeast prion Sup35 in guanidine-HCl-treated [PSI] cells.
The yeast prion [PSI(+)] is a protein-based heritable element, in which aggregates of Sup35 protein are transmitted to daughter cells in a non-Mendelian manner. To elucidate the mechanism of the transmission, we have developed methods to directly analyse the dynamics of Sup35 fused with GFP in single mother-daughter pairs. As it is known that the treatment of yeast cells with guanidine hydrochloride (GuHCl) cures [PSI(+)] by perturbing Hsp104, a prion-remodelling factor, we analysed the diffusion profiles of Sup35-GFP in GuHCl-treated [PSI(+)] cells using fluorescence correlation spectroscopy (FCS). FCS analyses revealed that Sup35-GFP diffusion in the daughter cells was faster; that is, the Sup35-GFP particle was smaller, than that in the mother [PSI(+)] cells, and it eventually reached the diffusion profiles in [psi(-)] cells. We then analysed the flux of Sup35-GFP oligomers from mother to daughter [PSI(+)] cells in the presence of GuHCl, using a modified fluorescent recovery after photobleaching technique, and found that the flux of the diffuse oligomers was completely inhibited. The noninvasive methods described here can be applied to other protein-based transmissible systems inside living cells.
Kawai-Noma
Shigeko
S
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan.
Pack
Chan-Gi
CG
Tsuji
Toshikazu
T
Kinjo
Masataka
M
Taguchi
Hideki
H
D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D019791:Guanidine / Q000378:metabolism / Q000494:pharmacology*; D010454:Peptide Termination Factors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
D010454:Peptide Termination Factors; D011993:Recombinant Fusion Proteins; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D019791:Guanidine