[Estimating of changes in the amyloid and prion content of yeast cells].
An attempt was made at estimating the overall amyloid content of yeast cells by treating crude cellular lysates with thioflavin T, the agent specifically staining amyloid fibrils. We demonstrated that overproduction of the yeast chaperone Hsp104p, as well as GuHCI treatment of the [PSI+] cells led both to elimination of the [PSI+] factor and to a stable decrease of the overall amyloid content estimated by intensity of fluorescence (IF) of the thioflavin T. At the same time, overexpression of gene SUP35, coding the protein prionizable to [PSI+], led to generation of [PSI+] clones with higher IF of thioflavin T. Cytoduction in the crosses involving PSI factor leads to considerable enhancement of IF; cytoductants with the nucleus of the recipient [psi-] strain not only got [PSI+] factor from the donor strain but also increased their amyloid content. In these model experiments all treatments modifying one of the yeast prions, [PSI+] factor, led to a predictable shift of IF of thioflavin T that behaved like a cytoplasmic hereditary determinant. The data obtained show that IF of thioflavin T staining gives reliable estimates of cellular amyloid content and that mitotically stable shift of IF after a battery of treatments modifying cellular prion set provides quantitative estimate of the input of prionizable protein molecules to the amyloid pool. The combination of thioflavin staining and prionotropic treatments applied here can be possibly used for future attempts of checking yeast strains for cryptic prions.
Nevzgliadova
O V
OV
Kuznetsova
I M
IM
Artemov
A V
AV
Mikhaĭlova
E V
EV
Turoverov
K K
KK
Soĭdla
T R
TR
D000682:Amyloid / Q000032:analysis / Q000378:metabolism*; D052160:Benzothiazoles; D003433:Crosses, Genetic; D019791:Guanidine; D010454:Peptide Termination Factors; D011328:Prions / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D013844:Thiazoles
D000682:Amyloid; D052160:Benzothiazoles; D010454:Peptide Termination Factors; D011328:Prions; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013844:Thiazoles; C009462:thioflavin T; D019791:Guanidine