Effects of mutations in yeast prion [PSI+] on amyloid toxicity manifested in Escherichia coli strain BL21.
We previously showed that over production of a fusion protein in which the prion domain of Saccharomyces cerevisiae [PSI(+)] is connected to glutathione S-transferase (GST-Sup35NM) causes a marked decrease in the colony forming ability of Escherichia coli strain BL21 after reaching stationary phase. Evidence indicated that the observed toxicity was attributable to intracellular formation of fibrous aggregates of GST-Sup35NM. In this report, we describe the isolation of plasmids that encode mutant forms of GST-Sup35NM which do not confer the toxicity to E. coli strain BL21. Each of the four spontaneous mutant-forms of GST-Sup35NM obtained revealed amino acid substitutions. One substitution was located in the N domain, and the others in the M domain. Congo red binding assay indicated that none of these mutant proteins underwent conformational alteration in vitro. From these results, we conclude that the M domain, in collaboration with the N domain, plays an essential role in aggregation of Sup35NM. In addition, our data demonstrate the usefulness of the E. coli expression system in studying aggregate-forming proteins.
Ono
Bun-ichiro
B
Frontier Science Course, Ritsumeikan University Graduate School, Ritsumeikan University, Shiga, Japan. ono@se.ritsumei.ac.jp
Kawaminami
Hiroshi
H
Kobayashi
Hironori
H
Kubota
Masashi
M
Murakami
Yoshikazu
Y
D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D010454:Peptide Termination Factors; D011328:Prions / Q000096:biosynthesis* / Q000235:genetics; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics
D010454:Peptide Termination Factors; D011328:Prions; D011993:Recombinant Fusion Proteins; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D005982:Glutathione Transferase