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. E...

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Bibliographic Details
Published in:Prion 2008-01, Vol.2 (1), p.37-41
Main Authors: Ono, Bun-ichiro, Kawaminami, Hiroshi, Kobayashi, Hironori, Kubota, Masashi, Murakami, Yoshikazu
Format: Article
Language:English
Subjects:
Binding
Biology
Bioscience
Calcium
Cancer
Cell
Cycle
Escherichia coli - genetics
Escherichia coli - growth & development
Glutathione Transferase - biosynthesis
Glutathione Transferase - genetics
Landes
Mutation
Organogenesis
Peptide Termination Factors
Prions - biosynthesis
Prions - genetics
Protein Structure, Quaternary
Protein Structure, Tertiary - genetics
Proteins
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - genetics
Research Paper
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - biosynthesis
Saccharomyces cerevisiae Proteins - genetics
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Summary: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 protein 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.
ISSN:1933-6896
1933-690X
DOI:10.4161/pri.2.1.6436