Prion forming
abilities of interspecies recombinant Sup35p.
Julia V. Sopova,
Sergey P. Zadorsky, Sergey G. Inge-Vechtomov
Dept. of Genetics and Breeding, St-Petersburg State University,
Universitetskaya nab, St-Petersburg, 199034, Russia (sopova@hotmail.com)
The SUP35 gene of yeast Saccharomyces cerevisiae codes for translation termination factor eRF3. The Sup35 protein can undergo prion conversion and its prion form is known as omnipotent nonsense suppressor [PSI+]. The homologous Sup35 from yeast Pichia methanolica (Sup35P) is nonsense suppressor in S. cerevisiae strains lacking its own Sup35. We constructed recombinant gene SUP35SP, coding for NM-domain of S. cerevisiae Sup35 and C-domain of P. methanolica Sup35 and substituted authentic SUP35S for it in S. cerevisiae strains. Obtained transgenic strains with SUP35SP in the chromosome had increased level of nonsense-suppression and low viability. SUP35SP gene and [PSI+] factor were incompatible, like the strong suppressor sup35 mutations. Recombinant protein Sup35SP fused with GFP formed visible aggregates in [psi-] [PIN+] strains with SUP35S, SUP35SΔNM or SUP35SP in the chromosome. This indicates increased prion forming abilities of recombinant protein. We studied the ability of Sup35SP to convert the Sup35S into prion form. We transformed the non-transgenic strains with the SUP35SP gene on centromeric plasmid. Effective suppression in transformants was [PIN+]-dependent and GuHCl-curable, suggesting that [PSI+]-like prion form was induced. But after the loss of the inducing plasmid we did not observe any [PSI+] clones. This means that the induced [PSI+]-like factor is unstable in the absence of Sup35SP inducer. This work is supported by CRDF ST-012-0 and CRDF RB1-2336-ST-02.