The essential SUP35 gene encodes yeast translation termination factor Sup35/eRF3. The N-terminal domain of Sup35 is also responsible for Sup35 prionization that leads to generation of the [ PSI +] prion. Previously we isolated different types of sup35 mutations (missense and nonsense) and demonstrated that sup35 nonsense mutations ( sup35-n) are incompatible with the [ PSI +] prion, leading to lethality of sup35-n [ PSI +] haploid cells. Here, we show that sup35 missense mutations ( sup35-m) within conservative regions of the Sup35 C-domain result in lethality of [ PSI +] cells because of weak activity of Sup35/eRF3 as a translation termination factor. Mutant Sup35 maintain their ability to be incorporated into pre-existing [ PSI +] aggregates and to form amyloid aggregates in vitro, while sup35-m mutations do not influence the [ PSI +] prion induction and stability. All these mutations (D363N, R372K, T378I) are located in the conservative GTPase region of Sup35, decreasing the GTPase activity of mutated proteins. We propose that such low activity of mutant Sup35 combined with aggregation of Sup35 constituting the [ PSI +] prion is not sufficient to maintain the viability of yeast cells.