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Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for
S V Paushkin1, V V Kushnirov, V N Smirnov
1Institute of Experimental Cardiology, Cardiology Research Center, Moscow, Russia.
Abstract:
The SUP45 and SUP35 genes of Saccharomyces cerevisiae encode polypeptide chain release factors eRF1 and eRF3, respectively. It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian [PSI+] nonsense suppressor determinant. In a [PSI+] state, Sup35p forms high-molecular-weight aggregates which may inhibit Sup35p activity, leading to the [PSI+] phenotype. Sup35p is composed of the N-terminal domain (N) required for [PSI+] maintenance, the presumably nonfunctional middle region (M), and the C-terminal domain (C) essential for translation termination. In this study, we observed that the N domain, alone or as a part of larger fragments, can form aggregates in [PSI+] cells. Two sites for Sup45p binding were found within Sup35p: one is formed by the N and M domains, and the other is located within the C domain. Similarly to Sup35p, in [PSI+] cells Sup45p was found in aggregates. The aggregation of Sup45p is caused by its binding to Sup35p and was not observed when the aggregated Sup35p fragments did not contain sites for Sup45p binding. The incorporation of Sup45p into the aggregates should inhibit its activity. The N domain of Sup35p, responsible for its aggregation in [PSI+] cells, may thus act as a repressor of another polypeptide chain release factor, Sup45p. This phenomenon represents a novel mechanism of regulation of gene expression at the posttranslational level.
Insights
The prion-like Sup35 protein aggregates in yeast, sequestering the release factor Sup45 protein. This aggregation, driven by Sup35p's N-domain, suggests a novel post-translational gene regulation mechanism.
Area of Science:
- Molecular Biology
- Genetics
- Prion Biology
Background:
- Saccharomyces cerevisiae SUP45 and SUP35 genes encode polypeptide chain release factors eRF1 and eRF3.
- The Sup35 protein (Sup35p) may undergo a prion-like conformational switch, forming aggregates and causing the [PSI+] phenotype.
- Sup35p has distinct domains: N (for [PSI+] maintenance), M (middle), and C (for translation termination).
Purpose of the Study:
- To investigate the aggregation mechanism of Sup35p and its interaction with Sup45p in [PSI+] cells.
- To determine the role of Sup35p domains in aggregation and Sup45p binding.
- To elucidate a potential novel gene regulation mechanism.
Main Methods:
- Analysis of Sup35p aggregation in [PSI+] Saccharomyces cerevisiae cells.
- Identification of Sup45p binding sites within Sup35p.
- Characterization of aggregated Sup35p fragments and their effect on Sup45p.
Main Results:
- The N-terminal domain of Sup35p is responsible for its aggregation in [PSI+] cells.
- Sup45p binds to Sup35p at two sites (N/M domains and C domain) and co-aggregates with Sup35p.
- Aggregation of Sup35p sequesters Sup45p, potentially inhibiting its activity.
Conclusions:
- Sup35p aggregation in [PSI+] cells involves its N-domain and leads to Sup45p co-aggregation.
- The N-domain of aggregated Sup35p may act as a repressor of Sup45p activity.
- This represents a novel post-translational gene expression regulation mechanism.