Translation termination factors function outside of translation: yeast eRF1 interacts with myosin light chain, Mlc1p, to effect cytokinesis.

The translation termination factor eRF1 recognizes stop codons at the A site of the ribosome and induces peptidyl-tRNA hydrolysis at the peptidyl transferase centre. Recent data show that, besides translation, yeast eRF1 is also involved in cell cycle regulation. To clarify the mechanisms of non-translational functions of eRF1, we performed a genetic screen for its novel partner proteins. This screen revealed the gene for myosin light chain, Mlc1p, acting as a dosage suppressor of a temperature-sensitive mutation in the SUP45 gene encoding eRF1. eRF1 and Mlc1p are able to interact with each other and, similarly to depletion of Mlc1p, mutations in the SUP45 gene may affect cytokinesis. Immunofluorescent staining performed to determine localization of Mlc1p has shown that the sup45 mutation, which arrests cytokinesis, redistributed Mlc1p, causing its disappearance from the bud tip and the bud neck. The data obtained demonstrate that yeast eRF1 has an important non-translational function effecting cytokinesis via interaction with Mlc1p.

Ssb1 chaperone is a [PSI+] prion-curing factor.

Yeast SUP7' or SUP11 nonsense suppressors have no phenotypic expression in strains deficient in the isopentenylation of A37 in tRNA. Here we show that such strains spontaneously produce cells with a nonsense suppressor phenotype which is related to the cytoplasmically inherited determinant and manifests all the key features of the [PSI+] prion. A screen of a multicopy yeast genomic library for genes that inactivate the [PSI+]-related suppressor phenotype resulted in the isolation of the SSB1 gene. Moreover, we demonstrate that multicopy plasmid encoding the Ssb1 chaperone cures cells of the [PSI+] prion.

[Psi(+)] prion generation in yeast: characterization of the 'strain' difference.

The yeast cytoplasmically-inherited nonsense suppressor [PSI(+)] determinant is presumed to be a manifestation of the aggregated prion-like state of the Sup35 protein. Overexpression of the Sup35 protein induces generation of [PSI(+)] determinants with various suppressor efficiency and mitotic stabilities. Here, we demonstrate that the relative frequency of appearance of [PSI(+)] with different properties depends on the SUP35 allele used to induce their generation. The difference in properties of [PSI(+)] determinants was preserved after their transmission from one yeast strain to another. This difference correlated with variation in properties of the Sup35 protein. A novel type of prion instability was observed: some [PSI(+)] with weak suppressor efficiency could convert spontaneously into strong suppressor determinants.

Prion properties of the Sup35 protein of yeast Pichia methanolica.

The Sup35 protein (Sup35p) of Saccharomyces cerevisiae is a translation termination factor of the eRF3 family. The proteins of this family possess a conservative C-terminal domain responsible for translation termination and N-terminal extensions of different structure. The N-terminal domain of Sup35p defines its ability to undergo a heritable prion-like conformational switch, which is manifested as the cytoplasmically inherited [PSI(+)] determinant. Here, we replaced the N-terminal domain of S.cerevisiae Sup35p with an analogous domain from Pichia methanolica. Overexpression of hybrid Sup35p induced the de novo appearance of cytoplasmically inherited suppressor determinants manifesting key genetic and biochemical traits of [PSI(+)]. In contrast to the conventional [PSI(+)], 'hybrid' [PSI(+)] showed lower mitotic stability and preserved their suppressor phenotype upon overexpression of the Hsp104 chaperone protein. The lack of Hsp104 eliminated both types of [PSI(+)]. No transfer of prion state between the two Sup35p variants was observed, which reveals a 'species barrier' for the [PSI(+)] prions. The data obtained show that prion properties are conserved within at least a part of this protein family.

Mechanism of inhibition of Psi+ prion determinant propagation by a mutation of the N-terminus of the yeast Sup35 protein.

The SUP35 gene of Saccharomyces cerevisiae encodes the polypeptide chain release factor eRF3. This protein (also called Sup35p) is thought to be able to undergo a heritable conformational switch, similarly to mammalian prions, giving rise to the cytoplasmically inherited Psi+ determinant. A dominant mutation (PNM2 allele) in the SUP35 gene causing a Gly58-->Asp change in the Sup35p N-terminal domain eliminates Psi+. Here we observed that the mutant Sup35p can be converted to the prion-like form in vitro, but such conversion proceeds slower than that of wild-type Sup35p. The overexpression of mutant Sup35p induced the de novo appearance of Psi+ cells containing the prion-like form of mutant Sup35p, which was able to transmit its properties to wild-type Sup35p both in vitro and in vivo. Our data indicate that this Psi+-eliminating mutation does not alter the initial binding of Sup35p molecules to the Sup35p Psi+-specific aggregates, but rather inhibits its subsequent prion-like rearrangement and/or binding of the next Sup35p molecule to the growing prion-like Sup35p aggregate.

C-terminal truncation of the Sup35 protein increases the frequency of de novo generation of a prion-based [PSI+] determinant in Saccharomyces cerevisiae.

The yeast non-Mendelian [PSI+] determinant is presumed to be the manifestation of the aggregated prion-like state of the Sup35 protein. Plasmid-mediated amplification of the SUP35 gene greatly increases the frequency of Sup35p transition to this prion-like state. Here we show that the 3'-deletions of plasmid SUP35, leading to the C-terminal truncation of Sup35p, further increase the frequency of [PSI+] induction despite a marked decrease in Sup35p expression levels. The data suggest that the presence of Sup35p N-terminal proteolytic fragments can cause [PSI+] appearance in wild-type yeast cells.