A novel mutant of the Sup35 protein of Saccharomyces cerevisiae defective in translation termination and in GTPase activity still supports cell viability

Céline Fabret¹^,2 , Bruno Cosnier¹^,2 , Sergey Lekomtsev³ , Sylvie Gillet⁴ , Isabelle Hatin¹^,2 , Pierre Le Maréchal²^,4 and Jean Pierre Rousset¹^,2

¹IGM, Univ Paris-Sud, UMR 8621, Orsay, F 91405, France

²CNRS, Orsay, F 91405, France

³Engelhardt Institute of Molecular Biology, The Russian Academy of Sciences, 119991 Moscow, Russia

⁴IBBMC, Univ Paris-Sud, UMR 8619, Orsay, F 91405, France

author email corresponding author email

BMC Molecular Biology 2008, 9:22doi:10.1186/1471-2199-9-22


Published:	11 February 2008

Abstract

Background

When a stop codon is located in the ribosomal A-site, the termination complex promotes release of the polypeptide and dissociation of the 80S ribosome. In eukaryotes two proteins eRF1 and eRF3 play a crucial function in the termination process. The essential GTPase Sup35p, the eRF3 release factor of Saccharomyces cerevisiae is highly conserved. In particular, we observed that all eRF3 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. The goal of this study was to determine whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein.

Results

We detected no phosphorylation of the Sup35 protein in vivo. However, we show that it is phosphorylated by the cAMP-dependent protein kinase A on T341 in vitro. T341 was mutated to either alanine or to aspartic acid to assess the role of this residue in the activity of the protein. Both mutant proteins showed a large decrease of GTPase activity and a reduced interaction with eRF1/Sup45p. This was correlated with an increase of translational readthrough in cells carrying the mutant alleles. We also show that this residue is involved in functional interaction between the N- and C-domains of the protein.

Conclusion

Our results point to a new critical residue involved in the translation termination activity of Sup35 and in functional interaction between the N- and C-domains of the protein. They also raise interesting questions about the relation between GTPase activity of Sup35 and its essential function in yeast.