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


CONCLUSIONS
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.
Fabret
Céline
C
IGM, Univ Paris-Sud, UMR 8621, Orsay, F 91405, France. celine.fabret@igmors.u-psud.fr
Cosnier
Bruno
B
Lekomtsev
Sergey
S
Gillet
Sylvie
S
Hatin
Isabelle
I
Le Maréchal
Pierre
P
Rousset
Jean Pierre
JP
D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010443:Peptide Chain Termination, Translational / Q000235:genetics*; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011328:Prions / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
D010454:Peptide Termination Factors; D011328:Prions; C068068:SUP35 protein, S cerevisiae; C080775:SUP45 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D020558:GTP Phosphohydrolases