A role for the Ppz Ser/Thr protein phosphatases in the regulation of translation elongation factor 1Balpha.
In vivo 32P-labeled yeast proteins from wild type and ppz1 ppz2 phosphatase mutants were resolved by bidimensional electrophoresis. A prominent phosphoprotein, which in ppz mutants showed a marked shift to acidic regions, was identified by mixed peptide sequencing as the translation elongation factor 1Balpha (formerly eEF1beta). An equivalent shift was detected in cells overexpressing HAL3, a inhibitory regulatory subunit of Ppz1. Subsequent analysis identified the conserved Ser-86 as the in vivo phosphorylatable residue and showed that its phosphorylation was increased in ppz cells. Pull-down experiments using a glutathione S-transferase (GST)-EF1Balpha fusion version allowed to identify Ppz1 as an in vivo interacting protein. Cells lacking Ppz display a higher tolerance to known translation inhibitors, such as hygromycin and paromomycin, and enhanced readthrough at all three nonsense codons, suggesting that translational fidelity might be affected. Overexpression of a GST-EF1Balpha fusion counteracted the growth defect associated to high levels of Ppz1 and this effect was essentially lost when the phosphorylatable Ser-86 is replaced by Ala. Therefore, the Ppz phosphatases appear to regulate the phosphorylation state of EF1Balpha in yeast, and this may result in modification of the translational accuracy.
de Nadal
E
E
Departament de Bioquímica, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain.
Fadden
R P
RP
Ruiz
A
A
Haystead
T
T
Ariño
J
J
D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D017931:DNA Primers; D020648:Peptide Elongation Factor 1; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010749:Phosphoprotein Phosphatases