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.
Departament de Bioquímica, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain.
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de Nadal
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Fadden
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Ruiz
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Haystead
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Ariño
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