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.
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Related Subject Headings
- Recombinant Fusion Proteins
- Phosphorylation
- Phosphoprotein Phosphatases
- Peptide Elongation Factor 1
- Glutathione Transferase
- Escherichia coli
- DNA Primers
- Biochemistry & Molecular Biology
- Base Sequence
- 11 Medical and Health Sciences
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Recombinant Fusion Proteins
- Phosphorylation
- Phosphoprotein Phosphatases
- Peptide Elongation Factor 1
- Glutathione Transferase
- Escherichia coli
- DNA Primers
- Biochemistry & Molecular Biology
- Base Sequence
- 11 Medical and Health Sciences