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The phosphorylation site for Ste20p-like protein kinases is essential for the function of myosin-I in yeast
1Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada.
Abstract:
The budding yeast Saccharomyces cerevisiae has two functionally redundant myosin-I isoforms encoded by the MYO3 and MYO5 genes. The function shared by these myosin proteins is required for proper yeast budding. Serine residue 357 in the head domain of Myo3p, conserved among myosin-I proteins including yeast Myo5p, was identified as a unique phosphorylation site for the serine/threonine protein kinase Ste20p and its closely related isoform Cla4p. These protein kinases share a function that is also essential for budding. Replacement of serine 357 with alanine disrupted the in vivo function of Myo3p, whereas this function was maintained by changing the serine residue to aspartate. This mutant version failed to compensate the growth defect of cells which lack both Ste20p and Cla4p, suggesting that myosin-I is not the only essential target of these protein kinases. Our results suggest that phosphorylation of the head domain by Ste20p-like protein kinases plays an essential role in the function of myosin-I in yeast cells.
Insights
Phosphorylation of the Myo3p protein by Ste20p-like kinases is crucial for yeast budding. This specific serine residue modification is essential for myosin-I function in Saccharomyces cerevisiae.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Saccharomyces cerevisiae possesses two redundant myosin-I isoforms, Myo3p and Myo5p, vital for budding.
- Myosin-I function is essential for proper yeast cell division and morphology.
Purpose of the Study:
- To investigate the role of serine 357 phosphorylation in Myo3p function.
- To identify the protein kinases responsible for phosphorylating Myo3p at serine 357.
- To elucidate the functional consequences of this phosphorylation event in yeast budding.
Main Methods:
- Site-directed mutagenesis to replace serine 357 with alanine or aspartate.
- In vivo functional assays to assess Myo3p activity in budding yeast.
- Analysis of kinase-mutant interactions and complementation studies.
Main Results:
- Serine 357 in the Myo3p head domain is a unique phosphorylation site for Ste20p and Cla4p.
- Mutation of serine 357 to alanine abolished Myo3p function, while aspartate substitution partially maintained it.
- Myosin-I is not the sole essential target of Ste20p/Cla4p, as evidenced by complementation failures.
Conclusions:
- Phosphorylation of the myosin-I head domain by Ste20p-like kinases is critical for its function in yeast.
- This phosphorylation event is integral to the regulation of myosin-I during the budding process.
- Ste20p and Cla4p kinases have essential targets beyond myosin-I for cell budding.