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Characterization of functional regions in the Schizosaccharomyces pombe mei3 developmental activator
1Department of Microbiology and Immunology, Morse Institute for Molecular Biology and Genetics, Health Science Center, State University of New York, Brooklyn, New York 11203, USA.
Abstract:
The Schizosaccharomyces pombe mei3(+) gene is expressed only in diploid cells undergoing meiosis. Ectopic expression of mei3(+) in haploid cells causes meiotic catastrophe. Mei3 is an inhibitor of Ran1/Pat1 kinase and contains a nine-amino-acid motif, Mei3-RKDIII, that resembles two regions in the Ste11 substrate for Ran1/Pat1. Substitution of serine for Arg-81 within Mei3-RKDIII transforms the inhibitor into a substrate for Ran1/Pat1. Thus, it is likely that Mei3-RKDIII defines a pseudosubstrate sequence. In this study, we constructed a series of mei3 deletion mutations and assayed each for activity. This analysis indicates that the carboxy-terminal domain of Mei3 is sufficient for function in vivo. Alanine-scanning mutagenesis identifies critical residues within the inhibitory domain. Two mutations, SM1 and SM8, fail to cause meiotic catastrophe. The SM1 mutation contains alterations of amino acid residues in Mei3-RKDIII. Recombinant SM1 protein exhibits reduced ability to inhibit Ran1/Pat1 kinase in vitro and interacts inefficiently with the kinase in a two-hybrid assay. The SM8 protein binds to Ran1/Pat1 in a two-hybrid assay but fails to inhibit Ran1/Pat1 substrate phosphorylation in vitro. These findings provide evidence that Mei3-RKDIII defines a Ran1/Pat1-binding site that is necessary but not sufficient for inhibition of the kinase. Using fusions to green fluorescent protein, the cellular localization of Ran1 and Mei3 was examined in living cells. Ran1 is concentrated in the nucleus. Mei3 is also enriched in the nucleus and, consistent with the genetic and biochemical results, the inhibitory domain of Mei3 is sufficient for nuclear localization.
Insights
Mei3, a meiosis regulator in fission yeast, inhibits Ran1/Pat1 kinase. Its inhibitory domain binds the kinase but is insufficient for full inhibition, revealing a necessary binding site for meiotic control.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The mei3(+) gene in Schizosaccharomyces pombe is crucial for meiosis, exclusively expressed in diploid cells.
- Ectopic expression of mei3(+) in haploid cells leads to meiotic catastrophe, highlighting its regulatory role.
- Mei3 functions as an inhibitor of the Ran1/Pat1 kinase, a key enzyme in meiotic regulation.
Purpose of the Study:
- To elucidate the functional domains and critical residues of the Mei3 protein responsible for inhibiting Ran1/Pat1 kinase.
- To investigate the interaction between Mei3 and Ran1/Pat1 kinase at the molecular level.
- To determine the subcellular localization of Mei3 and Ran1 during meiosis.
Main Methods:
- Construction and functional analysis of mei3 deletion and alanine-scanning mutants.
- In vitro kinase inhibition assays using recombinant Mei3 proteins.
- Yeast two-hybrid assays to assess Mei3-Ran1/Pat1 interactions.
- Green fluorescent protein (GFP) tagging for live-cell imaging of protein localization.
Main Results:
- The carboxy-terminal domain of Mei3 is sufficient for its meiotic function in vivo.
- Mutations within the Mei3-RKDIII motif (SM1) impaired kinase inhibition and interaction.
- The SM8 mutation allowed binding to Ran1/Pat1 but abolished kinase inhibition, indicating binding is necessary but not sufficient.
- Both Ran1 and Mei3 are concentrated in the nucleus, with the inhibitory domain of Mei3 sufficient for nuclear localization.
Conclusions:
- The Mei3-RKDIII motif represents a Ran1/Pat1-binding site essential for kinase inhibition.
- While necessary, the binding site alone is insufficient for complete inhibition, suggesting additional regulatory mechanisms.
- The carboxy-terminal domain and nuclear localization of Mei3 are critical for its meiotic regulatory function.