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Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast
Published on: February 20, 2017
The budding yeast RasGEF Cdc25 reveals an unexpected nuclear localization
Renata Tisi1, Fiorella Belotti, Chiara Paiardi
1Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy.
Biochimica Et Biophysica Acta
|October 22, 2008
Summary
The yeast protein Cdc25, a Ras-GEF, is unexpectedly found in the nucleus, not the cell membrane. Specific domains within Cdc25 drive this nuclear localization, suggesting a novel regulatory role.
Area of Science:
- Cell Biology
- Molecular Biology
- Yeast Genetics
Background:
- The regulatory mechanisms of Saccharomyces cerevisiae Ras-guanine nucleotide exchange factor (Ras-GEF) Cdc25 are not fully understood.
- While the C-terminal domain's catalytic function is well-characterized, the N-terminal domain's role in negative regulation is a recent discovery.
Purpose of the Study:
- To investigate the cellular localization of Cdc25 and the functional roles of its distinct domains.
- To explore the potential nuclear import and export mechanisms influencing Cdc25 activity.
Main Methods:
- Construction of fusion proteins using full-length Cdc25 or its fragments with green fluorescent protein (GFP).
- Localization studies of fusion proteins within Saccharomyces cerevisiae cells.
- Analysis of endogenous Cdc25 localization using HA-tagged protein in nuclear extracts.
Main Results:
- Full-length Cdc25, even when slightly overexpressed, accumulates intracellularly and within the nucleus, not at the plasma membrane.
- Endogenous HA-tagged Cdc25 is detected in purified nuclear extracts.
- Cdc25 fragments (aa 353-875, 876-1100, 353-1100) show strong nuclear localization, particularly in the peripheral nuclear region, distinct from the nucleolus.
- Nuclear localization signals (NLS) at positions 547 and 806, and the region 876-1100, are implicated in this nuclear import.
- Fusion proteins demonstrate efficient nuclear export and re-import, indicating dynamic localization.
Conclusions:
- Saccharomyces cerevisiae Cdc25 predominantly localizes to the nucleus, challenging previous assumptions of plasma membrane localization.
- Specific domains and predicted nuclear localization signals within Cdc25 are critical for its nuclear import.
- The dynamic nuclear localization suggests a physiological role for Cdc25 within the nucleus, potentially linked to its regulatory functions.
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