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Nuclear accumulation of Saccharomyces cerevisiae Mcm3 is dependent on its nuclear localization sequence
1Department of Biological Science, Faculty of Science, Hiroshima University, Kagamiyama, Japan.
Background:
The proteins of the Mcm2-7 family are required for the initiation of DNA replication. In Saccharomyces cerevisiae the nuclear envelope does not break down during the mitotic phase of the cell cycle. Large nuclear proteins, such as the Mcm proteins, which accumulate in the nucleus during specific portions of the cell cycle, must have regulated mechanisms to direct their entry into the nucleus.
Results:
We have identified a nuclear localization sequence (NLS) in Mcm3, and demonstrated that it is necessary for the translocation of Mcm3 into the nucleus and sufficient for directing Escherichia coli beta-galactosidase to the nucleus. Immediately adjacent to the nuclear localization sequence are four potential sites for phosphorylation by Cdc28. Mutagenesis of all four sites has no immediate phenotypic effect on cell growth or viability, nor does it affect nuclear accumulation of Mcm3, although two-dimensional protein gel analysis has shown that at least some of these sites are normally phosphorylated in vivo. Substitution of the Mcm3 NLS by the SV40 large T-antigen NLS also directs the nuclear accumulation of the Mcm3-T-antigen protein, although cell growth is compromised. Replication activity in cells bearing either the Mcm3-Cdc28 phosphorylation site mutations or the Mcm3 T-antigen NLS substitution, as measured by plasmid stability assays, is comparable to activity in wild-type cells.
Conclusions:
The Mcm3 protein is imported into the nucleus by a specific NLS. The cell cycle specific nuclear accumulation of Mcm3 appears to be a result of nuclear retention or nuclear targeting, rather than nuclear import regulated through the NLS.
Insights
Researchers identified a nuclear localization sequence (NLS) in Mcm3, crucial for its nuclear entry in yeast. This NLS directs Mcm3 into the nucleus, but cell cycle-specific accumulation likely involves nuclear retention mechanisms.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The Mcm2-7 protein family is essential for DNA replication initiation.
- In Saccharomyces cerevisiae, the nuclear envelope remains intact during mitosis, necessitating regulated nuclear import of large proteins like Mcm.
- Understanding the mechanisms of nuclear transport for cell cycle-regulated proteins is critical.
Purpose of the Study:
- To identify and characterize the nuclear localization sequence (NLS) of the Mcm3 protein.
- To investigate the role of potential phosphorylation sites near the Mcm3 NLS in nuclear import and function.
- To determine the contribution of the NLS to cell cycle-specific nuclear accumulation of Mcm3.
Main Methods:
- Identification and mutagenesis of a putative NLS in Mcm3.
- Functional assays using beta-galactosidase reporter system to assess NLS activity.
- Analysis of Mcm3 phosphorylation status using two-dimensional gel electrophoresis.
- Complementation assays and plasmid stability assays to evaluate replication activity.
Main Results:
- A functional NLS was identified in Mcm3, essential for its nuclear translocation.
- The identified NLS is sufficient to direct a reporter protein (beta-galactosidase) into the nucleus.
- Mutations in four adjacent potential phosphorylation sites did not affect Mcm3 nuclear accumulation or cell viability.
- Substitution of the Mcm3 NLS with the SV40 large T-antigen NLS led to nuclear accumulation but compromised cell growth.
Conclusions:
- Mcm3 protein is imported into the nucleus via a specific NLS.
- Cell cycle-specific nuclear accumulation of Mcm3 is likely mediated by nuclear retention or targeting, not solely by NLS-regulated import.
- The identified NLS plays a key role in Mcm3 nuclear import, but other mechanisms contribute to its regulated nuclear presence.