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A nuclear localization signal binding protein in the nucleolus
1Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York 10021.
The Journal of Cell Biology
|December 1, 1990
Summary
Researchers identified a novel nucleolar protein, p140, using synthetic nuclear localization signal peptides. This protein may shuttle between the nucleolus and cytoplasm, potentially acting as a nuclear import carrier.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nuclear localization signals (NLS) are crucial for protein import into the nucleus.
- SV-40 T antigen NLS peptides are widely used to study nuclear transport mechanisms.
Purpose of the Study:
- To identify nuclear proteins that bind to SV-40 T antigen NLS peptides.
- To characterize the identified proteins and determine their cellular localization and function.
Main Methods:
- Western blotting using synthetic NLS peptides conjugated to human serum albumin.
- Competition assays with free wild-type and mutant NLS peptides.
- Protein purification and antibody generation.
- Indirect immunofluorescence microscopy.
Main Results:
- Wild-type NLS peptide conjugates specifically bound to two nuclear proteins of 140 kD (p140) and 55 kD (p55).
- Binding was competed by free wild-type peptides but less effectively by mutant peptides.
- p140 was purified and antibodies confirmed its specificity and 140 kD size.
- Immunofluorescence revealed p140 localizes to the nucleolus with a punctate pattern, similar to NLS peptide conjugate binding.
Conclusions:
- p140 is a novel nucleolar protein.
- p140 may shuttle between the nucleolus and cytoplasm.
- p140 might function as a nuclear import carrier.