The long term goal of this work is to determine the molecular mechanism for the transport of nuclear proteins from the cytoplasm to the nucleus. The nuclear localization of all nuclear proteins occurs by selective mediated mechanisms, and therefore is likely to play a crucial role in normal cellular function. In addition, nucleo-cytoplasmic transport is an important regulatory point in embryonic development, regulation of cell metabolism by extracellular factors, viral infection and the toxic and carcinogenic effects of environmental pollutants. The main focus of this work is the molecular characterization of a cytoplasmic receptor for nuclear location sequences and its interaction with other cytoplasmic import components. A CDNA for the receptor will be isolated and sequenced. Regions of bacterially expressed protein will be subcloned to define regions of the protein involved in NLS recognition. Site directed mutagenesis will be used to identify amino acid residues important for NLS-binding and for the observed sensitivity of the receptor to inactivation by sulfhydryly alkylating reagents. The bacterially expressed receptor will be used to prepare an immobilized affinity matrix for isolation of cytoplasmic and nuclear envelope proteins that interact with the receptor. Additional cytoplasmic factors required for protein import will be isolated and characterized. Their interaction with the NLS receptor and role in protein import will be investigated. Monoclonal and polyclonal antibodies to the receptor and other cytoplasmic import factors will be made and used to dissect the cytoplasmic events in nuclear protein import.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047866-03
Application #
2185269
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1992-08-01
Project End
1997-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Biology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
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