BSAP (Pax5)-importin alpha 1 (Rch1) interaction identifies a nuclear localization sequence.
BSAP (Pax5) is an essential transcription factor for early B cell and central nervous system development. In later B cell development, BSAP has been implicated in the regulation of 3' Ig enhancers and a number of B cell-specific genes. Previous studies have suggested a role for BSAP-interacting proteins in the regulation of the function of BSAP. Using the yeast two-hybrid system, we identified importin alpha1 (Rch1) as a BSAP-interacting protein. Importin alpha proteins have been shown to escort proteins into the nucleus through interaction with a nuclear localization signal (NLS), composed of short stretches of basic amino acids. A predicted NLS in BSAP (NKRKRDE, located at amino acids 195-201 in the central domain) was confirmed to be essential for interaction with importin alpha1 by the yeast two-hybrid assay. Physical interaction between BSAP and importin alpha1 was detected in vitro by a glutathione S-transferase (GST) pulldown assay. The NLS sequence in BSAP conferred nuclear localization to green fluorescent protein (GFP)-BSAP fusion proteins. Although the N-terminal paired (DNA-binding) domain of BSAP also conferred nuclear localization when coupled to green fluorescent protein, this domain did not bind to importin alpha1 in the yeast two-hybrid assay. The NLS sequence in the central domain of BSAP binds to the C-terminal 98-amino acid fragment of importin alpha1.
Kovac
C R
CR
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Emelyanov
A
A
Singh
M
M
Ashouian
N
N
Birshtein
B K
BK
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000378:metabolism*; D051757:PAX5 Transcription Factor; D011485:Protein Binding; D014157:Transcription Factors; D028901:alpha Karyopherins
D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human; D014157:Transcription Factors; D028901:alpha Karyopherins; C087978:karyopherin alpha 2; D005982:Glutathione Transferase