Abstract
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.
Highlights
BSAP (Pax5) is a transcription factor expressed exclusively in the developing midbrain, testes, pro-B cells, pre-B cells, and mature B cells [1]
nuclear localization signal (NLS) motifs bound by importins are composed of short stretches of highly basic amino acids, some of which are a single stretch of amino acids, others of which comprise two short stretches separated by a spacer region
The physical interaction between BSAP and importin ␣1 was confirmed in glutathione S-transferase (GST) pulldown assays
Summary
BSAP (Pax5) is a transcription factor expressed exclusively in the developing midbrain, testes, pro-B cells, pre-B cells, and mature B cells [1]. 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 ␣1 by the yeast two-hybrid assay.
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