Abstract
BackgroundNuclear import of proteins is typically mediated by their physical interaction with soluble cytosolic receptor proteins via a nuclear localization signal (NLS). A simple genetic assay to detect active NLSs based on their function in the yeast Saccharomyces cerevisiae has been previously described. In that system, a chimera consisting of a modified bacterial LexA DNA binding domain and the transcriptional activation domain of the yeast Gal4 protein is fused to a candidate NLS. A functional NLS will redirect the chimeric fusion to the yeast cell nucleus and activate transcription of a reporter gene.ResultsWe have reengineered this nuclear import system to expand its utility and tested it using known NLS sequences from adenovirus E1A. Firstly, the vector has been reconstructed to reduce the level of chimera expression. Secondly, an irrelevant "stuffer" sequence from the E. coli maltose binding protein was used to increase the size of the chimera above the passive diffusion limit of the nuclear pore complex. The improved vector also contains an expanded multiple cloning site and a hemagglutinin epitope tag to allow confirmation of expression.ConclusionThe alterations in expression level and composition of the fusions used in this nuclear import system greatly reduce background activity in β-galactosidase assays, improving sensitivity and allowing more quantitative analysis of NLS bearing sequences.
Highlights
Nuclear import of proteins is typically mediated by their physical interaction with soluble cytosolic receptor proteins via a nuclear localization signal (NLS)
We evaluated nuclear import conferred by fusing the full length adenovirus type 5 (Ad5) 289 amino acid residues (289R) early region 1A (E1A) protein or the N-terminal 82 amino acids (1–82) in frame to the modified LexA DBDGal4 activation domain (AD) chimera (Fig. 2)
A substantial increase in β-galactosidase activity was detected with E1A 1–82 as compared to the empty pNIA vector, surprisingly little change was observed with the full length 289R E1A protein or the well studied SV40 large T antigen NLS
Summary
Nuclear import of proteins is typically mediated by their physical interaction with soluble cytosolic receptor proteins via a nuclear localization signal (NLS). Nuclear import of many cellular and viral proteins is typically mediated by their physical interaction with soluble cytosolic receptor proteins via nuclear localization signals (NLS) [1]. A canonical NLS is characterized by a short single stretch of basic amino acids, as exemplified by the NLS sequence of the large T antigen of the simian virus 40 (PKKKRKV) [2]. Proteins containing NLSs are imported into the nucleus by interacting in the cytosol with members of the importin α family of NLS receptors ( known as karyopherin α). At least in part, an extraordinarily diverse range of cellular processes including cell cycle, signal transduction, apoptosis and circadian rhythm, the identification of mechanisms regulating nuclear import is a valuable area of investigation
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