An improved genetic system for detection and analysis of protein nuclear import signals.
BACKGROUND
Nuclear 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.


RESULTS
We 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.


CONCLUSIONS
The alterations in expression level and composition of the fusions used in this nuclear import system greatly reduce background activity in beta-galactosidase assays, improving sensitivity and allowing more quantitative analysis of NLS bearing sequences.
Marshall
Kris S
KS
Department of Microbiology & Immunology, The University of Western Ontario, London Regional Cancer Program, London, Ontario, N6A 4L6, Canada. kmarsh23@uwo.ca
Zhang
Zhiying
Z
Curran
Jennifer
J
Derbyshire
Stephanie
S
Mymryk
Joe S
JS
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D017500:Adenovirus E1A Proteins / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D016384:Consensus Sequence; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins / Q000235:genetics; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis / Q000235:genetics; D028884:Karyopherins / Q000502:physiology; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033902:Periplasmic Binding Proteins / Q000235:genetics; D021382:Protein Sorting Signals / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics; D014170:Transformation, Genetic; D000099160:LexA Repressor Protein
D017500:Adenovirus E1A Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D028884:Karyopherins; D050506:Mutant Chimeric Proteins; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C052130:GAL4 protein, S cerevisiae; C461619:MalE protein, E coli