The nuclear localization signal of NGFI-A is located within the zinc finger DNA binding domain.
NGFI-A is an immediate-early gene that encodes a transcription factor whose DNA binding domain is composed of three C2H2 zinc fingers. To identify its nuclear localization signal (NLS), wild type NGFI-A and various mutants were transfected into COS cells and their cellular location assayed by indirect immunofluorescence. Although wild type NGFI-A was located exclusively within the nucleus, deletions lacking the highly basic zinc finger region were not efficiently translocated to the nucleus. However, DNA binding per se is not required for nuclear localization, as an NGFI-A mutant (A Y339G), which does not bind DNA, is still faithfully directed to the nucleus. To determine the minimal region(s) of NGFI-A sufficient to direct nuclear localization, the cellular location of various NGFI-A/beta-galactosidase fusion proteins was examined. Fusion proteins containing all three zinc fingers were found in the nucleus, but those containing only two zinc fingers were predominantly cytoplasmic. When the zinc finger structure was altered by mutating a zinc-chelating cysteine residue in any one of the three zinc fingers, the resulting domain was no longer capable of directing beta-galactosidase to the nucleus. Furthermore, the mutation of an arginine residue in the third zinc finger of NGFI-A, a position which is occupied by a leucine residue in most C2H2 zinc fingers, abolished nuclear localization, but had no effect on DNA binding. These studies suggest that NGFI-A contains a novel NLS which is dependent on the overall structure of the DNA binding domain and not solely upon its highly basic nature.
Matheny
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Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Day
M L
ML
Milbrandt
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D051766:Early Growth Response Protein 1; D017781:Genes, Immediate-Early; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D016335:Zinc Fingers / Q000235:genetics*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D017874:Immediate-Early Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D001120:Arginine; D001616:beta-Galactosidase