Intracellular localization of GBF proteins and blue light-induced import of GBF2 fusion proteins into the nucleus of cultured Arabidopsis and soybean cells.
The G-box is an important regulatory element found in the promoters of many different genes. Four members of an Arabidopsis gene family encoding basic leucine zipper proteins (GBFs) which bind the G-box have previously been cloned. To study GBFs, a polyclonal antibody was raised against GBF1 expressed in bacteria. This antibody also recognized GBF2 and GBF3. Immunoblot analysis of nuclear and cytoplasmic fractions from Arabidopsis and soybean (SB-M) cell cultures indicated that over 90% of proteins detected with anti-GBF1 were cytoplasmic. Electrophoretic mobility shift assays indicated that over 90% of G-box binding activity was cytoplasmic. DNA affinity chromatography demonstrated that each protein detected with anti-GBF1 specifically bound the G-box. To study individual GBFs, DNA constructs fusing GBF1, GBF2 and GBF4 to GUS were made and assayed by transient expression in SB-M protoplasts. Of GUS:GBF1 proteins, 50-62% were localized in the cytoplasm under all conditions tested, while 97% of GUS:GBF4 was localized in the nucleus. By contrast, whereas about 50% of GUS:GBF2 was found in the cytoplasm of dark-grown cells, over 80% of this protein was found in the nucleus in cells cultured under blue light. Deletion analysis of GBF1 identified a region between amino acids 112 and 164 apparently required for cytoplasmic retention. These results suggest the intriguing possibility that limitation of nuclear access may be an important control on GBF activity. In particular, GBF2 is apparently specifically imported into the nucleus in response to light.
Terzaghi
W B
WB
Department of Biology, University of Pennsylvania, Philadelphia 19104, USA. terzaghi@Wilkes1.wilkes.edu
Bertekap
R L
RL
Cashmore
A R
AR
D000918:Antibody Specificity; D017360:Arabidopsis / Q000166:cytology; D050976:Basic-Leucine Zipper Transcription Factors; D001692:Biological Transport / Q000528:radiation effects; D002451:Cell Compartmentation / Q000528:radiation effects*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003624:Darkness; D051790:G-Box Binding Factors; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics; D049452:Green Fluorescent Proteins; D016350:Leucine Zippers; D008027:Light; D008164:Luminescent Proteins / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013025:Glycine max / Q000166:cytology; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D008164:Luminescent Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D005966:Glucuronidase