The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization.
Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.
Heine
M A
MA
Department of Biochemistry, Louisiana State University, Baton Rouge 70810.
Rankin
M L
ML
DiMario
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D002466:Cell Nucleolus / Q000737:chemistry / Q000378:metabolism*; D002854:Chromatography, Paper; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis; D000939:Epitopes; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D016259:Genes, myc; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D009865:Oocytes; D010750:Phosphoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014981:Xenopus; D000097796:Nucleolin
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000939:Epitopes; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016271:Proto-Oncogene Proteins c-myc; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D000097796:Nucleolin