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 cel ls 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 nucleoli n were tagged at their amino termini with five tandem human c-myc epit opes. Whether produced in E. coli or in Xenopus, epitope-tagged full-l ength nucleolin bound nucleic acid probes in in vitro filter binding a ssays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation mai ntained 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 oocyt e nuclei. The epitope-tagged GAR truncation also translocated to the o ocyte nuclei, but it failed to efficiently localize to the nucleoli. O ur results show that the carboxy GAR domain must be present for nucleo lin to efficiently bind nucleic acids in vitro and to associate with n ucleoli in vivo.