NUCLEOLAR PROTEIN B23 STIMULATES NUCLEAR IMPORT OF THE HIV-1 REV PROTEIN AND NLS-CONJUGATED ALBUMIN

Nucleolar phosphoprotein B23 is a putative ribosome assembly factor wi th a relatively high affinity for peptides containing sequences of nuc lear localization signals (NLSs) of the SV40 T-antigen type [Szebeni, A., Herrera, J. E., & Olson, O. J. (1995) Biochemistry 34, 8037-8042]. The effects of protein B23 on nuclear import were determined by an in vitro assay [Dean, D. A., & Kasamatsu, H. (1994) J. Biol. Chem. 269, 4910-4916] using NLS peptide-conjugated bovine serum albumin (NLS-BSA) or the HIV-1 Rev protein as substrates for import into isolated rat l iver nuclei. The import was ATP-dependent and inhibited by wheat germ agglutinin or by an antibody against p97, a component of the nuclear i mport system. The rate of import of either substrate was increased if protein B23 was added to the incubation medium. Similar enhancements o f import were seen with both isoforms (B23.1 and B23.2). The stimulato ry effect on Rev protein import was saturable with maximum stimulation (2-3-fold) at a molar ratio of protein B23:Rev of approximately 1:1. Phosphorylation of protein B23.1 by casein kinase II produced an addit ional doubling of the import rate. This effect was not seen if protein B23.1 was phosphorylated with a cdc2 type protein kinase. Mutant form s of protein B23.1 in which the nuclear localization signal was either deleted or altered did not stimulate import of the substrates. These results suggest that protein B23 plays a role as an accessory factor i n the nuclear import of the NLS-containing proteins and that phosphory lation at sites in the highly acidic segments of the protein enhances the stimulatory effect.