CELL CYCLE-DEPENDENT TRANSLOCATIONS OF A MAJOR NUCLEOLAR PHOSPHOPROTEIN, B23, AND SOME CHARACTERISTICS OF ITS VARIANTS

A major nucleolar phosphoprotein, B23, is thought to play several appa rently unrelated roles and appears to be associated with other cell co mpartments besides tile nucleolus, However, characteristic properties of B23 variants still remain to be established, Here, we raised a new monoclonal antibody against B23 (20B2) and used it to address the issu e particularly focusing can the events during mitosis. Also, we made a n attempt to generalize the data on tile cell cycle-dependent transloc ations of B23 by the use of three mammalian cell lines (HeLa, PK, RAMT ) which were found to be immunoreactive for 20B2. In all tile cell str ains studied, B23 was chiefly located within the nucleolus at interpha se, and was associated with a felv cellular domains during mitosis. Th ey were: the nucleoplasm (at prophase before the nuclear envelope brea kdown), the cytoplasm (from prometaphase until mid telophase), the per ichromosomal layer (from prometaphase till early telophase), cytoplasm ic B23-containing bodies (at anaphase and telophase) and prenucleolar bodies, PNBs (at telophase). On Western blots, electrophoretic mobilit y of B23 was found to be the same at G1, S and G2 periods of interphas e, but became slower at mitosis. In situ and cell extraction experimen ts shelved that like the nucleolar B23, B23 of the perichromosomal lay er and that of PNBs was highly resistant to extraction with Triton X-1 00, but could be released with Triton X-100/RNase A. These findings in dicated that these portions of B23 mere most likely to be associated v iith RNA. The cytoplasmic B23 was the major intracellular variant of B 23 during mitosis. It had a slightly lower electrophoretic mobility th an the perichromosomal B23 and could readily he extracted with Triton X-100 without addition of RNase A, a fact indicating that the cytoplas mic B23 was mainly in an RNA-free state. Mitosis-like translocations o f B23 from the nucleolus to the nucleoplasm induced by actinomycin D i ncreased its extractability but did not affect the electrophoretic mob ility The phosphorylation status of different B23 variants at interpha se and mitosis both in controls and following the drug, is discussed.