Overexpression of Human Nucleolar Proteins in Insect Cells: Characterization of Nucleolar Protein p120

Nucleolar p120 is a proliferation-associated protein, which becomes detectable early in the G1phase of the cell cycle and peaks early in the S phase. A variety of human malignant tumor cells contain much higher levels of p120 than normal resting cells. The cellular functions of p120 are unknown, and little information is available on the structural characteristics of the human p120 protein. For biochemical characterization, human p120 protein was expressed in a baculovirus system and purified to approximately 95% purity. By indirect immunofluorescence, most of the recombinant human p120 as well as recombinant human B23, C23, or fibrillarin were localized to insect cell nucleoli and to large globular nuclear inclusions. Like endogenous p120 in HeLa cells, recombinant p120 expressed in insect cells was phosphorylated. On sucrose density gradients, p120 from HeLa cells sedimented in the 60–80S region, in which preribosomal particles sedimented using similar extraction and centrifugation procedures. The sedimentation of p120 shifted to the 5–10S region by treatment with 1MKCl or with RNAse which suggests that p120 is bound to RNA.