A high-molecular-mass nucleolar protein (100-kDa protein) is associated with nascent pre-rRNA and preribosomes in Chinese hamster ovary cells. We have prepared antiserum against the 100-kDa protein and we have used it to study the intracellular localization and the possible processing of this protein.
Serologically related proteins were detected in the nucleolus and in ribosomes. Proteins of various subcellular fractions were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, reacted with serum and the protein-immunoglobulin complexes were revealed by 125I-labeled protein A. In the nucleolus, four proteins with molecular masses of 100 kDa, 95 kDa, 76 kDa and 70 kDa were thus visualized. In the ribosomes, two proteins (of 100 kDa and 76 kDa) gave a strong reaction while six others (of 70 kDa, 60 kDa, 50 kDa, 30 kDa, 21 kDa, 18 kDa) reacted slightly. By immunological precipitation of total cell extracts, we have shown that the 100-kDa protein antiserum cross-reacts with five proteins with molecular masses of 120 kDa, 100 kDa, 95 kDa, 70 kDa and 60 kDa.
Specific degradation of the 100-kDa protein into similar peptides with molecular masses of 95 kDa, 76 kDa, 70 kDa, 60 kDa and 50 kDa can be achieved by incubation of isolated nucleoli or of purified 100-kDa protein in vitro. Cleavage of the protein is due to a thiol endoprotease which is tightly bound to the 100-kDa protein. Possible relations between the maturation of this preribosomal protein into ribosomal proteins and the processing of preribosomal RNA into mature ribosomal RNA are discussed.
- CHO cells
Chinese hamster ovary cells
sodium dodecyl sulfate
phosphate-buffered dine (8 g NaCl/1, 1.15 g Na2HPO4/1, 0.2 g KCl/1, 0.2 g KH2PO4/1, pH 8.2