Studies on the origin of proteins in the nucleoli of mouse ascites tumour cells: A test for protein transfer to the nucleoli

Abstract

Since it has been reported from this laboratory that the nucleoli of mouse ascites tumour cells are incapable of synthesizing proteins via a typical ribosomal system, the possibility of transfer of newly synthesized protein molecules to the nucleoli from other cellular components was tested. 1. 1. Pulse-chase experiments of amino acids were carried out on ascites tumour cells cultivated temporarily in a defined medium, RPMI 1640 supplemented with 5 to 10 % calf serum. Virtually complete inhibition of amino acid incorporation into subcellular components was attained by administration of both cycloheximide (100–200 μg/ml) and an excess of unlabeled amino acids (7500 times). 2. 2. After a 1- to 2-min pulse, radioactivity in the nucleoli was increased by 70 to 100 % during a 20-min chase. Accumulation of the labeled protein during the chase was also observed, but to a smaller extent, in an extranucleolar nuclear fraction. These increments found in the subnuclear fractions were always accompanied by a significant decline of the radioactivity in the cytoplasmic 25 000 × g supernatant fraction. 3. 3. Polyacrylamide gel electrophoresis demonstrated that the pattern of basic proteins labeled by a 1-min pulse and a subsequent 20-min chase was exactly the same as that obtained by a 1-min pulse only. 4. 4. The decline of radioactivity in cytoplasmic 25 000 × g supernatant fraction during the chase was shown to be the result of a remarkable decrease in the 105 000 × g sediment, microsomes and a concomitant but smaller increase in the 105 000 × g supernatant fraction. 5. 5. Protein transfer to the nucleoli was affected by temperature and by both actinomycin D and 2,4-dinitrophenol. These effects suggest that the protein transfer may not be a simple diffusion process, but an active one mediated by RNA content and by ATP level. 6. 6. These results strongly suggest that active incorporation of amino acids into the nucleoli in vivo is largely, if not entirely, based on the uptake of protein molecules synthesized on the outside of this organelle, probably in the cytoplasmic polysomes.