Cytoplasmic synthesis of nuclear proteins. Kinetics of accumulation of radioactive proteins in various cell fractions after brief pulses.
The synthesis of cytoplasmic and nuclear proteins has been studied in HeLa cells by examining the amount of radioactive protein appearing in the various subcellular fractions after labeling for brief periods. Due to the rapid equilibration of the amino acid pool, the total radioactivity in cytoplasmic protein increases linearly. The radioactivity observed in the cytoplasm is the sum of two components, the nascent proteins on the ribosomes and the completed proteins. At very short labeling times the specific activity of newly formed proteins found in the soluble supernatant fraction (completed protein) increases as the square of time, whereas the specific activity of the ribosomal fraction (nascent protein) reaches a plateau after 100 sec. The kinetics of accumulation of radioactive protein in the nucleus and the nucleolus is very similar to that of completed cytoplasmic protein, which suggests that the proteins are of similar origin. The rate of release and migration of proteins from the ribosomes into the nucleus requires less time than the synthesis of a polypeptide, which is about 80 sec. The uptake of label into nucleolar proteins is as rapid as the uptake of label into proteins of the soluble fraction of the cytoplasm, while nuclear proteins, including histones, tend to be labeled more slowly. The same results are obtained if protein synthesis is slowed with low concentrations of cycloheximide. The kinetics of incorporation of amino acids into various fractions of the cell indicates that the nucleus and the nucleolus contain few if any growing polypeptide chains, and thus do not synthesize their own proteins.
D000704:Analysis of Variance; D002247:Carbon Isotopes; D002458:Cell Fractionation; D002466:Cell Nucleolus / Q000032:analysis / Q000378:metabolism; D002467:Cell Nucleus / Q000032:analysis / Q000378:metabolism; D046508:Culture Techniques; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008861:Microsomes / Q000032:analysis; D009363:Neoplasm Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D009698:Nucleoproteins / Q000096:biosynthesis; D012270:Ribosomes / Q000378:metabolism; D013997:Time Factors; D014238:Trichloroacetic Acid; D014316:Tritium
D002247:Carbon Isotopes; D006657:Histones; D009363:Neoplasm Proteins; D009698:Nucleoproteins; D014316:Tritium; D014238:Trichloroacetic Acid; D003513:Cycloheximide; D007930:Leucine