title: Are highly phosphorylated 40-S subunits preferentially utilized during protein synthesis in a cell-free system from HeLa cells?
abstract: It has been concluded from circumstantial evidence obtained with HeLa cells in vivo that the phosphorylation of ribosomal protein S6 increases the affinity of 40S particles for mRNP [Duncan, R. and McConkey, E. H. (1982) Eur. J. Biochem. 123, 535-538; Thomas, G., Martin-Pérez, J., Siegmann, M. and Otto, A.M. (1982) Cell 30, 235-242]. This conclusion needs to be tested in vitro in a reinitiating cell-free translation system from growth-competent cells. We have prepared such a system from HeLa cells and have compared the capacity of homologous 40S subunits of various degrees of phosphorylation to enter the existing polysome pool. The 40S subunits' degree of phosphorylation was manipulated by exposing aliquots of growth-stimulated HeLa cells to hyperthermia (see accompanying paper). 40S subunits from heat-shocked and control cells, despite differences in S6 phosphorylation level as verified by two-dimensional electrophoresis, did not differ with respect to their recruitment into the existing polysome fraction. Owing to the reinitiation activity of the translation system, assay times could be kept sufficiently short, to avoid any serious interference by the S6 phosphatase activities of the system. Our results suggest that increased S6 phosphorylation by itself is not sufficient to accelerate the participation of 40S subunits in protein synthesis.
mesh_terms: D002474:Cell-Free System; D006367:HeLa Cells / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism
chemical_list: D012335:RNA, Ribosomal; D012261:Ribonucleoproteins; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; C026452:messenger ribonucleoprotein