Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis

Abstract

To mimic what might happen in cells exposed to hypertonicity, the effects of increased osmolarity and ionic strength on cell-free protein synthesis have been examined. Translation of globin mRNA by rabbit reticulocyte lysate decreased by 30—60% when osmolality was increased from 0.35 to 0.53osmol/kg of water by the addition of NaCl, KCl, CH3CO2Na or CH3CO2K. In contrast, equivalent additions of the compatible osmolytes betaine or myo-inositol caused a 40—50% increase in the rate of translation, whereas amino acids (50—135mM) that are transported via system A had no significant effect. Addition of 75mM KCl caused a dramatic fall in the amount of the 43S pre-initiation complex, whereas it was totally preserved when osmolarity was similarly increased by the addition of 150mM betaine. The formation of a non-enzymic initiation complex between rabbit [3H]Phe-tRNA, poly(U) and the 80S ribosomes was unaffected by the addition of 75mM NaCl or KCl, but translation of the complex decreased by 70%. Density-gradient centrifugation of reticulocyte extracts translating endogenous mRNA revealed that addition of 150mM betaine had no effect, whereas addition of 75mM KCl caused a marked decrease in the polysome peak, concomitant with an increase in the proportion of 80S ribosomes and ribosomal subunits, even when elongation was inhibited with fragment A of diphtheria toxin. These results are consistent with the notion that both initiation and elongation are inhibited by unusually high concentrations of inorganic ions, but not by the compatible osmolytes betaine or myo-inositol.