Initiation of protein synthesis in Ehrlich ascites tumour cells. Evidence for physiological variation in the association of methionyl-tRNAf with native 40-S ribosomal subunits in vivo

Abstract

Binding of methionyl-tRNAf to native 40-S ribosomal subunits is thought to be an early stage in the process of polypeptide chain initiation, and [35S]Met-tRNAf - 40-S-subunit complexes can be isolated from Ehrlich ascites tumour cells following a brief incubation with [35S]methionine. To determine whether this step is subject to modulation by physiological conditions, we have estimated the extent of binding of Met-tRNAf to native- 40S ribosomal subunits in Ehrlich cells under nutritional conditions known to affect the rate of protein synthesis in these cells. Deprivation of either an essential amino acid, lysine, or of glucose, results in a substantial reduction in the proportion of native 40-S subunits which have Met-tRNAf associated with them, and refeeding of lysine to cells deprived of this amino acid partially reverses this effect within 10 min. These effects on the concentration of Met-tRNA - 40-S-subunit complexes are paralleled by changes of similar magnitude in the rate of protein synthesis and in polyribosome profiles. Native 40-S subunits can be spearated by equilibrium density gradient analysis on caesium chloride into two species, with buoyant densities approximately 1.40 and 1.49 g X cm-3. In cells deprived of either lysine or glucose, the radioactivity from [35S]methionine is bound exclusively to the particle of buoyant density 1.40 g X cm-3. In well-fed cells, or in starved cells shortly after refeeding, a significant proportion of the label is associated with a region of the CsCl gradient corresponding to a particle of higher density. The results suggest that the binding of Met-tRNAf to native 40-S ribosomal subunits can be greatly affected by physiological conditions which alter the rate of protein synthesis. This is consistent with a regulatory role for this step in the sequence of reactions involved in initiation of translation.