MODULATION OF PROTEIN SYNTHESIS AND EUKARYOTIC INITIATION FACTOR 2 (eIF-2) FUNCTION DURING NUTRIENT DEPRIVAL IN THE EHRLICH ASCITES TUMOR CELL

Abstract

ABSTRACT We have shown previously that initiation is inhibited in Ehrlich cells deprived of an essential amino acid or of glucose, and that an inhibited step in initiation is the binding of Met-tRNA f to the native 40s ribosomal subunit. This step is mediated by eukaryotic initiation factor 2 (eIF-2). In the present work we have studied the amount and degree of phosphorylation of eIF-2 in the ribosome-bound (KCl wash) and soluble (S-100) fractions of fed, glucose-deprived, and amino acid-deprived cells. The amount of eIF-2, measured by the Millipore filter binding assay, is similar in fed and deprived cells, in both the KCl wash and the S-100 fractions. In cells exposed to 32 Pi for two hours, the 38,000 dalton subunit of eIF-2 is labeled in eIF-2 purified from total ribosomes or from native 40s subunits, but not in eIF-2 purified from the soluble fraction. Function of eIF-2 requires cycling from the free to the ribosome-bound state, and these data suggest that phosphorylation of eIF-2 may play a role in its cycling. The degree of labeling of the 38,000 dalton subunit is similar in eIF-2 of fed compared to nutrient-deprived cells, whether extracted from the total ribosomal KCl wash fraction or from the native 40s subunits. The 48,000 dalton subunit is also phosphorylated in vivo and differences were not seen between fed and deprived cells. If the degree of labeling reflects the extent of phosphorylation of the proteins, these data indicate that phosphorylation of eIF-2 is not the mechanism regulating initiation in glucose- or amino acid-deprived Ehrlich cells. A ribosomal protein of molecular weight 36,000, presumably S6, is much more highly labeled by 32 Pi in ribosomes of fed than of deprived cells. Monomeric ribosomes and polyribosomes are equally labeled.