Protein Synthesis In Mammalian Systems Research Articles

Wheat Germ Initiation Factor 2 (WG·eIF2) Decreases the Inhibition in Protein Synthesis and eIF2B Activity of Reticulocyte Lysates Mediated by eIF2α Phosphorylation

Phosphorylation of serine 51 residue in the α-subunit of eukaryotic initiation factor 2 (eIF2α) impairs the guanine nucleotide exchange (GNE) activity of eIF2B protein and thereby inhibits protein synthesis in mammalian systems, insects and yeast. It is not known if phosphorylation of plant eIF2 can inhibit an eIF2B-like activity. Interestingly purified wheat germ eIF2 (WG·eIF2) can exchange guanine nucleotidesin vitrowithout the addition of any protein factor like eIF2B. It is not clear if this is due to a contaminant eIF2B-like activity associated with WG·eIF2 or because the affinity of WG·eIF2 for GDP and GTP is not markedly different. Our observations here indicate that the GNE activity of WG·eIF2 is not inhibited upon phosphorylation of the p41–42 doublet subunit in WG·eIF2 by reticulocyte eIF2α kinases, or in the presence of reticulocyte eIF2(αP) in which serine 51 residue is phosphorylated. Further, addition of WG·eIF2 reduces the inhibition in eIF2B activity, protein synthesis, and also the formation of 15S complex that occurs between reticulocyte eIF2(αP) and eIF2B protein in heme-deficient or poly(IC)-treated reticulocyte lysates, presumably by a mechanism of competition between wheat germ and reticulocyte eIF2 for phosphorylation. Unlike reticulocyte eIF2(αP), phosphorylated WG·eIF2 is unable to interact with reticulocyte eIF2B to form a 15S complex. The ability of WG·eIF2 to exchange guanine nucleotides independent of an eIF2B like protein and the inability of phosphorylated WG·eIF2 to interact with reticulocyte eIF2B suggests that WG·eIF2 is different from mammalian eIF2 and these differences may have occurred in evolution probably due to some changes in the amino acid sequences around the phosphorylation site in eIF2α.

Phosphorylation of Wheat Germ Initiation Factor 2 (eIF-2) byN-Ethylmaleimide-Treated Wheat Germ Lysates and by Purified Casein Kinase II Does Not Affect the Guanine Nucleotide Exchange on eIF-2

Phosphorylation of the small subunit of eukaryotic initiation factor-2 (eIF-2α) impairs protein synthesis in mammalian systems. It is not known, however, if a similar regulatory mechanism exists in plants. Previous reports indicate that one of the wheat germ eIF-2 subunits, the p40–41 doublet, is phosphorylated by heterologous eIF-2α kinases. Here we report that phosphorylation of the small subunit in wheat germ eIF-2, p36, occurs in translating wheat germ lysates which are pretreated withN-ethylmaleimide (NEM) and dithiothreitol. Also, a purified sea star casein kinase II (CKII) phosphorylates the p41–42 doublet and p36 subunits of wheat germ eIF-2. While heme-regulated eIF-2α kinase from reticulocyte lysates does not inhibit wheat germ protein synthesis, CKII and NEM are found to be inhibitory. To determine whether phosphorylation of the small subunit (p36) is the cause for protein synthesis inhibition, we have further studied the exchange of labeled GDP for unlabeled GDP in the preformed eIF-2·[3H]GDP complexin vitroin the presence of CKII and ATP. The GDP exchange in eIF-2·GDP complex can occur without the addition of any protein factor and the exchange reaction is marginally inhibited by CKII. A 0–70% ammonium sulfate cut fraction, prepared from NEM-treated wheat germ lysate, also does not inhibit the guanine nucleotide exchange reaction. These findings suggest that the protein synthesis inhibition in these cases is not mediated by eIF-2 phosphorylation.

Characterization of Yeast Translation Initiation Factor 1A and Cloning of Its Essential Gene

Translation initiation factor eIF1A is required in vitro for maximal rates of protein synthesis in mammalian systems. It functions primarily by dissociating ribosomes and stabilizing 40 S preinitiation complexes. To better elucidate its precise role in promoting the translation initiation process, the yeast form of eIF1A has been identified in Saccharomyces cerevisiae and purified to homogeneity on the basis of its cross-reaction with antibodies prepared against mammalian eIF1A. The apparent mass of yeast eIF1A (22 kDa) resembles that of the mammalian homolog (20 kDa), and the yeast factor is active in stimulating methionyl-puromycin synthesis in an assay composed of mammalian components. The gene encoding yeast eIF1A, named TIF11, was cloned and shown to be single copy. TIF11 encodes a protein comprising 153 amino acids (17.4 kDa); the deduced amino acid sequence exhibits 65% identity with the sequence of human eIF1A. Both human and yeast eIF1A contain clusters of positive residues at the N terminus and negative residues at the C terminus. Deletion/disruption of TIF11 demonstrates that eIF1A is essential for cell growth. Expression of human eIF1A cDNA rescues the growth defect of TIF11-disrupted cells, indicating that the structure/function of yeast and mammalian eIF1A is highly conserved.

Initiation of the polypeptide chain by reticulocyte cell-free systems. Survey of different inhibitors of translation.

In order to elucidate the mechanism of action of inhibitors that block the initiation of protein synthesis in mammalian systems, we have studied the following steps: (a) formation of the ternary complex Met-tRNAr-IF-E2-GTP, (b) binding of the initiator Met-tRNAf to the 40-S ribosomal subunit in the presence of initiation factors and dependent or not on the addition of mRNA, (c) formation of the initiation complex with 80-S ribosomes and (d) formation of the first peptide bond. Adrenochrome, aurintricarboxylic acid, polydextran sulphate, pyrochatechol violet and showdomycin block the formation of the ternary complex Met-tRNAf-IF-E2-GTP. Edeine A1, aurintricarboxylic acid and polydextran sulphate block the binding of the mRNA to the 40-S ribosomal subunit. Pactamycin induces the formation of stable smaller initiation complexes which are unable to go through the subsequent steps of initiation. Stimulation of the binding of the initiator Met-tRNAf to the 80-S ribosome in the presence of initiation factors is observed with sparsomycin and antibiotics of the sesquiterpene family (verrucarin A, trichodermin and trichothecin). However, these antibiotics block the reaction of the bound Met-tRNAf with puromycin. Narciclasine has no effect on the binding of the initiator to the ribosome but strongly blocks its reaction with puromycin. We have developed a simple technique to detect the Met-tRNAf-40-S-subunit-poly(A, G, U) initiation complexes by chromatography on Sepharose 6B columns. The requirements for the formation of such complexes measured by this technique and its comparison with the sucrose gradient centrifugation method are described.

Polyglutamate Forms of Folate in Resting and Proliferating Mammalian Tissues

1. Tritiated folic acid (pteroylglutamic acid) was injected into rats whose livers were undergoing regeneration after partial hepatectomy, and into normal control rats. 2. The major form of labelled liver folate was found to be pteroylpentaglutamate in both hepatectomized and normal rats and there was no difference in the proportion of pteroylpentaglutamate to other forms of folate in the regenerating or resting liver. 3. Folate concentrations were also measured in leucocytes from normal and leukaemic patients. 4. Pteroylpolyglutamates constituted the main form of folate in both sets of cells. There was again no difference in proportion of pteroylpolyglutamate to pteroylmonoglutamate forms of folate between normal mature cells and primitive blast cells. 5. It is therefore suggested that pteroylpolyglutamates, which constitute nearly all the folates in cells actively synthesizing DNA and protein as well as in resting cells, probably act as the coenzymes in vivo for the folate-dependent reactions in DNA, RNA and protein synthesis in mammalian systems.