Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins. Many proteins are also N alpha-acetylated. The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation. The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions. In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid. Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure. All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained. These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.

Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.

Partially purified Met-tRNAf binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography. Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNAf and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg2+ and dissociated by Co-eIF-2B at 5 mM Mg2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-eIF-2C. Also under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNAf binding to 40S ribosomes.However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNAfr40S-AUG complex to the 60S ribosomal subunit to form Met-tRNAfr80S*AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRIphosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation. During heme deficiency, protein synthesis in reticulocyte lysates is inhibited due to activation of a latent protein synthesis inhibitor, heme-regulated inhibitor (HRI) (7-9). HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of Met-tRNAf binding factor, eIF-2 (10-14). HRI also inhibits the following partial initiation reactions and in each case, the inhibition requires ATP: (i) Co-eIF-2B-promoted dissociation of Met-tRNAf-eIF-2-GTP complex at high Mg2+ and low temperature (0°C) (TDF activity) (3, 15, 16); (fi) CoeIF-2C stimulation of ternary complex formation at 1 mM Mg2+ (5,[16][17][18]; and (iii) Met-tRNAf binding to 40S ribosomes (15,16,19). Based on these results, it has been proposed that HRI phosphorylation causes conformational modification of eIF-2 and that phosphorylated eIF-2 [eIF-2(P)] does not interact with two factors, Co-eIF-2B and Co-eIF-2C, and is inactive in peptide chain intiation. However, these studies do not conclusively establish the precise role of eIF-2(P) in the inhibition process and the possibility that the phosphorylation of other components such as HRI, Co-eIF-2B, and Co-eIF-2C also is involved in this inhibition has not been ruled out. Moreover, Trachsel and Staehelin (20) used prephosphorylated eIF-2 (HRI catalyzed) and observed that both eIF-2(P) and eIF-2 were equally active in all the partial initiation reactions studied including Met-tRNAf binding to 40S ribosomes. Also, the possibility that HRI can inhibit more than one step in peptide chain initiation has been indicated (21).To clearly establish the mechanism of HRI inhibition of peptide chain initiation and the precise role of HRI-phosphorylated eIF-2 in this inhibition process, we prep...

Purified preparations of epidermal growth factor (EGF) receptor were used to test hen oviduct progesterone receptor subunits as substrates for phosphorylation catalyzed by EGF receptor. Both the 80-kilodalton (kDa) (A) and the 105-kDa (B) progesterone receptor subunits were phosphorylated in a reaction that required EGF and EGF receptor. No phosphorylation of progesterone receptor subunits was observed in the absence of EGF receptor, even when Ca2+ was substituted for Mg2+ and Mn2 . Phospho amino acid analysis revealed phosphorylation at tyrosine residues, with no phosphorylation detectable at serine or threonine residues. Two-dimensional maps of phosphopeptides generated from phosphorylated 80-or 105-kDa subunits by tryptic digestion revealed similar patterns, with resolution of two major, several minor, and a number of very minor phosphopeptides. The Km of progesterone receptor for phosphorylation by EGF-activated EGF receptor was 100 nM and the Vmax was 2.5 nmol/ min per mg of EGF receptor protein at 0°C. The stoichiometry of phosphorylation/hormone binding for progesterone receptor subunits was 0.31 at ice-bath temperature and approximately 1.0 at 22°C.Tyrosine phosphorylation has become recognized as a protein modification reaction with potential roles in regulation of cell metabolism, reproduction, and differentiation. Viral transforming gene products and their normal cell homologues (1-4) and receptors for epidermal growth factor (EGF) (5), platelet-derived growth factor (6-8), and insulin (9-10) all catalyze transfer of phosphate from ATP to tyrosine residues of proteins. The best-characterized substrates for these kinases are the kinase proteins themselves; all undergo autophosphorylation. In addition, viral transformation of cells or binding of ligands to specific cell surface receptors stimulates tyrosine phosphorylation of cellular proteins (11-13). With the exception of vinculin (14) (20). To test for possible involvement of tyrosine phosphorylation in regulation of steroid receptor function, we examined purified hen oviduct progesterone receptor as a substrate for purified EGF receptor kinase. EXPERIMENTAL PROCEDURESPurification of Progesterone Receptor. The transformed progesterone receptor was purified from hen oviducts by a procedure similar to that of Chang et al. and B (105-kDa) subunits (kDa, kilodaltons). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/ PAGE) analysis of purified preparations showed minor impurities migrating at 280, 150, and 45 kDa on gels overloaded for receptor protein. In addition, there were faint bands immediately below the 80-and 105-kDa receptor bands that could arise from limited proteolysis; these increased when the serine protease inhibitor phenylmethylsulfonyl fluoride was omitted during receptor preparation. Receptor preparations were concentrated at the final step by precipitation with (NH4)2SO4 and dissolved in, and dialyzed against, 20 mM Hepes, pH 7.4. Photoaffinity labeling of the receptor subunits with 3H-labeled 17a, 21-dimethy...

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