Specificity of cotranslational amino-terminal processing of proteins in yeast.

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.

[1]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[2]  S. Moore,et al.  The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. , 1963, The Journal of biological chemistry.

[3]  H. Bunn,et al.  Amino-terminal processing of proteins: hemoglobin South Florida, a variant with retention of initiator methionine and N alpha-acetylation. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[4]  K. Narita,et al.  Isolation of acetylseryltyrosine from the chymotryptic digests of proteins of five strains of tobacco mosaic virus. , 1958, Biochimica et biophysica acta.

[5]  G. Fink,et al.  Transformation of yeast. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[6]  S. Cohen,et al.  Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. , 1980, Journal of molecular biology.

[7]  H. van der Wel,et al.  The complete amino-acid sequence of the sweet protein thaumatin I. , 1979, European journal of biochemistry.

[8]  H. Bloemendal.  The vertebrate eye lens. , 1977, Science.

[9]  J. Stewart,et al.  Amino-terminal processing of mutant forms of yeast iso-1-cytochrome c. The specificities of methionine aminopeptidase and acetyltransferase. , 1985, The Journal of biological chemistry.

[10]  G. Kreil,et al.  Micro method for detection of formyl and acetyl groups in proteins. , 1969, Analytical biochemistry.

[11]  F Sherman,et al.  Methionine or not methionine at the beginning of a protein , 1985, BioEssays : news and reviews in molecular, cellular and developmental biology.

[12]  C. Verrips,et al.  Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli. , 1982, Gene.

[13]  G. Blobel,et al.  Intracellular protein topogenesis. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[14]  W. D. de Jong,et al.  The mechanism of N-terminal acetylation of proteins. , 1985, CRC critical reviews in biochemistry.

[15]  R. Doolittle,et al.  Amino acid sequence studies on factor XIII and the peptide released during its activation by thrombin. , 1974, Biochemistry.

[16]  S. Kim,et al.  Three-dimensional structure of thaumatin I, an intensely sweet protein. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[17]  H. Jörnvall,et al.  Acetylation of Protein N-terminal amino groups structural observations on alpha-amino acetylated proteins. , 1975, Journal of theoretical biology.

[18]  R. A. Butow,et al.  How are proteins imported into mitochondria? , 1983, Cell.

[19]  Y. Kan,et al.  Hemoglobin Long Island is caused by a single mutation (adenine to cytosine) resulting in a failure to cleave amino-terminal methionine. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Lin,et al.  NH 2 -terminal formylmethionine- and NH 2 -terminal methionine-cleaving enzymes in rabbits. , 1972, The Journal of biological chemistry.

[21]  K. Itakura,et al.  Solid phase synthesis of polynucleotides. VI. Further studies on polystyrene copolymers for the solid support. , 1982, Nucleic acids research.

[22]  Finn Wold,et al.  How much sequence information is needed for the regulation of amino-terminal acetylation of eukaryotic proteins? , 1986 .

[23]  G von Heijne,et al.  Structures of N-terminally acetylated proteins. , 1985, European journal of biochemistry.

[24]  Y. Burstein,et al.  Primary structures of N-terminal extra peptide segments linked to the variable and constant regions of immunoglobulin light chain precursors: implications on the organization and controlled expression of immunoglobulin genes. , 1978, Biochemistry.

[25]  F. Wold.  Acetylated N-terminals in proteins — a perennial enigma , 1984 .

[26]  H C Pitot,et al.  Acetylation of nascent polypeptide chains on rat liver polyribosomes in vivo and in vitro. , 1975, Biochemistry.

[27]  T. Hunter,et al.  Role of Methionine in the Initiation of Haemoglobin Synthesis , 1970, Nature.