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.
Huang
S
S
INGENE, Inc., Santa Monica, California 90404.
Elliott
R C
RC
Liu
P S
PS
Koduri
R K
RK
Weickmann
J L
JL
Lee
J H
JH
Blair
L C
LC
Ghosh-Dastidar
P
P
Bradshaw
R A
RA
Bryan
K M
KM
D000123:Acetyltransferases / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010443:Peptide Chain Termination, Translational; D010940:Plant Proteins / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013549:Sweetening Agents
D010940:Plant Proteins; D013549:Sweetening Agents; C003427:thaumatin protein, plant; D000123:Acetyltransferases