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(1) Protein N-terminal modifications
Although there are only 20 primary amino acids that are encoded during translation, it is found that more than 100 different enzymatically modified amino acid residues are known from different proteins. The two cotranslational processes, cleavage of N-terminal methionine and N-terminal acetylation (N- acetylation), are by far the most common modifications, occurring on the vast majority of eukaryotic proteins.
N-acetylation is an enzyme-catalyzed reaction in which the protein N-terminal residues, such as a-Ser, a-Ala, a-Met, etc., accepts the acetyl group from acetyl-CoA. This modification neutralizes positive charges that may influence the protein function, stability, interaction with other molecules, or other subsequent modifications. The reaction is catalyzed by a number of acetyltransferases (NATs) that have been found in all kingdoms, prokaryotes, archaea and eukaryotes. N-acetylation is occurring on approximately 80-90% of the different varieties of cytosolic mammalian proteins, on about 50% of yeast proteins, but rarely on prokaryotic or archaeal proteins. It is believed that N-acetylation is cotranslational only in eukaryotes but not in prokaryotes, where it is posttranslational. In vitro studies indicated that NATs act on the newly synthesized polypeptide when there are between 25 to 50 residues extruding from the ribosome.
In our studies with yeast Saccharomyces cerevisiae we revealed that N-terminal protein acetylation occurs mainly by action of three NATs, NatA, NatB and NatC, which contain Ard1p, Nat3p and Mak3p catalytic subunits, respectively, and which act on groups of substrates, each containing degenerate motifs. NatA acetylates a subclasses of proteins with Ser-, Ala-, Gly- and Thr- termini; NatB acetylates Met-Glu- and Met-Asp- termini; and NatC acetylates a rare class of Met- termini. Recently, an additional NAT, Nat4p (NatD) was shown to acetylate the N-termini of histones H2A and H4, Ser-Gly-Gly-Lys-Gly- and Ser-Gly-Arg-Gly-Arg-, respectively. However, only subsets of proteins with any of these N-terminal residues are acetylated, and none of these residues guarantee acetylation, indicating that the enzymes recognize some structural characteristics of the N-terminal portion in addition to a particular amino acid sequence. Overall, the patterns of N-terminally acetylated proteins and orthologous genes possibly encoding NATs suggest that yeast and higher eukaryotes have the same or very similar system for N-terminal acetylation.
Three major NATs, NatA, NatB and NatC are heteromeric protein complexes containing at least one auxiliary subunit in addition to catalytic subunit, in contrast to NatD that appears to have no additional subunit. Interestingly, NatA contains two potential catalytic subunits, Ard1p and hypothetical acetyltransferase Nat5p, presumably with different substrate specificities. It has been shown recently that Nat1p is attached to the ribosome. In our experiments we demonstrated that Ard1p, Mak3p and Nat4p are cytoplasmic proteins, which were co-localized with polyribosomes in sucrose gradient. We suggested that the three auxiliary subunits, Nat1p, Mdm20p and Mak10p, may play a role in NAT attachment to the ribosome and recognition of a proper protein substrate.
(2) Protein methylation
A wide range of proteins are posttranslationally methylated, including histones, ribosomal proteins, and cytochromes c. Methylation reaction occurs as a result of enzymatic transfer of methyl group of S-adenosine methionine to the free amino groups of several amino acid residues.
Previously we identified cytochrome c methyltransferase, Ctm1p, which is responsible for trimethylating lysine at position 72 of iso-1-cytochrome c (Cyc1p). Ctm1p is located in cytosol and corresponding gene is coordinately regulated with CYC1 expression during anaerobic to aerobic transition.
Methylation of ribosomal proteins has been observed in diverse organisms, from bacteria to mammals, although the enzymes responsible for such modification were not clearly identified. Methylation occurs primarily on lysine, arginine, and glutamine residues, however the biological significance of these modifications is unknown. Possibly, it may play a role in proper functioning of the ribosome or its hierarchical assembly.
Currently we are working on identification of the methyltransferases responsible for methylating ribosomal proteins by using overexpressed candidate genes and hypomethylated ribosomal proteins as a substrate. Detection of the methylated proteins and modification sites will help us to understand the function of these protein methylations.
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