Members and sponsors are encouraged to share ideas, applications research and experiences, or technical problems by contributing to this column. In addition, short reviews covering aspects of biotechnology that are of interest to members are also encouraged. All contributions will be subject to peer review and the deadline for submitting articles for the June issue is May 1, 1993. Periodically, articles will also be solicited from non-members by the editors. Contributions may be mailed or faxed to the Editor, Ken Williams, W.M. Keck Foundation Biotechnology Resource Laboratory, P.O. Box 9812, 295 Congress Avenue, New Haven, CT 06536-0812, (201) 737-2206, Fax (203) 737-2638. Contributions will not necessarily reflect views of the editorial board or the Association.
NH2-terminal modifications of nascent polypeptides are by far the most common processing events, occurring on nearly all proteins (1). In eukaryotes, all proteins are initiated with methionine, whereas in prokaryotes, mitochondria and chloroplasts, translation of proteins is initiated with N-formyl methionine. The formyl group is usually removed co-translationally from proteins by a deformylase, leaving methionine bearing a free NH2 group. In eukaryotic as well as in prokaryotic cells, the NH2-terminal methionine may then be removed by a methionine aminopeptidase (MAP), if the penultimate amino acid is small and uncharged. The removal of methionine is essential for further NH2-terminal modifications (e.g., acetylation by N-alpha-acetyltransferase and myristoylation of glycine by N-myristoyltransferase). The retention of methionine, on the other hand, may protect short-lived proteins from premature protein degradation, according to the N-end rule, proposed by Varshavsky and coworkers.
The prokaryotic genes encoding MAPs from E. coli, S. typhimurium and B. subtilis are cloned and sequenced, and it is known that MAP genes from E. coli and S. typhimurium are essential for cell growth (2). A eukaryotic MAP from Saccharomyces cerevisiae is also purified, and its in vitro specificity agrees with the specificity observed in vivo for yeast (3). However, its primary structure differs from the prokaryotic enzyme by the addition of a NH2-terminal domain ( ~ 120 residues) containing two zinc-finger motifs, which likely contribute to MAP's interaction with ribosomes (4). Interestingly, the deletion of the yeast MAP1 gene is not lethal, suggesting that there are additional, as yet undetermined, processing pathways for the removal of NH2-terminal methionines from nascent eukaryotic proteins.
N-acetylation is the major co-translational modification of the alpha-amino group of eukaryotic proteins, and it markedly affects the biological function of proteins and peptides. For example, N-alpha-acetylation has a profound effect on the activity of two products of proopiomelanocortin (beta-endophin and alpha-MSH), affinity of hemoglobin for oxygen, heat-stability of proteins, growth and mating of yeast, and protein turnover mediated by the ubiquitin-dependent degradation system.
The acetyl moiety was initially identified as an NH2-terminal blocking group in tobacco mosaic virus coat protein (5), and it was later found that ~ 80% of the soluble proteins in higher eukaryotic cells are N-acetylated. This percentage was calculated from the data of Brown & Roberts (6) indicating the amount of acetate derived from N-alpha-acetylated amino acids originating from the soluble proteins in mouse L-cells and Ehrlich ascites cells and an estimate of the average Mr of the soluble proteins present in these cells. However, it was later demonstrated by comparing the 2-D gel electrophoresis patterns of the proteins from wild type (AAA1; also called NATl (7)) and mutant (aaal; also called natl (7)) yeast, which lacks one of the two known N-alpha-acetyltransferases in yeast (see below), that 20deg. o of the soluble proteins from yeast are N-alpha-acetylated (8). Out of 855 proteins identified in the 2-D gel pattern of the wild type yeast, 48 (~6%) shifted to a higher pI with no detectable change in Mr in the mutant yeast. Such a result is expected for proteins lacking an NH2-terminal acetyl group. Another 144 proteins observed in the wild type yeast disappeared from the 2-D gel pattern of the mutant strain, and we have suggested that these proteins are rapidly degraded, because they are not N-alpha-acetylated. However, since other proteins in the wild type strain are also N-acetylated by methionine N-acetyltransferase (M-NYAT, see below), our estimate of the percentage of acetylated proteins in yeast modified by N-acetyltransferase is clearly a lower limit, and it is not currently known whether there are significant differences in the extent of Ndeg.'-acetylation between lower and higher eukaryotes.
N-acetylation is the principal cause for the "blocked proteins" identified by Edman degradation. Unfortunately, there is no chemical or enzyme methods, which will specifically remove the acetyl group from the alpha-amino group of a protein, although it is possible to remove the acetylated, NH2-terminal residue from short (< 15 residues) peptides by acylpeptide hydrolase.
N-acetylated proteins exist as two distinct classes: (i) those for which the penultimate residue is acetylated after cleavage of the initiator methionine and (ii) those for which the initiator methionine is acetylated, when the initiator methionine is followed by Asn, Asp, Gln, or Glu (Fig. 1).
From an analysis of eukaryotic protein sequences, it was previously demonstrated that five amino acid residues (i.e., alanine, serine, methionine, glycine and threonine) account for 95deg.70 of all acetylated residues in eukaryotic proteins (9). Although this predominant use of certain amino acid residues suggests that an NH2-terminal residue is the primary recognition signal for N-acetyltransferases, there are numerous examples of proteins having these amino acids at their NH2-termini that are not acetylated. Thus, it is likely that the enzymes may also recognize adjacent residues (e.g., N-acetylated methionine is usually followed by Asn, Asp, Gln, or Glu), distal residues, and conformational features within the NH2-terminal region of a protein or peptide.
Ndeg.'-Acetylation is mediated by at least two N-acetyltransferases, which catalyze the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides.
N-acetyltransferase (NAT) was purified to homogeneity from yeast (10) and is known to have broad specificity and to catalyze Ndeg.'-acetylation of peptide substrates bearing any NH2-terminal residue, except Arg, Lys, Gln, lle, Leu, and Trp (11). At present, it is not known whether additional NAT's with specificity for any of these residues exist in eukaryotic cells.
M-NAT with specificity for methionine was later identified (12). Using two series of synthetic peptide substrates mimicking the first 24 residues of yeast proteinase A inhibitor 3 (PAI), the only endogenous yeast protein proven biochemically to be N-acetylated at its NH2-terminal methionine, and iso-l-cytochrome c variants (previously established to be N-acetylated in vivo (13)), N-acetylation of methionine occurred only for substrates containing Asn, Asp, Gln, or Glu as the penultimate residue (12; F.-J.S. Lee, L.-W. Lin & J.A. Smith, unpublished). Furthermore, the differences in the relative activity for acetylation of the NH2-terminal Met of PAI ( 1-24) and its analogs indicated that the penultimate residue has a major role in controlling N-acetylation by M-NAT. Although a natural protein containing a N-acetylated NH2-terminal methionine followed by Gln is not observed in the major protein databases, its absence may be due to the limited number of N-acetylated proteins that have been biochemically characterized .
What role do NAT's play in controlling cell physiology? Clearly, they play an important role in controlling cell growth and mating in yeast, as was shown by disrupting the NAT gene (AAA1) (14). The yeast mutant (aaal), lacking all NAT activity, was also sporulation defective, sensitive to heat shock, and a-type mating function defective (14). However, which acetylated proteins control these critical growth, mating, and response functions is still unknown.
Are NAT genes regulated transcriptionally? In the case of the AAA1 gene, the answer is no, since it is constitutively expressed (15). No conclusion can be made regarding AAA2 gene, because it has not yet been cloned.
Are NAT activities regulated translationally? Interestingly, another yeast mutant, ard1 (7), also lacks NAT activity (i.e., can not acetylate NH2-terminal Ser- and Ala-containing synthetic peptide substrates), but the ard1 mutant still possesses M-NAT activity (F-J.S. Lee, L.-W. Lin, and J.A. Smith, unpublished). Furthermore, the 2-,D gel electrophoretic patterns of the proteins from aaal and ard1 mutants are very similar but not identical (F-J.S. Lee, L.-W. Lin, and J.A. Smith, unpublished). These results suggest that the ard1 gene product may be a regulatory protein for NAT, as we previously proposed (14), and that it is not a regulatory protein for M-NAT. The ard1 gene product is unlikely to be a subunit of NAT, since the biophysical evidence indicates that NAT is a homodimer (10).
Although much is known about the intracellular role of aminopeptidases and N-acetytransferases in NH2-terminal processing of eukaryotic proteins, there is still much that remains to be understood.
References
1. Arfin, S.M., & Bradshaw, R.A., (1988) Biochemistry 27, 7979. 2. Chang, S.-Y.P., McGary, E.C., & Chang, S. (1989) J. Bacteriol. 171, 751.
3. Chang, Y.-H., Teichert, U., & Smith, J.A. (1990) J. Biol. Chem. 265, 19892.
4. Chang, Y.-H., Teichert, U., & Smith, J.A. (1992) J. Biol. Chem. 267, 8007.
5. Narita, K. (1958) Biochim. Biophys. Acta 28, 184.
6. Brown, J.L. & Roberts, W.K. (1976) J. Biol. Chem. 251, 1009
7. Mullen, J.R., Kayne, P.S., Moerschell, R.P., Tsunasawa, S., Gribskov, M., Sherman, F., & Sternglanz, R. (1989) EMBO J. 8, 2067.
8. Lee, F.-J.S., Lin, L.-W., & Smith, J.A. (1988) FEBS Lett. 256, 139.
9. Persson, B., Flinta, C., Heijne, G., & Jornvall, H. (1985) Eur. J. Biochem. 152, 523
10. Lee, F.-J.S., Lin, L.-W., & Smith, J.A. (1988) J. Biol. Chem. 263, 14948.
11. Lee, F.-J.S., Lin, L.-W., & Smith, J.A. (1990) J. Biol. Chem. 265, 11576.
12. Lee, F.-J.S., Lin, L.-W., & Smith, J.A. (1990) J. Biol. Chem. 265, 3603.
13. Tsunasawa, S., Stewart, J.W., & Sherman, F. (1985) J. Biol. Chem. 260, 5382.
14. Lee, F.-J.S., Lin, L.-W., & Smith, J.A. (1988) J. Bacteriol. 171, 5795.
15. Lee, F.-J.S., Lin, L.-W., & Smith, J.A. (1989) J. Biol. Chem. 264, 12339.
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