The mechanism of N-terminal acetylation of proteins

N alpha-acetylation is almost exclusively restricted to eukaryotic structural proteins. As a rule it is a post-initiational process, requiring the presence of the enzyme N alpha-acetyltransferase and the acetyl donor acetylcoenzyme A. N alpha-acetyltransferases appear to have a narrow substrate specificity, which is very similar for enzymes from different tissues and species. Amino acids predominantly present at the N terminus of N alpha-acetylated proteins are alanine, serine, and methionine. The occurrence of these residues is apparently a prerequisite for acetylation. The region following these amino acids is also important. If methionine is at the N terminus, the second position is always occupied by a strongly hydrophilic amino acid. Two- and three-dimensional structural characteristics of the protein do not seem to play a major role in N alpha-acetylation. Up to now the exact function for N alpha-acetylation is not known.     

Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell-free synthesis systems

Heterologous expression of a bacterial light-harvesting (LH) integral membrane protein was attempted using Escherichia coli cells and cell-free synthesis systems prepared from E. coli extracts. The alpha-apoprotein of LH1 complex from purple photosynthetic bacterium Rhodospirillum rubrum was overexpressed as a recombinant protein with a histidine (His6) tag added to the carboxyl terminus. Both of the expression systems produced alpha-apoprotein in a fully functional form as can judged by its ability to form a structural subunit with native beta-apoprotein and the pigment molecule bacteriochlorophyll a. The expression product in E. coli appears to be located in the inner cell membrane and can be almost completely extracted by 0.5% (w/v) Triton X-100. Circular dichroism measurement indicated that the expressed alpha-apoproteins from both systems had alpha-helical contents essentially identical with that of the native one. About two thirds of the alpha-apoprotein expressed in E. coli was found to have the amino terminal methionine residue modified by a formyl group. About one third of the alpha-apoprotein expressed in the cell-free system was found to be oxidized at the side chain of the amino terminal methionine residue. Functional expression of the alpha-apoprotein using the cell-free system provides an useful example for producing highly hydrophobic integral membrane proteins with relatively large quantities sufficient for biophysical and structural analysis.     

Identification of N-terminal methionine in the precursor of immunoglobulin light chain. Initiation of translation of messenger ribonucleic acid in plants and animals.

The proteins programmed in the wheat-germ cell-free system by the mRNA coding for the MOPC-321 mouse myeloma L (light) chain were labelled with [35S]methionine, [4,5-3H]leucine or [3-3H]serine, and were subjected to amino acid-sequence analyses. Over 95% of the total cell-free product was sequenced as one homogeneous protein, which corresponds to the precursor of the L-chain protein. In the precursor, 20 amino acid residues precede the N-terminus of the mature protein. This extra piece contains one methionine residue at the N-terminus, one serine residue at position 18, and six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13. The identification of methionine at the N-terminus of the precursor is in agreement with the evidence showing that unblocked methionine is the initiator residue for protein synthesis in eukaryotes. The absence of methionine at position 20, which precedes the N-terminal residue of the mature protein, suggests that myeloma cells synthesize the precursor. However, within the cell the precursor should be rapidly processed to the mature L chain, since precursor molecules have not yet been found in the intact animal. The abundance (30%) of leucine residues indicates that the extra-piece moiety is quite hydrophobic. The extra piece of the MOPC-321 L-chain precursor synthesized with the aid of the Krebs II ascites cell-free system is of identical size and it has the same leucine sequence [Schechter et al. (1975) Science 188, 160-162]. This indicates that cell-free systems derived from the plant and animal kingdom initiate mRNA translation from the same point. It is shown that the amino acid sequence of minute amounts of a highly labelled protein (0.1 pmol) can be faithfully determined in the presence of a large excess (over 2000 000-fold) of unrelated non-radioactive proteins.     

Identification of methionine Nalpha-acetyltransferase from Saccharomyces cerevisiae

N alpha-Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eukaryotic proteins and was believed until now to be catalyzed by a single N alpha-acetyltransferase. The transfer of an acetyl group from acetyl coenzyme A to the alpha-amino group of five NH2-terminal residues (serine, alanine, methionine, glycine, and threonine) in proteins accounts for approximately 95% of acetylated residues. We have found that a crude lysate from Saccharomyces cerevisiae mutant (aaa1) deficient in N alpha-acetyltransferase activity can effectively transfer an acetyl group to peptides containing NH2-terminal methionine but not to serine or alanine. This methionine N alpha-acetyltransferase has been extensively purified, and this purified enzyme can selectively transfer an acetyl group to various model peptides containing an NH2-terminal methionine residue and a penultimate aspartyl, asparaginyl, or glutamyl residue. Such specificity of N alpha-acetylation of methionine has been previously observed based on the analysis of eukaryotic protein sequences (Persson, B., Flinta, C., Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527; Arfin, S.M., and Bradshaw, R. A. (1988) Biochemistry 27, 7979-7984). The indentification of this methionine N alpha-acetyltransferase provides an explanation as to why two distinct classes of N alpha-acetylated proteins exist in nature: (i) those whose initiator methionine is acetylated and (ii) those whose penultimate residue is acetylated after cleavage of the initiator methionine.     

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.     

Translation rate of human tyrosinase determines its N-linked glycosylation level

Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or seven N-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2-3-fold faster (7.7-10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinase N-linked glycosylation, as well as other potential cotranslational maturation events.     

Ultrasensitive fluorescence-based detection of nascent proteins in gels

The most common method of analysis of proteins synthesized in a cell-free translation system (e.g., nascent proteins) involves the use of radioactive amino acids such as [(35)S]methionine or [(14)C]leucine. We report a sensitive, nonisotopic, fluorescence-based method for the detection of nascent proteins directly in polyacrylamide gels. A fluorescent reporter group is incorporated at the N-terminus of nascent proteins using an Escherichia coli initiator tRNA(fmet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, we find that the protein translational machinery accepts BODIPY-FL, a relatively small fluorophore with a high fluorescent quantum yield, as an N-terminal modification. Under the optimal conditions, fluorescent bands from nanogram levels of in vitro-produced proteins could be detected directly in gels using a conventional UV-transilluminator. Higher sensitivity ( approximately 100-fold) could be obtained using a laser-based fluorescent gel scanner. The major advantages of this approach include elimination of radioactivity and the rapid detection of the protein bands immediately after electrophoresis without any downstream processing. The ability to rapidly synthesize nascent proteins containing an N-terminal tag facilitates many biotechnological applications including functional analysis of gene products, drug discovery, and mutation screening.     

Purification and characterization of hen oviduct N alpha-acetyltransferase

Hen oviduct N alpha-acetyltransferase was purified to homogeneity by ammonium sulfate fractionation and DEAE-cellulose, Sepharose 6B, hydroxylapatite, and CoA affinity chromatography. The molecular weights of the native N alpha-acetyltransferase and its protein subunit were estimated as 240,000 and 79,000, respectively. The purified enzyme exhibited a narrow pH optimum centered at 7.8. The enzyme was activated by dithiothreitol, cysteine, glutathione, and beta-mercaptoethanol, but inhibited by Fe2+, Mn2+, Zn2+, Ca2+, Mg2+, and all thiol-specific reagents tested. These findings suggest that a thiol group(s) is essential to the enzyme activity. Substrate specificity experiments of the purified enzyme revealed that (i) the minimal length of a peptide chain required for N alpha-acetylation is 10 residues, (ii) the amino acids, Ala, Ser, Met, and Gly, which are predominantly found in the N termini of N alpha-acetylated proteins, are not the sole determinant of N alpha-acetylation for 10 and more residue peptides, and (iii) N alpha-acetyltransferase recognizes a minute difference in the side chain structure at the N termini of ACTH1-18-NH2 and [Gly1]ACTH1-18-NH2, a productive and a nonproductive substrate, respectively.     

Fluorescent labeling of cell-free synthesized proteins by incorporation of fluorophore-conjugated nonnatural amino acids

Although fluorescent dyes, such as fluorescein derivatives, have bulky and complex structures, nonnatural amino acids carrying these fluorescein derivatives are acceptable by the Escherichia coli ribosome and are useful for the cotranslational fluorescent labeling of cell-free synthesized proteins. Surprisingly, the incorporation efficiency of nonnatural amino acids carrying fluorescein derivatives into translated proteins depends on the source of the translational machinery used in cell-free protein synthesis. That is, whereas the E. coli ribosome efficiently supported the incorporation of nonnatural amino acids carrying fluorescein derivatives into a protein structure, no detectable fluorescent signal was observed from the protein expressed in the eukaryotic cell-free protein synthesis system performed in the presence of fluorescein-conjugated aminoacylated transfer RNA (tRNA).     

N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry

To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications.     

Archaeal N-terminal protein maturation commonly involves N-terminal acetylation: a large-scale proteomics survey

We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.     

Rat liver polysome N alpha-acetyltransferase: substrate specificity.

The substrate specificity of polysome rat liver N alpha-acetyltransferase (NAT) has been examined by utilizing a series of synthetic and natural substrates that has been systematically altered with respect to N-terminal sequence and length. Families of peptides of the structure S-Y-S-G-G-L-L-L were generated by successively replacing the N-terminal serine, the penultimate tyrosine, and the antepenultimate serine with all 19 commonly occurring amino acids, which were then assessed for their reactivity with the rat liver enzyme. Only peptides with N-terminal serine, alanine, methionine, leucine, and phenylalanine were modified. Glycine, lysine, arginine, valine, isoleucine, and tryptophan in the second position are (with N-terminal serine) strongly inhibitory, and proline completely blocks modification. Third-position substitutions have less of an effect on NAT activity with glycine, aspartic acid, glutamic acid, and tryptophan being most inhibiting (with N-terminal Ser-Tyr). These observations are generally in agreement with in situ modifications although there are some significant differences particularly with respect to the amino-terminal residues. Optimal chain length was determined to be 10-11 residues with either synthetic peptides of the structure S-Y-S-(G)n-L-L-L or adrenocorticotropin (ACTH) sequences ranging from 8 to 39 residues. The ACTH peptides were generally found to be severalfold better substrates than the corresponding synthetic ones. Activity was not affected by increased chain length beyond approximately 17 residues. These data support the view that polysome-catalyzed N alpha-acetylation occurs as a cotranslational event on nascent chains of about 20-40 amino acids in length.     

Oxidation inhibits amyloid fibril formation of transthyretin

The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated. Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on protein structures. Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing protein structures. Transthyretin (TTR) is a soluble human plasma protein. The wild-type (WT) and several of its variants are prone to fibril formation, which leads to amyloidosis associated with many clinical syndromes. The effects of amino acid side chain oxidations were investigated by comparing the kinetics of fibril formation of oxidized and unoxidized proteins. The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species. In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins. Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments. Side chain modification of methionine residues at position 13 and 30 (the latter for V30M TTR only) were dominant oxidative products. Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach. Oxidation inhibited the initial rates and extent of fibril formation for both the WT and V30M TTR proteins. In the case of WT TTR, oxidation inhibited fibril growth by approximately 76%, and for the V30M TTR by nearly 90%. These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures.     

A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.     

Tolerance for random recombination of domains in prokaryotic and eukaryotic translation systems: Limited interdomain misfolding in a eukaryotic translation system

It has been proposed that eukaryotic translation systems have a greater capacity for cotranslational folding of domains than prokaryotic translation systems, which reduces interdomain misfolding in multidomain proteins and, therefore, leads to tolerance for random recombination of domains. However, there has been a controversy as to whether prokaryotic and eukaryotic translation systems differ in the capacity for cotranslational domain folding. Here, to examine whether these systems differ in the tolerance for the random domain recombination, we systematically combined six proteins, out of which four are soluble and two are insoluble when produced in an Escherichia coli and a wheat germ cell-free protein synthesis systems, to construct a fusion protein library. Forty out of 60 two-domain proteins and 114 out of 120 three-domain proteins were more soluble when produced in the wheat system than in the E. coli system. Statistical analyses of the solubilities and the activities indicated that, in the wheat system but not in the E. coli system, the two soluble domains comprised mainly of beta-sheets tend to avoid interdomain misfolding and to fold properly even at the neighbor of the misfolded domains. These results demonstrate that a eukaryotic system permits the concomitance of a wider variety of domains within a single polypeptide chain than a prokaryotic system, which is probably due to the difference in the capacity for cotranslational folding. This difference is likely to be related to the postulated difference in the tolerance for random recombination of domains.     

Specificity of lens initiator tRNA for N-terminal recognition

The optimal magnesium ion concentration for chain initiation in a cell-free system derived from bovine eye lens which synthesizes 4 classes of crystallins appears to be 5 mM. In the synthesis of -crystallin polypeptides which contain one internal methionine residue and the second one in N-terminal position, Met-RNA^fMet functions exclusively as initiator. On the other hand at 5 mM Mg²⁺ Met-tRNA^Met inserts its methionine into the internal position. However, at higher magnesium ion concentrations the initiator tRNA also donates methionine for chain elongation while at the same time the cell-free system loses its capacity to initiate new polypeptides.     

Control of ribosomal protein synthesis during wheat embryo imbibition

Dry wheat embryos contain large quantities of ribosomes, synthesized and assembled during embryogenesis. When messenger RNA isolated from dry embryos is translated, in vitro, a significant proportion of the total translation products (approx. 10%) is identifiable as ribosomal proteins, by electrophoresis in two distinct two-dimensional polyacrylamide gel electrophoretic systems. When germinating embryos are labelled with [³⁵S]methionine, during the first 24 h of imbibition, the appearance of newly synthesized ribosomal proteins in the cytosolic fraction is barely detectable. However, this low level (< 1% of total cytosolic protein synthesis) of observed ribosomal protein synthesis is not correlated with a correspondingly low level of ribosomal protein mRNA. Ribosomal proteins constitute at least 10% of the products of translation, in vitro, of mRNA isolated from germinating wheat embryos. Ribosomal proteins are also conspicuous products of translation when polyribosomes isolated from imbibing embryos are used to direct protein synthesis in a cell-free ‘run-off’ system, and newly synthesized ribosomal proteins can be detected in the nuclei isolated from germinating embryos. It is proposed that their absence from the cytosolic fraction is a consequence of post-translational regulatory events.     

Site-specific modification of Candida antarctica lipase B via residue-specific incorporation of a non-canonical amino acid

In order to modify proteins in a controlled way, new functionalities need to be introduced in a defined manner. One way to accomplish this is by the incorporation of a non-natural amino acid of which the side chain can selectively be reacted to other molecules. We have investigated whether the relatively simple method of residue-specific replacement of methionine by azidohomoalanine can be used to achieve monofunctionalization of the model enzyme Candida antarctica lipase B. A protein variant was engineered with one additional methionine residue. Due to the high hydrophobicity and low abundance of methionine, this was the only residue out of five that was exposed to the solvent. The use of the Cu (I)-catalyzed [3 + 2] cycloaddition under native conditions resulted in a monofunctionalized enzyme which retained hydrolytic activity. The strategy can be considered a convenient tool to modify proteins at a single position as long as one solvent-exposed methionine is available.     

Initiation of histone synthesis by f-met-tRNAf in an ascites cell-free system

Formyl-[³⁵S]methionine is incorporated into histones synthesized by a mouse ascites cell-free system supplemented with histone mRNA and f-[³⁵S]met-tRNA_f from yeast. Most of the [³⁵S]methionine incorporated can be shown to be at the N-terminus by Edman degradation after deformylation. This indicates that methionine can be used as the initiator amino acid for histones in this cell-free system.     

Switching the sorting mode of membrane proteins from cotranslational endoplasmic reticulum targeting to posttranslational mitochondrial import

Hydrophobic membrane proteins are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, mediated by hydrophobic signal sequence. Mitochondrial membrane proteins escape this mechanism despite their hydrophobic character. We examined sorting of membrane proteins into the mitochondria, by using mitochondrial ATP-binding cassette (ABC) transporter isoform (ABC-me). In the absence of 135-residue N-terminal hydrophilic segment (N135), the membrane domain was integrated into the ER membrane in COS7 cells. Other sequences that were sufficient to import soluble protein into mitochondria could not import the membrane domain. N135 imports other membrane proteins into mitochondria. N135 prevents cotranslational targeting of the membrane domain to ER and in turn achieves posttranslational import into mitochondria. In a cell-free system, N135 suppresses targeting to the ER membranes, although it does not affect recognition of hydrophobic segments by signal recognition particle. We conclude that the N135 segment blocks the ER targeting of membrane proteins even in the absence of mitochondria and switches the sorting mode from cotranslational ER integration to posttranslational mitochondrial import.