NH2-terminal processing of Drosophila melanogaster actin. Sequential removal of two amino acids

Class II actins, such as Drosophila and mammalian skeletal muscle actins, have genes that code for a Met-X-Asp NH2 terminus where X is usually cysteine. These actins have an Ac-Asp NH2 terminus so two amino acids must be removed. To determine the nature of this processing, we labeled Drosophila Schneider L-2 cells with [35S]methionine or cysteine, isolated the actin, and analyzed the NH2-terminal actin tryptic peptides and their thermolysin digestion products. After a 4-h labeling period, we detected completed actin polypeptide chains with either an unblocked Asp or an Ac-Asp NH2 terminus. No intermediate with an NH2-terminal Cys or Met could be demonstrated. If, however, Drosophila mRNA was translated in a mRNA-dependent rabbit reticulocyte lysate system, an additional 43-kDa actin intermediate was observed. On the basis of thermolysin digestion studies and experiments using mild acid hydrolysis of a labeled actin NH2-terminal tryptic peptide fragment, we identified this intermediate as having an Ac-Cys-Asp NH2 terminus. In a time-dependent fashion, Ac-Cys was removed generating actin with an exposed NH2-terminal Asp which was subsequently acetylated to produce the mature form of actin. The removal of Met and the acetylation of Cys may occur early in translation while the nascent polypeptide chain is still attached to the ribosome. Subsequent processing occurs following completion of the synthesis of the actin polypeptide. The removal of Ac-Cys from Drosophila actin is thus similar to removal of Ac-Met from the NH2 terminus of class I actins although in the case of the class II actins, it is the second amino acid that is removed as an acetylated species.     

Correct NH2-terminal processing of cardiac muscle alpha-isoactin (class II) in a nonmuscle mouse cell

Both mammalian nonmuscle and muscle actins possess an AcAsp(Glu)NH2 terminus. The nonmuscle actin genes code for a polypeptide with a Met-Asp NH2 terminus (class I) whereas the muscle actin genes code for a polypeptide with a Met-Cys-Asp NH2 terminus (class II). Two amino acids must be removed for mature muscle actin synthesis, whereas only the Met must be removed for nonmuscle actin synthesis. We wished to know whether a nonmuscle cell which normally does not synthesize a class I actin can correctly process a muscle actin with its extra NH2-terminal amino acid in vivo. To answer this question we have used L/LK165 cells, a mouse L-cell transfected with a human cardiac muscle actin gene. When these cells were labeled overnight with [35S]Cys, an actin with an NH2-terminal tryptic peptide corresponding to that of mature cardiac muscle actin was detected. When the cells were pulse-labeled for 20 min, a new actin intermediate containing an AcCys-Asp amino terminus was observed which then disappeared with time. Furthermore, the muscle actin was processed as fast if not faster than the nonmuscle actin in these cells. This actin intermediate was also seen in chick myotube cultures. Our results show that the ability to correctly process muscle specific actins is not tissue specific. Furthermore, these results confirm a processing pathway for class II actins proposed by us earlier on the basis of experiments with a cell-free translation system.     

Unusual metabolism of the yeast actin amino terminus

In this paper we have examined the post-translational modifications of the NH2 terminus of actin from the yeast Saccharomyces cerevisiae. Like actins examined previously, this actin contains an acetylated NH2 terminus. Actins in other organisms undergo a unique post-translational processing event in which the initial amino acid(s) are removed by an actin-specific processing enzyme in an acetylation-dependent reaction. This is defined as actin processing. In yeast, actin retains its initiator Met in vivo and is thus not processed even though a rat liver actin processing enzyme can process yeast actin in vitro. This lack of actin processing appears to be a general property of fungi, as the actin from three other species, Aspergillus nidulans, Schizosaccharomyces pombe, and Candida albicans are not NH2 terminally processed either. Yeast actin is a class I actin; its initiator Met directly precedes an acidic residue. We converted yeast actin to a class II species by inserting a Cys codon between the Met-1 and Asp-2 codons. In normal class II actins the Cys residue is removed as acetyl-Cys during processing. Neither the mutant actin nor chick beta-actin (a class I actin) are processed when expressed in yeast. S. cerevisiae thus appears to be also incapable of processing exogenous actins. Further study of the mutant actin containing a Cys at position 2 shows that 30-40% of this actin is stably unacetylated. This unacetylated actin does not have a shorter half-life than the acetylated form. From these studies we conclude that 1) NH2-terminal actin-specific processing is not required for actin function in yeast and three other fungi, 2) yeast are apparently incapable of processing any type of actin precursor, and 3) the stability of a yeast pseudo-class II actin is not affected by the acetylation state of the NH2 terminus.     

Removal of the amino-terminal acidic residues of yeast actin. Studies in vitro and in vivo.

We have examined the role of the acidic residues Asp2 and Glu4 at the NH2 terminus of Saccharomyces cerevisiae actin through site-directed mutagenesis. In DNEQ actin, these residues have been changed to Asn2 and Gln4, whereas in delta DSE actin, the Asp2-Ser-Glu tripeptide has been deleted. Both mutant actins can replace wild type yeast actin. Peptide mapping studies reveal that DNEQ, like wild type actin, retains the initiator Met and is NH2 terminally acetylated, whereas delta DSE has a free NH2 terminus and has lost the initiator Met. Interestingly, microscopic examination of filaments of these two actins reveal the appearance of bundled filaments. The DNEQ bundles are smaller and more ordered, whereas the delta DSE bundles are larger and more loosely organized. Additionally, both mutant actins activate the ATPase activity of rabbit muscle myosin S1 fragment to a lesser extent than wild type. We have also developed a sensitive assay for actin function in vivo that enabled us to detect a slight defect in the ability of these mutant actins to support secretion, an important function in yeast. Thus, although the mutant actins resulted in no gross phenotypic changes, we were able to detect a defect in actin function through this assay. From these studies we can conclude that 1) although NH2-terminal negative charges are not essential to yeast life, the loss of such charges does result in a slight defect in the actins' ability to support secretion, 2) removal of the NH2-terminal negative charges promotes the bundling of actin filaments, and 3) actins lacking NH2-terminal negative charges are unable to activate the myosin S1 ATPase activity as well as wild type actin.     

Amino-terminal processing of actins mutagenized at the Cys-1 residue.

Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.     

Drosophila ACT88F indirect flight muscle-specific actin is not N-terminally acetylated: a mutation in N-terminal processing affects actin function

Many eukaryotic proteins are co and post-translationally modified at their N termini by removal of one or two amino acid residues and N(alpha)-acetylation. Actins show two different forms of N-terminal processing dependent on their N-terminal sequence. In class II actins, which include muscle actins, the common primary sequence of Met-Cys-Asp-actin is processed to acetyl-Asp-actin. The functional significance of this in vivo is unknown. We have studied the indirect flight muscle-specific actin, ACT88F, of Drosophila melanogaster. Our results show that ACT88F is N-terminally processed in vivo as a class II actin by removal of the first two amino acid residues (Met and Cys), but that uniquely the N terminus is not acetylated. In addition we show that ACT88F is methylated, probably at His73.Flies carrying the mod(-) mutation fail to complete post-translational processing of ACT88F. We propose that the mod gene product is normally responsible for removing N-acetyl-cysteine from actin. The biological significance of this process is demonstrated by observations that retention of the N-acetyl-cysteine in ACT88F affects the flight muscle function of mod(-) flies. This suggests that the extreme N terminus affects actomyosin interactions in vivo, a proposal we have examined by in vitro motility assays of ACT88F F-actin from mod(-) flies. The mod(-) actin only moves in the presence of methylcellulose, a viscosity-enhancing agent, where it moves at velocities slightly, but significantly, reduced compared to wild-type. These data confirm that N-acetyl-cysteine at the N terminus affects actomyosin interactions, probably by reducing formation of the initial actomyosin collision complex, a process known to involve the actin N terminus.     

NH2-terminal processing of actin in mouse L-cells in vivo

When Dictyostellium discoideum actin is synthesized in vitro, it is made as a 43,000-dalton polypeptide with an NH2-terminal N-acetylmethionine. The acetylmethionine is then cleaved post-translationally, and the new NH2-terminal aspartic acid is acetylated to give the mature form of actin. Inhibition of methionine acetylation prevents methionine cleavage (Redman, K., and Rubenstein, P. (1981) J. Biol. Chem. 256, 13226-13229). In this paper, we describe the results of experiments designed to discover whether this novel actin processing pathway is peculiar to the rabbit reticulocyte lysate system or whether it is utilized by mammalian cells in vivo as well. We show that in mouse L-929 cells, actin is made as a 43,000-dalton protein with an NH2-terminal N-acylmethionine residue. Experiments using thin layer chromatography and digestion of the acylmethionine residue with hog kidney acylase I demonstrate that the acyl group is an acetyl residue. Pulse-chase experiments show that over the course of 1 h, this precursor is transformed first to an actin with a free NH2-terminal aspartic acid and is subsequently converted to mature L-cell actin with an acetylaspartic acid NH2 terminus. The half-life of the initial actin precursor in the cell appears to be approximately 12-15 min. These studies demonstrate the existence of this novel actin processing pathway in vivo and suggest that it is used for those actins where, in the gene, the initiator methionine codon directly precedes the codon for aspartic or glutamic acids, the residues normally found at the actin NH2 terminus.     

Amino acid sequence of Acanthamoeba actin

By amino acid sequence studies, only one form of cytoplasmic actin was detected in Acanthamoeba castellanii. Its amino acid sequence is very similar to the sequences of Dictyostelium and Physarum actins, from which Acanthamoeba actin differs in only nine and seven residues, respectively, including the deletion of the first residue. Acanthamoeba actin is unique in containing a blocked NH2-terminal neutral amino acid (glycine), while all other actins sequenced thus far have a blocked acidic amino acid (aspartic or glutamic) at the NH2 terminus. Acanthamoeba actin is also unique in that it contains an N epsilon-trimethyllysine residue at position 326. Like other actins, Acanthamoeba actin contains an NT-methylhistidine residue at position 73. The protein sequence is in complete agreement with the sequence derived from the nucleotide sequence of an expressed actin gene.     

Post-translational processing of the amino terminus affects actin function

We have studied the importance of N-terminal processing for normal actin function using the Drosophila Act88F actin gene transcribed and translated in vitro. Despite having different charges as determined by two-dimensional (2D) gel electrophoresis, Act88F expressed in vivo and in vitro in rabbit reticulocyte lysate bind to DNase I with equal affinity and are able to copolymerise with bulk rabbit actin equally well. Using peptide mapping and thin-layer electrophoresis we have shown that bestatin [( 3-amino-2-hydroxy-4-phenyl-butanoyl]-L-leucine), an inhibitor of aminopeptidases, can inhibit actin N-terminal processing in rabbit reticulocyte lysate. Although processed and unprocessed actins translated in vitro are able to bind to DNase I equally well, unprocessed actins are less able to copolymerise with bulk actins. This effect is more pronounced when bulk rabbit actin is used but is still seen with bulk Lethocerus actin. Also, the unprocessed actins reduce the polymerisation of the processed actin translated in vitro with the bulk rabbit actin. This suggests that individual actins do interact, even in non-polymerising conditions. The reduced ability of unprocessed actin to polymerise shows that correct post-translational modification of the N terminus is required for normal actin function.     

The actin genes of Drosophila: protein coding regions are highly conserved but intron positions are not

The entire set of six closely related Drosophila actin genes was isolated using recombinant DNA methodology, and the structures of the respective coding regions were characterized by gene mapping techniques and by nucleotide sequencing of selected portions. Structural comparisons of these genes have resulted in several unexpected findings. Most striking is the nonconservation of the positions of intervening sequences within the protein-encoding regions of these genes. One of the Drosophila actin genes, DmA4, is split within a glycine codon at position 13; none of the remaining five genes is interrupted in the analogous position. Another gene, DmA6, is split within a glycine codon at position 307; at least two of the Drosophila actin genes are not split in the analogous position. Additionally, none of the Drosophila actin genes is split within codon four, where the yeast actin gene is interrupted. The six Drosophila actin genes encode several different proteins, but the amino acid sequence of each is similar to that of vertebrate cytoplasmic actins. None of the genes encodes a protein comparable in primary sequence to vertebrate skeletal muscle actin. Surprisingly, in each of these derived actin amino acid sequences in the initiator methionine is directly followed by a cysteine residue, which in turn precedes the string of three acidic amino acids characteristic of the amino termini of mature vertebrate cytoplasmic actins. We discuss these findings in the context of actin gene evolution and function.     

Signal peptide of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by the removal of the NH2-terminal 41 amino acid sequence (41 amino acid leader sequence). DNA fragments coding for short sequences consisting of 28 (Pro as the COOH terminus) 29 (Ala), 31 (Ala), and 33 (Ala) amino acids from the translation initiator, Met, in the leader sequence were prepared and fused in frame to the DNA encoding the mature alpha-amylase. The secretion activity of the 33 amino acid sequence was nearly twice as high as that of the parental 41 amino acid sequence, whereas the activity of the 31 amino acid sequence was 75% of that of the parent. In contrast, almost no secretion activity was observed with the 28 and 29 amino acid sequences. The signal peptide cleavage site of the precursor expressed from the plasmid encoding the 33 amino acid sequence was located between Ala and Leu at positions 33 and 34 and that from the 31 amino acid sequence between Thr and Ala at positions 33 and 34. The NH2-terminal amino acid from the latter corresponded to the 3rd amino acid of the mature enzyme. These results indicated that the functional signal peptide of the B. subtilis beta-amylase consists of the first 33 amino acids from the initiator, Met.     

Organization of the actin multigene family of Dictyostelium discoideum and analysis of variability in the protein coding regions

There are 17 to 20 actin genes in the genome of the cellular slime mold Dictyostelium discoideum. Genomic clones of 15 of the genes have been isolated. Extensive nucleotide sequence within the protein-coding regions has been determined, including the complete nucleotide sequence of four genes representing the three distinct evolutionary groups of Dictyostelium actin genes. All are similar to mammalian cytoplasmic actins at diagnostic amino acid positions, and there is generally less variability among Dictyostelium actin genes than among Drosophila actin genes. Two genes, Actins 3-sub 1 and 3-sub 2 differ substantially from all the rest in terms of replacement amino acid substitutions and probably encode actin-related proteins rather than bona fide actins. Each contains several amino acid substitutions that should alter the secondary structure of the resulting proteins, and Actin 3-sub 2 encodes four additional amino acids at the C terminus. This gene is as divergent from other Dictyostelium actin genes as is the yeast or a soybean actin gene. At present, evidence suggests that all 15 genes examined are expressed, except the previously identified Actin 2-sub 2. We suggest that Dictyostelium might maintain a high number of functional actin genes for the purpose of regulating the level of actin synthesis within narrow limits, rather than because most genes perform different functions.     

Studies on the role of actin's N tau-methylhistidine using oligodeoxynucleotide-directed site-specific mutagenesis

The primary structure of all actins except that isolated from Naegleria gruberi contains a unique N tau-methylhistidine (MeHis) at position 73. This modified residue has been implicated as possibly being important for the post-translational processing of actin's amino terminus, the binding of actin to DNase I, and in the polymerization of G-actin. We have investigated the potential role of MeHis in each of these processes by utilizing site-directed mutagenesis to change His-73 of skeletal muscle actin to Arg and Tyr. Wild type and mutant actins were synthesized in vivo, using non-muscle cells transfected with mutant cDNAs, and in vitro by translating mutant RNAs synthesized using SP6 RNA polymerase in a rabbit reticulocyte lysate. We have found that actins containing Arg or Tyr at position 73 undergo amino-terminal processing, bind to DNase I-agarose, and become incorporated into the cytoskeleton of a nonmuscle cell as efficiently as wild type actin. Furthermore, using an in vitro copolymerization assay we have found that although there is no difference between the Arg mutant and the wild type actins, the Tyr mutant has a slightly greater critical concentration for polymerization. These results show that MeHis is not absolutely required for any of these processes.     

Structure of chicken 16-kDa beta-galactoside-binding lectin. Complete amino acid sequence, cloning of cDNA, and production of recombinant lectin

The complete primary structure of chicken 16-kDa beta-galactoside-binding lectin (C-16) was determined. It was composed of 134 amino acid residues and has an acetylated NH2 terminus. A cDNA was also cloned, but no signal sequence was found in the initiator region. The initiator methionine remained as the NH2 terminus of the mature lectin. Although C-16 is distinct from chicken 14-kDa beta-galactoside-binding lectin (C-14), it proved to be a member of the vertebrate 14-kDa-type lectin family. Comparison of the primary structures between the vertebrate 14-kDa-type lectins suggests that C-14 and C-16 were produced by gene duplication of an ancestral lectin gene at a time close to the divergence of birds and mammals. Northern and Southern blot analysis indicated that these isolectins are encoded by individual genes which are differently regulated during the development of the embryo. A recombinant C-16 lectin was produced in Escherichia coli. The product was indistinguishable from the authentic C-16 lectin except that the NH2 terminus of the former was found to begin with free methionine.     

Multiple actins in drosophila melanogaster

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.     

Immunological characterization of an anti-actin antibody specific for cytoplasmic actins and its use for the immunocytological localization of actin in Aplysia nervous tissue

Previous immunochemical and immunocytochemical studies have shown that an antibody to actin prepared from body wall muscle of the marine mollusc Aplysia californica is specific for vertebrate cytoplasmic actins. The ability of this anti-actin to distinguish between different forms of actin most likely reflects the recognition of amino acid sequences unique to cytoplasmic actins. We have confirmed the specificity of this antibody for cytoplasmic actins using nervous tissue as a source of cytoplasmic actin in further immunochemical studies. In addition to binding cytoplasmic actin in purified preparations, the antibody removed actin selectively from crude extracts of nervous tissue of some but not all of the species tested. Our results also suggest that tissue-specific differences in the distribution of cytoplasmic actins may exist. Immunofluorescence studies of Aplysia nervous tissue stained with anti-actin revealed that actin is present in the cell body and axonal processes of Aplysia neurons. Although the function of actin in nerve cells is not understood, the observed pattern of immunofluorescence staining is consistent with the idea that actin may be involved in movement within the axoplasm.     

A cysteine-histidine-aspartate catalytic triad is involved in glutamine amide transfer function in purF-type glutamine amidotransferases

A family of four glutamine amidotransferases has a homologous glutamine amide transfer domain, designated purF-type, that is named after purF-encoded glutamine phosphoribosylpyrophosphate amidotransferase. The glutamine amide transfer domain of approximately 194 amino acid residues is at the NH2 terminus of the protein chain. Site-directed mutagenesis was used to replace several of the 9 invariant amino acids in the glutamine amide transfer domain of glutamine phosphoribosylpyrophosphate amidotransferase. The results indicate that a Cys1-His101-Asp29 catalytic triad is involved in the glutamine amide transfer function of this enzyme. The evidence suggests that His101 functions to increase the nucleophilicity of Cys1, which is used to form a glutamine-enzyme covalent intermediate. Asp29 has a role subsequent to formation of the covalent intermediate. The Cys-His-Asp catalytic triad is implicated in the glutamine amide transfer function of purF-type amidotransferases.     

Processing of pulmonary surfactant protein B by napsin and cathepsin H

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.