Studies on trypanosomatid actin. I. Immunochemical and biochemical identification

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai, actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.     

DNase-I-dependent dissociation of erythrocyte cytoskeletons

The human erythrocyte contains a complex of peripheral membrane proteins which forms an extensive network or cytoskeleton on the cytoplasmic membrane surface. When I treat erythrocyte cytoskeletons with deoxyribonuclease I (DNase I), the cytoskeletons dissociate and erythrocyte actin is solubilized. The dissociation of the cytoskeletons by DNase I parallels the disruption of actin filaments in vitro by DNase I and is blocked by the addition of action to the DNase I. Large protein complexes remain after DNase I disrupts the cytoskeletons, but these complexes are no longer visible in the light microscope nor sedimentable and are selectively depleted with respect to actin. From these studies, I suggest that DNase I binds to and solubilizes actin, which serves as a structural link between protein complexes in the erythrocyte cytoskeleton.     

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.     

Characterization of acidic actin in mouse sarcoma 180 cells

Mouse sarcoma 180 cells have a polypeptide that has the same molecular weight as actin but it is more acidic than alpha-actin. Its tryptic peptide pattern on reversed-phase HPLC was very similar to that of beta + gamma-actin, an actin sample prepared by affinity chromatography on DNase I-Sepharose contained the acidic polypeptide, and monoclonal anti-actin antibody reacted with it; therefore, the polypeptide is considered an actin isoform. The mRNA for this variant actin was identified by analyzing the polypeptides translated in vitro, which indicated that the variant actin is not a post-translationally modified form of any known actin. The variant actin was not stained by polyclonal anti-gizzard actin antibody which reacts with gamma-cytoplasmic, alpha-smooth and gamma-smooth muscle actins, nor by polyclonal anti-skeletal muscle actin antibody which reacts with skeletal, cardiac and alpha-smooth muscle actins. These results suggest that this variant actin is related to beta-cytoplasmic actin or, is a novel species whose N-terminal amino acid sequence is not Glu-Glu-Glu.     

MICROHETEROGENEITY OF BRAIN CYTOPLASMIC AND SYNAPTOPLASMIC ACTINS

Abstract— Actin present in whole rat brain cytoplasm and in synaptosomes was purified by DNase I affinity chromatography. By use of two-dimensional gels and one-dimensional isoelectric focusing gels, brain actin was shown to be composed of two isomeric forms. By comparison with muscle actins, brain actins were identified as the β and γ isomers. Muscle type α actin is not present in brain. Synaptosomal protein with high affinity for DNase I is primarily composed of β and γ actin, however, two minor synaptosomal proteins, S₁ and S₂, with similar DNase I affinity were also isolated. S₁1 and S₂ have the same apparent molecular weight as whole brain actin, are more acidic than the major actin forms and are distinct from a actin. Relative to β and γ actin, the content of S₁ and S₂ is 3-fOld greater in synaptosomes when compared to similar non-synaptosomal species. The results demonstrate heterogeneity of brain actins and compartmentalization of brain proteins with high affinity for DNase I at the synapse. It was also shown that tubulin has selective affinity for the DNase I-actin complex.     

Multiple forms of actin in Physarum polycephalum

Actin in the acellular slime mold Physarum polycephalum consists of three major forms closely spaced at isoelectric point (IP) 4.7 and a minor form at IP 5.1. Amino acid analysis has shown the IP 5.1 actin to be nearly identical to the 4.7 actins. In actin purified from acetone powder, both actin forms were present. Both forms bound to DNase I and have the same molecular weight of about 43 000 on sodium dodecyl sulfate (SDS) polyacrylamide gels. On 2-D gels of nuclear proteins, both forms of actin were present. The IP 4.7 actins account for 8.6% of total plasmodial protein, and the IP 5.1 form for about 0.7%. In the nucleus the IP 4.7 actins comprise 2.7% of total nuclear protein, and the 5.1 actin about 0.4%. No cell cycle-associated change in the concentration of actins was observed in either total plasmodial extracts or in isolated nuclei. Pulse-labelling experiments have shown that in total plasmodia actin synthesis occurs throughout the cell cycle, with no relative changes in the rate of synthesis. In isolated nuclei labelled during mitosis and early S-phase, there is about twice as much labelled actin as in nuclei labelled prior to mitosis. This result may indicate an increase in the transport of actin into the nucleus.     

An 100-kDa Ca2+-sensitive actin-fragmenting protein from unfertilized sea urchin egg

An actin-modulating protein was purified from unfertilized eggs of sea urchin, Hemicentrotus pulcherrimus, by means of DNase I affinity and DEAE-cellulose column chromatographies. This protein was a globular protein with a Stokes radius of 41-42 nm and consisted of a single polypeptide chain having an apparent molecular mass of 100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel filtration chromatography revealed that one 100-kDa protein molecule binds two or three actin monomers in the presence of Ca2+, but such binding was not observed in the absence of Ca2+. The effect of the 100-kDa protein on the polymerization of actin was studied by viscometry, spectrophotometry and electron microscopy. The initial rate of actin polymerization was decreased at a very low molar ratio of 100-kDa protein/actin. Acceleration of the initial rate of polymerization occurred at a relatively high, but still substoichiometric, molar ratio of 100-kDa protein/actin. The 100-kDa protein produced fragmentation of muscle actin filaments at Ca2+ concentrations greater than 0.3 microM as revealed by viscometry and electron microscopy. Evidence was also presented that the 100-kDa protein binds to the barbed end of the actin filament.     

Isolation of a minor species of actin from the nuclei of Acanthamoeba castellanii

Actin was extracted from isolated nuclei of Acanthamoeba castellanii and purified to homogeneity under nondenaturing conditions by diethylaminoethylcellulose and Sephadex G-100 chromatography. The pure protein has the same molecular weight as cytoplasmic Acanthamoeba actin and a very similar amino acid composition. Isoelectrofocusing shows that nuclear actin is slightly more acidic than the major cytoplasmic species, and comparative analysis of peptides from tryptic and cyanogen bromide digests shows that both actins are very similar but not chemically identical. In an assay that is specific for most actins, the inhibition of DNase I through the formation of a 1:1 G-actin-DNase I complex, the nuclear and cytoplasmic actins are equally effective. By use of a similar procedure for the purification of both actins, it is estimated that the amount of nuclear actin is about 1.5% of the amount of cytoplasmic actin, a major protein of the amoeba. It is concluded that a minor isoelectric species of actin associates selectively with the nuclei of A. castellanii.     

Actin of Histriculus cavicola: characteristics of the highly divergent hypotrich ciliate actins.

A macronuclear gene-sized molecule carrying an actin gene from the hypotrich ciliate, Histriculus cavicola, was characterized. Southern blot analysis using a coding region probe suggested that actin in H. cavicola is encoded by a single gene. A comparison of the promoter regions indicated that the H. cavicola actin gene has a TATA box in the 5' flanking region in a position identical to those in other oxytrich ciliates. The coding sequence of this gene is not interrupted by any introns, and codes for a protein of 375 amino acid residues. This protein shares a high degree of similarity with other oxytrichid actins, and a relatively low similarity with actins from other eukaryotes. Comparative analyses of sequences indicated that most of the amino acid substitutions in hypotrich actins are found in surface loops, while the core structures are well-conserved. The sites that interact with DNase I and several regions involved in actin-actin contact have diverged considerably in hypotrich actins, while nucleotide-binding sites are the best-conserved interaction motif.     

ADP-ribosylation of platelet actin by botulinum C2 toxin

Botulinum C2 toxin is a microbial toxin which possesses ADP-ribosyltransferase activity. In human platelet cytosol a 43-kDa protein was ADP-ribosylated by botulinum C2 toxin. Labelling of the 43-kDa protein using [32P]NAD as substrate was reduced by unlabelled NAD and nicotinamide. The label was removed by treatment with snake venom phosphodiesterase. Half-maximal and maximal ADP-ribosylation occurred at 0.1 microgram/ml and 3 micrograms/ml botulinum C2 toxin, respectively. The Km value of the ADP-ribosylation reaction for NAD was about 1 microM. The peptide map of the ADP-ribosylated 43-kDa protein was almost identical with platelet actin. The ADP-ribosylated 43-kDa substrate protein bound to and was eluted from immobilized DNase I in a manner similar to G-actin. Trypsin treatment of platelet cytosol decreased subsequent ADP-ribosylation of the 43-kDa protein without occurrence of smaller labelled polypeptides. Purified platelet actin was also ADP-ribosylated by botulinum C2 toxin with similar characteristics found with actin in platelet cytosol. Phalloidin decreased the ADP-ribosylation of actin in platelet cytosol and of isolated platelet actin. Half-maximal and maximal, about 90%, reduction of actin ADP-ribosylation was observed at 0.4 microM and 10 microM phalloidin, respectively. ADP-ribosylation of purified actin, induced by botulinum C2I toxin, abolished the formation of the typical microfilament network. The data indicate that platelet G-actin but not F-actin is a substrate of botulinum C2 toxin and that this covalent modification largely affects the functional properties of actin.     

A 60-kDa polypeptide in mammalian cells with epitopes related to actin

We have identified a novel actin-related 60-kDa polypeptide in mammalian cells. The relatedness of this polypeptide to actin is indicated by its affinity for DNase I, two monoclonal anti-actin antibodies, and two independent peptide-specific anti-actin antibodies which bind to actin at around amino acid 244. It is not incorporated into cytoskeletal stress fibers, although it is a stable protein. Its expression (60-kDa polypeptide, pI of 5.4 to 5.5) is inhibited by the K+ ionophore, nonactin, which is known to collapse the energy-dependent translocation of cytoplasmically synthesized proteins into mitochondria.     

Purification and biochemical characteristics of actin from the rat malignancy sarcoma-45]

Actin was purified from rat sarcoma-45 by using affinity chromatography on DNase I agarose. Actin was detected in the soluble and cytoskeletal fractions. The molecular mass of the protein was found to be equal to 45 kDa. The tumour actin specifically reacted with the antibody against skeletal muscle actin, inhibited the DNAase I activity and activated in the fibrillar state Mg(2+)-ATPases of sarcoma-45 and skeletal muscle myosins. The activating effect of the tumour protein was lower than that of its skeletal muscle counterpart. V8-protease peptide mapping revealed a similarity between tumour and brain actins. Sarcoma-45 actin was found to contain beta- and gamma-actin isoforms and an unusual isoform which appeared to be more acidic than the alpha-actin isoform.     

Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1

Homogeneous preparations of actin cleaved into two fragments, the N-terminal 9- and C-terminal 36-kDa peptides, were achieved by proteolysis of G-actin with subtilisin at 23 degrees C at a 1:1000 (w/w) ratio of enzyme to actin. The subtilisin cleavage site was identified by sequence analysis to be between Met-47 and Gly-48. Although under nondenaturing conditions the two fragments remained associated to one another, the cleavage affected macromolecular interactions of actin. The rates of cleaved actin polymerization by MgCl2, KCl, and myosin subfragment 1 (S-1) were slower and the critical concentrations for this process were higher than in intact protein. Intact and cleaved actin formed morphologically indistinguishable filaments and copolymerized in the presence of MgCl2. The affinity of actin for S-1 was decreased by about 10-fold due to subtilisin cleavage, but the S-1 ATPase activity was activated to the same Vmax value by both intact and cleaved actins. DNase I inhibition measurements revealed lower affinity of cleaved actin for DNase I than that of intact protein. These results are discussed in terms of actin's structure.     

Two monoclonal antibodies to actin: one muscle selective and one generally reactive

Two IgG1, kappa monoclonal antibodies (Mab) against actin have been obtained from a fusion in which chicken gizzard actin was used as the immunogen. One Mab, designated B4, shows a preferential reactivity toward enteric smooth muscle actin but also cross-reacts with skeletal, cardiac, and aorta actins on the basis of immunoblots, ELISA assays, and indirect immunofluorescence. However, this antibody does not react with either cytoplasmic actin in any of these assay systems. A second Mab, designated C4, reacts with all six known vertebrate isoactins as well as Dictyostelium discoideum and Physarum polycephalum actins. Thus B4 Mab appears to react with an epitope that is at least partially shared among the muscle actins but not found in cytoplasmic actins, while C4 Mab binds to an antigenic determinant that has been highly conserved among the actins. The binding sites of both Mabs on skeletal actin overlap that of pancreatic DNase I. Both antibodies bind a SV8 proteolytic product comprising the amino-terminal two-thirds of the actin molecule, and their epitopes appear to overlap since C4 can compete for the binding of B4 to skeletal actin. Neither antibody is able to prevent actin polymerization.     

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.     

A DNase I binding/immunoprecipitation assay for actin

An actin assay which employs the competition between labeled and unlabeled rabbit skeletal muscle actin for DNase I has been developed. Iodination of actin by the method of Bolton and Hunter results in the incorporation of approximately 0.5 mol of 125-iodine/incorporation of approximately 0.5 mol of 125-iodine/mol of actin. This 125I-actin retained the ability to bind to DNase I and inhibit enzymatic activity. The 125I-actin-DNase complex can be precipitated by the addition of a monospecific rabbit antibody to DNase I. The efficiency of this immunoprecipitation step is improved by the use of a second sheep anti-rabbit gamma-globulin. Using this immunoprecipitation assay, there is a linear displacement of the DNase I-bound 125I-actin by rabbit skeletal muscle actin standards or by the actin present in tissue and cell extracts. Using 17.5 ng of DNase I and approximately 500 pg of 125I-actin, 50% inhibition of binding was obtained with 23 ng of unlabeled actin. Reducing the amount of DNase I to 2 ng results in 50% inhibition of binding with 4 ng of unlabeled actin and an increase in the estimated sensitivity of the assay from 1.7 to 0.24 ng. The slopes of the displacement curves generated with both vertebrate and invertebrate non-muscle actins are parallel to rabbit skeletal muscle actin. This observation indicates approximately equal actin-DNase I binding affinities and suggests a high degree of conservation of the actin-DNase I binding site. The assay is useful for measuring the pools of F- and G-actin in a wide range of cells.     

Identification and purification of DBF-A, a double-stranded DNA-binding protein from Saccharomyces cerevisiae.

Using oligonucleotide affinity chromatography with DNase I footprinting as an assay we have looked for proteins that interact with sequence elements within the yeast origin of replication, autonomously replicating sequence 1 (ARS1). In this work we describe a protein that binds with high affinity to DNA but displays only moderate sequence specificity. It is eluted at 0.7 M salt from an ARS1 oligonucleotide column. Footprinting analysis on ARS1 at a high protein concentration revealed at least three sites of protection flanking element A and its repeats. Element A itself is rendered hypersensitive to DNase I digestion upon protein binding. This pattern is also observed for the H4 and HMR-E ARSs, suggesting that the protein alters the DNA conformation at element A and its repeats. The affinity-purified fraction is also capable of supercoiling a relaxed, covalently closed plasmid in the presence of topoisomerase. Highly purified preparations of the protein are enriched in an 18-kDa polypeptide which can be renatured from a denaturing gel and shown to bind ARS1 DNA. We have designated this protein DBF-A, DNA-binding factor A.     

Identification of L-cell growth stimulating antibody as anti-actin

Anti-L-cell antisera having potent cell growth stimulatory properties were shown by Western blotting to have predominant specificity toward a protein with a molecular weight of 42K which we identified as actin. Extractions of L cells, based upon the known insolubility of cytoskeletal proteins (including actin) in Triton X-100 and the solubility of actin in low ionic strength Ca2+ and ATP-containing buffer, led to actin-enriched preparations that retained immunoreactivity with the anti-L-cell antisera. The 42-kDa antigen binds to deoxyribonuclease I, has a pI = 5.2-5.4, and has an amino acid composition, including the presence of 3-methylhistidine, compatible with compositions determined for actins from other sources. Rabbit antiserum specific for this 42-kDa protein, isolated by SDS-PAGE, reproduced the cell growth stimulation by the anti-L-cell antisera and absorption of the antiserum with purified L-cell actin eliminated this stimulation. Moreover, these antibodies bind to the microfilaments of 3T3 fibroblasts. When purified actins were used as soluble antigen inhibitors of the immune reactivity of antiserum to 42-kDa protein with intact L cells, rabbit thymus actin competed with the surface molecules on L cells and reduced the stimulatory effect of the antiserum by 80% at an actin concentration of 150 micrograms/ml. Chicken muscle actin reduced the antibody stimulation effect by only 24% at the same protein concentration, and mouse muscle actin was ineffective as an inhibitor. The F(ab')2 fraction of anti-42K IgG was effective in stimulating L cells, thus documenting the immune nature of the actin-anti-42K interaction. We conclude that anti-actin antibodies, upon binding to actin-like cell surface determinants on L cells, stimulate cellular metabolism.     

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.     

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.