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In the Saccharomyces cerevisiae actin-profilin interface, Ala(167) of the actin barbed end W-loop and His(372) near the C terminus form a clamp around a profilin segment containing residue Arg(81) and Tyr(79). Modeling suggests that altering steric packing in this interface regulates actin activity. An actin A167E mutation could increase interface crowding and alter actin regulation, and A167E does cause growth defects and mitochondrial dysfunction. We assessed whether a profilin Y79S mutation with its decreased mass could compensate for actin A167E crowding and rescue the mutant phenotype. Y79S profilin alone caused no growth defect in WT actin cells under standard conditions in rich medium and rescued the mitochondrial phenotype resulting from both the A167E and H372R actin mutations in vivo consistent with our model. Rescue did not result from effects of profilin on actin nucleotide exchange or direct effects of profilin on actin polymerization. Polymerization of A167E actin was less stimulated by formin Bni1 FH1-FH2 fragment than was WT actin. Addition of WT profilin to mixtures of A167E actin and formin fragment significantly altered polymerization kinetics from hyperbolic to a decidedly more sigmoidal behavior. Substitution of Y79S profilin in this system produced A167E behavior nearly identical to that of WT actin. A167E actin caused more dynamic actin cable behavior in vivo than observed with WT actin. Introduction of Y79S restored cable movement to a more normal phenotype. Our studies implicate the importance of the actin-profilin interface for formin-dependent actin and point to the involvement of formin and profilin in the maintenance of mitochondrial integrity and function.

We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.

Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.] 347: 44-49) hypothesized that between subdomains 3 and 4 of actin is a loop of 10 amino acids including a four residue hydrophobic plug that inserts into a hydrophobic pocket formed by two adjacent monomers on the opposing strand thereby stabilizing the F-actin helix. To test this hypothesis we created a mutant yeast actin (L266D) by substituting Asp for Leu266 in the plug to disrupt this postulated hydrophobic interaction. Haploid cells expressing only this mutant actin were viable with no obvious altered phenotype at temperatures above 20 degrees C but were moderately cold-sensitive for growth compared with wild-type cells. The critical concentration for polymerization increased 10-fold at 4 degrees C compared with wild-type actin. The length of the nucleation phase of polymerization increased as the temperature decreased. At 4 degrees C nucleation was barely detectable. Addition of phalloidin-stabilized F-actin nuclei and phalloidin restored L266D actin's ability to polymerize at 4 degrees C. This mutation also affects the overall rate of elongation during polymerization. Small effects of the mutation were observed on the exchange rate of ATP from G-actin, the G-actin intrinsic ATPase activity, and the activation of myosin S1 ATPase activity. Circular dichroism measurements showed a 15 degrees C decrease in melting temperature for the mutant actin from 57 degrees C to 42 degrees C. Our results are consistent with the model of Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.]. 347:44-49) involving the role of the hydrophobic plug in actin filament stabilization.

We examined the effects of yeast actin NH2-terminal mutations on actomyosin interactions and the function of actin in vivo through measurements of actin-activated ATPase activity, cosedimentation with rabbit muscle myosin subfragment 1 (S-1), in vitro motility, and invertase secretion assays. As reported earlier (Cook, R. K., Blake, W., and Rubenstein, P. A. (1992) J. Biol. Chem. 267, 9430-9436), elimination of NH2-terminal acidic residues from yeast actin results in an increased actin bundling, decreased actin-activated S-1 ATPase, and complete inhibition of actin filament sliding over myosin. Here we show that the addition of 2 new acidic residues to the NH2 terminus of yeast actin increased the Vmax value and the catalytic efficiency of the actin-activated ATPase activity of S-1. However, the binding of actin to S-1 in the presence of ATP and the velocities of actin sliding over myosin in the in vitro motility assays were not affected by this mutation. Thus, the number of actin NH2-terminal negative charges is important for actin activation of myosin S-1 ATPase activity, while only a minimum number of acidic residues is required for actin sliding over myosin in vitro. The number of actin NH2-terminal negative charges therefore appears to determine the efficiency with which the energy from ATP hydrolysis is converted to filament sliding.

The nucleotide state of actin (ATP, ADP-P(i), or ADP) is known to impact its interactions with other actin molecules upon polymerization as well as with multiple actin binding proteins both in the monomeric and filamentous states of actin. Recently, molecular dynamics simulations predicted that a sequence located at the interface of subdomains 1 and 3 (W-loop; residues 165-172) changes from an unstructured loop to a beta-turn conformation upon ATP hydrolysis (Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277-1283). This region participates directly in the binding to other subunits in F-actin as well as to cofilin, profilin, and WH2 domain proteins and, therefore, could contribute to the nucleotide sensitivity of these interactions. The present study demonstrates a reciprocal communication between the W-loop region and the nucleotide binding cleft on actin. Point mutagenesis of residues 167, 169, and 170 and their site-specific labeling significantly affect the nucleotide release from the cleft region, whereas the ATP/ADP switch alters the fluorescence of probes located in the W-loop. In the ADP-P(i) state, the W-loop adopts a conformation similar to that in the ATP state but different from the ADP state. Binding of latrunculin A to the nucleotide cleft favors the ATP-like conformation of the W-loop, whereas ADP-ribosylation of Arg-177 forces the W-loop into a conformation distinct from those in the ADP and ATP-states. Overall, our experimental data suggest that the W-loop of actin is a nucleotide sensor, which may contribute to the nucleotide state-dependent changes in F-actin and nucleotide state-modulated interactions of both G- and F-actin with actin-binding proteins.

Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y single mutants as well as the Ile-76/Phe-79 and Ile-76/Tyr-79 double mutants. Tyr or Phe should decrease crowding and increase protein flexibility. Subsequent introduction of Ile should restore packing and dampen changes. All mutants showed decreased growth in liquid medium. W79Y alone was severely osmosensitive and exhibited vacuole abnormalities. Both properties were rescued by Ile-76. Phe-79 or Tyr decreased the thermostability of actin and increased its nucleotide exchange rate. These effects, generally greater for Tyr than for Phe, were reversed by introduction of Ile-76. HD exchange showed that the mutations caused propagated conformational changes to all four subdomains. Based on results from phosphate release and light-scattering assays, single mutations affected polymerization in the order of Ile, Phe, and Tyr from least to most. Introduction of Ile-76 partially rescued the polymerization defects caused by either Tyr-79 or Phe-79. Thus, alterations in crowding of the 76–79 residue pair can strongly affect actin conformation and behavior, and these results support the theory that the amino acid array in which they are located may play a central role in actin regulation.

Actins from most eukaryotes undergo a unique post-translational modification of the amino terminus called "processing." Processing consists of the removal of an amino-terminal Ac-Met or Ac-Cys to leave an acidic amino-terminal residue. We have previously demonstrated that this reaction is not catalyzed by the ribosomally associated methionine aminopeptidase or by other previously described acetylaminopeptidases. Here we present the isolation and characterization of the actin N-acetylaminopeptidase (ANAP) from rat liver. A five-step purification protocol achieves a 4100-fold purification of the enzyme with an overall 8% recovery of activity. ANAP is a 77-kDa monomer with a pI of 4.6. Using unprocessed yeast actin as a substrate, the Km of ANAP is 3.5 microM. Purified ANAP was used to generate a polyclonal antibody. The antibody has been used along with activity assays to demonstrate the presence of ANAP in a variety of rat tissues. Finally, evidence is presented that in mammals, ANAP may function with a second, as yet unpurified, component to process actin amino termini.

Previous studies have yielded conflicting results concerning the physiological role of profilin, a 12-15-kD actin- and phosphoinositide-binding protein, as a regulator of actin polymerization. We have addressed this question by directly microinjecting mammalian profilins, prepared either from an E. coli expression system or from bovine brain, into living normal rat kidney (NRK) cells. The microinjection causes a dose-dependent decrease in F-actin content, as indicated by staining with fluorescent phalloidin, and a dramatic reduction of actin and alpha-actinin along stress fibers. In addition, it has a strong inhibitory effect toward the extension of lamellipodia. However, the injection of profilin causes no detectable perturbation to the cell-substrate focal contact and no apparent depolymerization of filaments in either the nonlamellipodial circumferential band or the contractile ring of dividing cells. Furthermore, cytokinesis of injected cells occurs normally as in control cells. In contrast to pure profilin, high-affinity profilin-actin complexes from brain induce an increase in total cellular F-actin content and an enhanced ruffling activity, suggesting that the complex may dissociate readily in the cell and that there may be multiple states of profilin that differ in their ability to bind or release actin molecules. Our results indicate that profilin and profilactin can function as effective regulators for at least a subset of actin filaments in living cells.

Two known tissue-specific tropomyosin (TM) isoforms are produced from the rodent beta-TM gene. Skeletal muscle beta-TM uses the alternative exons 6b and 9a and the exon 9a-associated poly(A) site. Fibroblast and smooth muscle TM-1 use exons 6a and 9b and the exon-9b associated poly(A) site. We have identified a new skeletal muscle beta-TM isoform, beta-TM2. beta-TM2 contains exon 6b (muscle) and exon 9b (nonmuscle). Full-length beta-TM2 cDNA clones were isolated from a cDNA library of mouse muscle BC3H1 cells. Its mRNA was also found in mouse skeletal muscle tissue but not in other tissues. beta-TM2 mRNA level and protein synthesis are differentiation-dependent, with a transient high level in the early stages of myogenesis both in BC3H1 cells and in mouse embryo limbs. Trace amounts of beta-TM3 mRNA, the other hybrid form (exons 6a + 9a), were found in less differentiated BC3H1 cells, mouse uterus, heart, and 3T3 fibroblasts but not skeletal muscle tissue. Thus, the selection of the two alternative exons appears to be controlled independently. Furthermore, during myogenesis, there is a sequential switch in the internal alternative exon, the terminal exon, and the poly(A) site from the nonmuscle to the muscle type.

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.

Auditory hair cell function requires proper assembly and regulation of the nonmuscle gamma isoactin-rich cytoskeleton, and six point mutations in this isoactin cause a type of delayed onset autosomal dominant nonsyndromic progressive hearing loss, DFNA20/26. The molecular basis underlying this actin-dependent hearing loss is unknown. To address this problem, the mutations have been introduced into yeast actin, and their effects on actin function were assessed in vivo and in vitro. Because we previously showed that polymerization was unaffected in five of the six mutants, we have focused on proteins that regulate actin, in particular cofilin, which severs F-actin and sequesters actin monomers. The mutations do not affect the interaction of cofilin with G-actin. However, T89I and V370A mutant F-actins are much more susceptible to cofilin disassembly than WT filaments in vitro. Conversely, P332A filaments demonstrate enhanced resistance. Wild type actin solutions containing T89I, K118M, or P332A mutant actins at mole fractions similar to those found in the hair cell respond in vitro toward cofilin in a manner proportional to the level of the mutant present. Finally, depression of cofilin action in vivo by elimination of the cofilin-activating protein, Aip1p, rescues the inability to grow on glycerol caused by K118M, T278I, P332A, and V370A. These results suggest that a filament instability caused by these mutations can be balanced by decreasing a system in vivo that promotes increased filament turnover. Such mutant-dependent filament destabilization could easily result in hair cell malfunction leading to the late-onset hearing loss observed in these patients.

The budding yeast formins, Bnr1 and Bni1, behave very differently with respect to their interactions with muscle actin. However, the mechanisms underlying these differences are unclear, and these formins do not interact with muscle actin in vivo. We use yeast wild type and mutant actins to further assess these differences between Bnr1 and Bni1. Low ionic strength G-buffer does not promote actin polymerization. However, Bnr1, but not Bni1, causes the polymerization of pyrene-labeled Mg-G-actin in G-buffer into single filaments based on fluorometric and EM observations. Polymerization by Bnr1 does not occur with Ca-G-actin. By cosedimentation, maximum filament formation occurs at a Bnr1:actin ratio of 1:2. The interaction of Bnr1 with pyrene-labeled S265C Mg-actin yields a pyrene excimer peak, from the cross-strand interaction of pyrene probes, which only occurs in the context of F-actin. In F-buffer, Bnr1 promotes much faster yeast actin polymerization than Bni1. It also bundles the F-actin in contrast to the low ionic strength situation where only single filaments form. Thus, the differences previously observed with muscle actin are not actin isoform-specific. The binding of both formins to F-actin saturate at an equimolar ratio, but only about 30% of each formin cosediments with F-actin. Finally, addition of Bnr1 but not Bni1 to pyrene-labeled wild type and S265C Mg-F actins enhanced the pyrene- and pyrene-excimer fluorescence, respectively, suggesting Bnr1 also alters F-actin structure. These differences may facilitate the ability of Bnr1 to form the actin cables needed for polarized delivery of nutrients and organelles to the growing yeast bud.

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.

Muscle and yeast actins display distinct behavioral characteristics. To better understand the allosteric interactions that regulate actin function, we created a muscle/yeast hybrid actin containing a muscle-specific outer domain (subdomains 1 and 2) and a yeast inner domain (subdomains 3 and 4). Actin with muscle subdomain 1 and the two yeast N-terminal negative charges supported viability. The four negative charge muscle N terminus in a muscle subdomain 1 background caused death, but in the same background actin with three N-terminal acidic residues (3Ac/Sub1) led to sick but viable cells. Addition of three muscle subdomain 2 residues (3Ac/Sub12) produced no further deleterious effects. These hybrid actins caused depolarized cytoskeletons, abnormal vacuoles, and mitochondrial and endocytosis defects. 3Ac/Sub1 G-actin exchanged bound epsilonATP more slowly than wild type actin, and the exchange rate for 3Ac/Sub12 was even slower, similar to that for muscle actin. The mutant actins polymerized faster and produced less stable and shorter filaments than yeast actin, the opposite of that expected for muscle actin. Unlike wild type actin, in the absence of unbound ATP, polymerization led to ADP-F-actin, which rapidly depolymerized. Like yeast actin, the hybrid actins activated muscle myosin S1 ATPase activity only about one-eighth as well as muscle actin, despite having essentially a muscle actin-specific myosin-binding site. Finally, the hybrid actins behaved abnormally in a yeast Arp2/3-dependent polymerization assay. Our results demonstrate a unique sensitivity of yeast to actin N-terminal negative charge density. They also provide insight into the role of each domain in the control of the various functions of actin.

Actin, one of the most highly conserved and abundant eukaryotic proteins, is constantly being polymerized and depolymerized within cells as part of cellular motility, tissue formation and repair, and embryonic development. Many proteins exist that bind to monomeric or filamentous (F) forms of actin to regulate the polymerization state. It has become increasingly apparent that the ability of different proteins to bind to and regulate actin filament dynamics depends on the ability of the filament to exist in altered conformations. Yet, little is known about how these conformational changes occur at the molecular level. We have destabilized F-actin filaments by forming a disulfide that locks the “hydrophobic plug” to the body of the actin subunit or by altering the C terminus of actin with a tetramethylrhodamine label. We also examined F-actin filaments at short times after the initiation of polymerization. In all three cases, a substantial fraction of protomers can be found in a “tilted” state that also is induced by actin depolymerizing factor/cofilin proteins. These observations suggest that F-actin filaments are annealed over time into a stable filament and that actin-depolymerizing proteins can effect a time reversal of polymerization.

Neisserial porins may play a role in the invasion of the host cell by the bacterium. The protein translocates to the host cell membrane and then to the cytosol during the invasive process, and we have shown it interacts with actin in vitro. Here, we have examined the nucleotide-dependence of the interaction of Neisseria porin, P.IB, with fluorescently labeled yeast G actin. Increasing free ATP between 0 to 0.5 mM retards complex formation between the two proteins. The ATP effect probably results from binding of the nucleotide to actin rather than to porin. Complex formation results in a biphasic release of bound nucleoside triphosphate from actin in the absence of free nucleotide at a rate slower than that of complex formation, but it does not induce hydrolysis of the actin-bound nucleotide. ATP prevents the porin-induced distortion of F-actin structure, and addition of ATP to the complex formed in the absence of free nucleotide induces actin polymerization indicating that P.IB stabilizes nucleotide-free G-actin. Our results suggest that P.IB causes an actin conformation change leading to the production of a polymerization-competent nucleotide-free protein.

With yeast actin, contrary to other actins, filament formation, ATP hydrolysis, and Pi release are concurrent at low actin concentrations, the condition usually employed to assess actin polymerization. This observation leads to a question concerning the conformation of the filament barbed end that might be recognized by specific actin-binding proteins. To try to detect possible new actin polymer conformations that might be intermediate in the pathway leading to mature F-actin, we monitored the change in intrinsic tryptophan fluorescence of yeast and muscle actins polymerized at pH 6 to accelerate the rate of filament formation. This allowed temporal resolution of the Pi release process from the slower process of polymerization. With both actins, we detected a biphasic instead of the usual monophasic fluorescence change, a rapid decrease that tracks with filament formation followed by a slower rebound (the second phase). This second phase postpolymerization conformational change requires Pi release and occurs nearly coincident with its release. The addition of Pi causes this second phase response to disappear, and the inclusion of Pi during polymerization prevents its appearance. At pH 7.5, with higher yeast actin concentrations to accelerate polymerization, a two-phase fluorescence change is also observed. In this case, the second phase change lags substantially behind Pi release. Pi release could also be resolved from polymer formation. V159N yeast actin, hypothesized previously as remaining in a postpolymerization ATP-like state, exhibits the same two-phase intrinsic tryptophan fluorescence behavior as wild-type yeast actin. Together, these observations demonstrate the presence of an intermediate filament state between ADP-Pi and mature ADP-F-actin.

Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih−/−) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk−/−) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk−/− embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfks11 variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease., Cardiac anomolies can result from very early defects in heart development. In zebrafish, such defects have been shown to be caused by a new gene called cardiofunk

The zebrafish cardiofunk actin mutation, R177H, causes abnormal heart development. We have introduced this mutation into yeast actin to assess its biochemical consequences. R177H G-actin exhibited reduced thermal stability and an accelerated nucleotide exchange rate. R177H actin has an increased critical concentration and polymerizes with a greatly extended nucleation phase but a faster elongation process, suggesting that significant fragmentation accompanies filament formation. Pi release from R177H actin is tightly coupled to polymerization, as with wild type (WT) actin, suggesting that the R177H mutation does not affect ATPase activity and Pi release. R177H actin shows no polymerization-dependent decrease in intrinsic Trp fluorescence, and the fluorescence yield of a pyrene at Cys374 is decreased. An equivalent amount of WT actin significantly but not completely rescues the mutant's polymerization defect. Tropomyosin greatly exacerbates the elongation of the nucleation phase of R177H actin but slightly decreases its critical concentration. It has only a slight effect on a 1:1 WT/mutant mixture. The defects we observed with R177H actin in vitro indicate that Arg177 is crucial for the control of the structural integrity of the actin monomer and the actin filament and provide insight into the defects caused by this mutation in zebrafish cardiogenesis.

Many critical cellular processes, including proliferation, vesicle trafficking, and secretion, are regulated by both phospholipase D (PLD) and the actin microfilament system. Stimulation of human PLD1 results in its association with the detergent-insoluble actin cytoskeleton, but the molecular mechanisms and functional consequences of PLD-actin interactions remain incompletely defined. Biochemical and pharmacologic modulation of actin polymerization resulted in complex bidirectional effects on PLD activity, both in vitro and in vivo. Highly purified G-actin inhibited basal and stimulated PLD activity, whereas F-actin produced the opposite effects. Actin-induced modulation of PLD activity was independent of the activating stimulus. The efficacy and potency of the effects of actin were isoform-specific but broadly conserved among actin family members. Human betagamma-actin was only 45% as potent and 40% as efficacious as rabbit skeletal muscle alpha-actin, whereas its inhibitory profile was similar to the single actin species from the yeast, Saccharomyces cerevisiae. Use of actin polymerization-specific reagents indicated that PLD1 binds both monomeric G-actin, as well as actin filaments. These data are consistent with a model in which the physical state of the actin cytoskeleton is a critical determinant of its regulation of PLD activity.

Polymerization increases a low level G-actin ATPase activity yielding ADP-P(i) F-actin and then ADP F-actin following release of P(i). By monitoring P(i) release, we explored the relationship between the ATPase activity and polymerization characteristics of a mutant yeast actin, GG. In this mutant, two hydrophobic residues at the tip of a proposed hydrophobic plug between actin subdomains 3 and 4, Val(266) and Leu(267), were mutated to Gly. Although GG-actin does not polymerize by itself in vitro, GG cells are viable. We show that GG-actin ATPase activity increases under normal polymerization conditions, although stable filaments do not form. A plot of P(i) release rate versus actin concentration yields an apparent critical concentration, like that seen for actin polymerization, of approximately 8 microm for Mg(2+) GG-actin and 11 microm for Ca(2+) GG-actin. In contrast to WT-actin, P(i) release from GG-actin is cold-sensitive, reflecting the temperature sensitivity associated with mutations that decrease hydrophobicity in this region. Thus, under polymerization conditions, GG-actin exhibits a continuous F-actin-like ATPase activity resulting from the temperature-sensitive formation of unstable cycling F-actin oligomers. Tropomyosin limits the extent and rate of this activity and restores polymerization by capturing and stabilizing these oligomers rather than enhancing filament nucleation.

A major function of tropomyosin (TPM) in nonmuscle cells may be stabilization of F-actin by binding longitudinally along the actin filament axis. However, no clear evidence exists in vitro that TPM can significantly affect the critical concentration of actin. We previously made a polymerization-defective mutant actin, GG (V266G, L267G). This actin will not polymerize alone at 25 degrees C but will in the presence of phalloidin or beryllium fluoride. With beryllium fluoride, but not phalloidin, this polymerization rescue is cold-sensitive. We show here that GG-actin polymerizability was restored by cardiac tropomyosin and yeast TPM1 and TPM2 at 25 degrees C with rescue efficiency inversely proportional to TPM length (TPM2 > TPM1 > cardiac tropomyosin), indicating the importance of the ends in polymerization rescue. In the presence of TPM, the apparent critical concentration of actin is 5.5 microm, 10-15-fold higher than that of wild type actin but well below that of the GG-actin alone (>20 microm). Non N-acetylated TPMs did not rescue GG-actin polymerization. The TPMs did not prevent cold-induced depolymerization of GG F-actin. TPM-dependent GG-actin polymerization did not occur at temperatures below 20 degrees C. Polymerization rescue may depend initially on the capture of unstable GG-F-actin oligomers by the TPM, resulting in the strengthening of actin monomer-monomer contacts along the filament axis.

Force and motion generation by actomyosin involves the cyclic formation and transition between weakly and strongly bound complexes of these proteins. Actin's N-terminus is believed to play a greater role in the formation of the weakly bound actomyosin states than in the formation of the strongly bound actomyosin states. It has been the goal of this project to determine whether the interaction of actin's N-terminus with myosin changes upon transition between these two states. To this end, a yeast actin mutant, Cys-1, was constructed by the insertion of a cysteine residue at actin's N-terminus and replacement of the C-terminal cysteine with alanine. The N-terminal cysteine was labeled stoichiometrically with pyrene maleimide, and the properties of the modified mutant actin were examined prior to spectroscopic measurements. Among these properties, actin polymerization, strong S1 binding, and the activation of S1 ATPase by pyrenyl-Cys-1 actin were not significantly different from those of wild-type yeast actin, while small changes were observed in the weak S1 binding and the in vitro motility of actin filaments. Fluorescence changes upon binding of S1 to pyrenyl-Cys-1 actin were measured for the strongly (with or without ADP) and weakly (with ATP and ATPgammaS) bound acto-S1 states. The fluorescence increased in each case, but the increase was greater (by about 75%) in the presence of MgATP and MgATPgammaS than in the rigor state. This demonstrates a transition at the S1 contact with actin's N-terminus between the weakly and strongly bound states, and implies either a closer proximity of the pyrene probe on Cys-1 to structural elements on S1 (most likely the loop of residues 626-647) or greater S1-induced changes at the N-terminus of actin in the weakly bound acto-S1 states.

A mutant yeast actin (GG) has decreased hydrophobicity in a subdomain 3/4 hydrophobic plug believed to be involved in a hydrophobic cross-strand "plug-pocket" interaction necessary for actin filament stability. This actin will not polymerize in vitro but is compatible with cell viability. We have assessed the ability of Sac6p, the yeast homologue of the actin filament stabilizing and bundling protein fimbrin, to restore polymerization in vitro and to facilitate GG-actin function in vivo. Sac6p rescues GG-actin polymerization at 25 degrees C but not at 4 degrees C. The actin polymerizes into bundles at room temperature with a fimbrin:actin molar ratio of 1:4. At this ratio, every actin monomer contacts a Sac6p actin binding domain. Following cold-induced depolymerization, actin/Sac6p mixtures repolymerize beginning at 15 degrees C instead of the 25 degrees C required for de novo assembly, because of the presence of residual actin-Sac6p nuclei. Generation of haploid Deltasac6/GG-actin cells from either diploid or haploid cells was unsuccessful. The facile isolation of cells with either mutation alone indicates a synthetic lethal relationship between this actin allele and the SAC6 gene. Sac6p may allow GG-actin function in vivo by stabilizing the actin in bundles thereby helping maintain sufficient levels of an otherwise destabilized actin monomer within the cell.

To identify a protein histidine methyltransferase from Saccharomyces cerevisiae, we examined purified actin for the presence of the highly conserved 3-methylhistidine residue at position 73 by amino acid analysis of the whole protein and by amino acid analysis and mass spectrometry of the corresponding tryptic fragment. Surprisingly, we found that His-73 is not modified. A similar lack of modification was also found in actin from the yeast Candida albicans, while rabbit muscle actin revealed the expected 3-methylhistidine residue. Phylogenetic analysis of actin sequences suggests that this modification was introduced in evolution after the divergence of yeast from higher eukaryotic organisms, including unicellular eukaryotes such as Acanthamoeba, Dictyostelium, and Physarum, whose actins contain 3-methylhistidine. Our methodology for the analytical determination of 3-methylhistidine in actin offers an improved approach for investigating histidine methylation in proteins.

The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure.

Although the actin sequence is very highly conserved across evolution, tissue-specific expression of different isoforms in high eukaryotes suggests that different isoforms carry out different functions. However, little information exists about either the differences in filaments made from different actins or the effects on filament structure caused by the various mutations in actin that have been introduced to gain insight into actin function. Using electron microscopy and three-dimensional reconstruction, we have studied the differences in the filaments made by yeast and rabbit skeletal muscle actin, two proteins with 88% homologous sequences, and we have assessed the changes in filament structure caused by the introduction of the S14A mutation into yeast actin. Elimination of the S14 hydroxyl group, assumed to bind to the gamma-phosphate of actin-bound ATP, results in a 40 to 60-fold decrease in actin's affinity for ATP. We show that yeast actin displays less extensive contacts between the two long-pitch helical strands than does muscle actin, and displays the large cooperativity within filaments previously observed for muscle actin. Finally, we demonstrate that the S14A mutation narrows the cleft between the two lobes of the actin subunit and strengthens the inter-strand connections in F-actin.

The hydrophobicity of the subdomain 3/4 hydrophobic loop (262-274) has been implicated to be essential for actin's function. We previously showed (Kuang, B., and Rubenstein, P. A. (1997) J. Biol. Chem. 272, 1237-1247) that a mutant yeast actin (V266G/L267G) with markedly decreased hydrophobicity in this loop conferred severe cold sensitivity to its polymerization. Here we further tested the mutational effect on the conformation and function of G-actin. This GG mutation caused no significant changes in overall secondary structure or in the microenvironment around actin's tryptophan residues, nor did it alter the dissociation constant of G-actin for ATP. However, it lowers the intrinsic ATPase activity and the melting temperature for Mg-GG actin from 51 to 33 degrees C and transforms the conformation of subdomain 2 and the central cleft of G-actin into an F-monomer-like structure. The results suggest that the hydrophobic plug may not only play a role in actin filament stabilization but also may be important for controlling the stability of G-actin and for promoting the conformational change of the monomer needed for addition to a growing actin filament.

Holmes proposed that in F-actin, hydrophobic residues in a subdomain 3/4 loop interact with a hydrophobic pocket on the opposing strand resulting in helix stabilization. We have determined how a decreased hydrophobicity of this plug affects yeast actin function. Cells harboring only the V266G, V266D, V266F, L267G, L269D, or L269K actins appear normal, although V266G cells display an altered budding pattern. However, V266G,L267G (GG) double mutant cells are cold-sensitive with randomly oriented thick actin assemblies seen in rhodamine phalloidin-stained GG cells. V266D actin polymerizes slower than wild-type actin at room temperature. At 4 degrees C, not only is polymerization slowed, but there is also an effect on critical concentration. However, the polymerization defects are milder than those associated with substitution of Asp for the neighboring Leu267. Purified GG-actin does not polymerize in vitro alone or in the presence of wild-type F-actin seeds. GG-actin polymerization can be restored by larger amounts of wild-type actin, beryllium fluoride, or phalloidin at room temperature, although at 4 degrees C only phalloidin is effective. These results suggest that the diminished hydrophobicity of the plug in GG-actin leads to filament destabilization. However, the V266D actin results require a modification of the original Holmes filament model.

The Ser14 hydroxyl group of actin is one of six groups that potentially form hydrogen bonds with the gamma-phosphate of the ATP bound in the cleft separating the two domains of the protein. To understand the importance of this group in actin function, we mutated Ser14 of Saccharomyces cerevisiae actin and studied the effects of these mutations in vivo and in vitro. Substitution of Cys of Gly resulted in cell death. Substitution of Thr for Ser resulted in an actin with wild-type properties in vivo and in vitro. Cells carrying the Ser14--Ala (S14A) mutation were viable but displayed a temperature sensitive lethality at 37 degrees C preceded by delocalization of actin patches, the appearance of bar-like structures, and finally the disappearance of identifiable actin structures. The mutation caused no effect on the critical concentration of polymerization but resulted in an actin with an increased rate of polymerization, an altered protease susceptibility, and a decreased filament ATPase activity. At 37 degrees C, Mg-, but not Ca-S14A-actin irreversibly lost the ability to polymerize. These results demonstrate the importance of the ATP-Ser14 hydroxyl hydrogen bond in regulating actin function in vivo and in vitro and the magnification of the effects of the mutation when Mg2+ is substituted for Ca2+ in the protein.

N-Terminal yeast actin mutants were used to assess the role of N-terminal acidic residues in the interactions of caldesmon with actin. The yeast actins differed only in their N-terminal charge: wild type, two negative charges; 4Ac, four negative charges; DNEQ, neutral charge; delta DSE, one positive charge. Caldesmon inhibition of actomyosin subfragment 1 ATPase was affected by alterations in the N-terminus of actin. This inhibition was similar for skeletal muscle alpha-actin and the yeast 4Ac and wild-type actins (80%), but much smaller for the neutral and deletion mutants (15%). However, cosedimentation experiments revealed similar binding of caldesmon to polymerized rabbit skeletal muscle alpha-actin and each yeast actin. This result shows that the N-terminal acidic residues of actin are not required for the binding of caldesmon to F-actin. Caldesmon-actin interactions were also examined by monitoring the polymerization of G-actin induced by caldesmon. Although the final extent of polymerization was similar for all actins tested, the rates of polymerization differed. Skeletal muscle and 4Ac actins had similar rates of polymerization, and the wild-type actin polymerized at a slower rate. The neutral and deletion mutants had even slower rates of polymerization by caldesmon. The slow polymerization of DNEQ G-actin was traced to a greatly reduced binding of caldesmon to this mutant G-actin when compared to wild-type and alpha-actin. MgCl2-induced actin polymerization proceeded at identical rates for all actins.(ABSTRACT TRUNCATED AT 250 WORDS)

Profilin is a small G-actin binding protein implicated in sequestering actin monomers in vivo. We have quantitated profilin and actin expression in human hepatoma HepG-2 cells and in two mouse myogenic cell lines, BC3H1 and C2C12, to determine whether the expression of profilin and the expression of nonmuscle isoactin or total actin are co-regulated. During differentiation of both muscle cell types, profilin and nonmuscle actin expression decrease in a coordinate manner as shown by measurements of steady state mRNA and newly synthesized protein. In human hepatoma HepG-2 cells, the twofold increase in actin synthesis observed after 24 hours of exposure to cytochalasin D did not result in an increase in profilin synthesis. Thus, profilin and actin expression are not co-regulated in all cells. To determine if there is sufficient profilin to sequester a large portion of cellular G-actin, we measured total profilin and G-actin levels in the three cell types. In each case, profilin accounted for less than 10% of the total G-actin on a molar basis. Thus, profilin is not responsible for total G-actin sequestration in these cells. Finally, using poly-L-proline affinity chromatography, we showed that, in the cell types tested, less than 20% of the poly-L-proline purified profilin existed as a complex with G-actin. The profilin in these cells may be interacting with cellular components other than actin.

Actin is a protein that plays an important role in cell structure, cell motility, and the generation of contractile force in both muscle and nonmuscle cells. In many organisms, multiple forms of actin, or isoactins, are found. These are products of different genes and have different, although very similar, amino acid sequences. Furthermore, these isoactins are expressed in a tissue specific fashion that is conserved across species, suggesting that their presence is functionally important and their behavior can be distinguished quantitatively from one another in vitro. In muscle cells, they are differentially distributed within the cell and some are specifically associated with structures such as costameres, mitochondria, and neuromuscular junctions. There is also good evidence for specific isoactin function in microvascular pericytes and in the intestinal brush border. However, the necessity of specific isoactins for various functions has not yet been conclusively demonstrated.

We have carried out a two-dimensional gel analysis of the actin system of Dictyostelium discoideum. Our results show that on the basis of isoelectric focusing, there is a single major [35S]methionine-labeled species which corresponds both to the actin purified by Uyemura et al. (Uyemura, D., Brown, S.S., and Spudich, J.A. (1978) J. Biol. Chem. 253, 9088-9095) and to the Coomassie Blue staining species seen in whole cell lysates of the organism. We also detect a minor labeled actin species, x, which has no corresponding Coomassie Blue staining counterpart. This species turns over much more rapidly than the major actin and has one more positive charge. It is not labeled with [3H]acetate, whereas the major actin is. When D. discoideum RNA is added to a mRNA-dependent rabbit reticulocyte lysate protein translation system, only one major actin is seen, and this species corresponds to the major actin observed in vivo. If endogenous acetyl coenzyme A is removed from the translation system, a second major actin appears corresponding in position to x. These results indicate that in D. discoideum, there is present a single major actin species in addition to a small amount of a rapidly turning over actin which is a nonacetylated form of the major actin. Additional experiments examining these actins through the developmental cycle of the organism show no consistent differences with the results obtained using vegetative cells.

Vesicular stomatitis virus (VSV) plaque formation and replication are restricted in duck embryo cells compared to chick embryo cells. Mutants of VSV which replicate normally on both chick and duck cells were isolated. The duck-adapted mutant, when passed through chick cells, retains its ability to grow normally on duck cells. This indicates that the ability of this virus to grow on duck cells is due to mutation rather than to host-controlled modification. VSV is restricted in duck cells but not in quail or pheasant embryo cells which follows the evolutionary relationship of these species. Newcastle disease virus and Rous sarcoma virus are restricted in duck cells also. Virus-specific protein synthesis of the wild-type VSV is greatly reduced in duck cells compared to chick cells. A structural protein of the duck cell-adapted mutant is altered.

Turkey gizzard smooth muscle myofibrils, the actin of which is composed of 75% smooth muscle γ-isoactin and 25% nonmuscle β-isoactin, were separated into an actomyosin and a cytoskeletal fraction. Isoelectric focusing analysis of the actomyosin actin showed it was 80% γ-isoactin and 20% β-isoactin. It thus appears that the major actin in the tissue is also the major form involved in force generation. When the cytoskeletal material was extracted with low-ionic-strength solution for 18 h at 4 °C, the actin released was 95% γ and 5% β compared with the 75:25 ratio found in the original cytoskeletal material. The extracted material revealed the presence of F-actin filaments and high-molecular-weight aggregates. Little of the material was in a low-molecular-weight form. On the other hand, extraction of the cytoskeletal material with 0.6 m KI resulted in the two isoactins being extracted in the same proportions in which they were found in the original cytoskeletal material. However, when this KI-extracted material was subsequently chromatographed on Bio-Gel A-5m equilibrated with 0.6 m KCl, the γ-isoactin migrated predominantly as a very high molecular weight form while the β-isomer moved in the lower-molecular-weight range of the elution profile. This aggregation behavior displayed by the γ-isoactin was not observed with the γ-isoactin in the actomyosin fraction. These results show that the two gizzard isoactins in the cytoskeletal residue behave very differently in response to various extraction media, and are consistent with possible differential isoactin utilization in gizzard smooth muscle.

Genes for the various isoactins define two classes of actin. Class I actin genes code for Met-Asp(Glu)-actin, and class II actin genes code for Met-X-Asp(Glu)-actin where X is usually cysteine. Amino termini of both are removed in an acetylation-dependent processing reaction yielding acetyl-Asp(Glu)-actin. Both classes are processed at approximately equal rate (t1/2 = 15 min) in vivo. In vitro, class II actins are 90% processed by endogenous enzymes after 60 min in a rabbit reticulocyte lysate system, whereas class I actins are only minimally processed during this period. Using site-directed mutagenesis of a human skeletal muscle isoactin coupled with in vitro transcription and translation methods, we have synthesized a pseudo-class I actin in which the penultimate cysteine has been changed to an aspartic acid, thus placing a class I amino terminus on an otherwise class II actin molecule. The pseudo-class I actin was less than 20% processed during the translation period as determined by peptide mapping. It was further processed by exogenous processing enzyme at a rate compatible with a class I actin. These results indicate that the major actin determinant controlling differential actin-processing rates is the amino-terminal residue being cleaved, not the remaining structure of the actin molecule. We have also demonstrated for the first time that N-acetylmethionine is the immediately released product from the amino terminus of a pseudo-class I actin during processing.

A fully translated actin biosynthetic intermediate containing N-acetylcysteine at the NH2 terminus has been identified in homogenates of differentiated mouse BC3H1 cerebrovascular smooth muscle cells labeled with L-[35S]cysteine. Thermolysin digestion of the highly acidic NH2-terminal tryptic peptide of this intermediate and electrophoretic analysis of the resulting fragments indicated that the intermediate was a precursor of smooth muscle alpha- isoactin , the major isoactin species in vascular smooth muscle. Carboxypeptidase A digestion of the thermolysin cleavage product corresponding to the first eight amino acid residues of the NH2-terminal tryptic peptide demonstrated an acetylcysteine-glutamate residue at the NH2 terminus. These results imply that the gene for smooth muscle alpha- isoactin , like genes coding for skeletal and cardiac alpha- isoactins , contains a cysteine codon immediately following the initiator methionine codon. Both the methionine and cysteine residues must be removed from the NH2 terminus of the intermediate to yield the mature form of smooth muscle alpha- isoactin . The removal of the cysteine residue in vivo is not direct but apparently involves acetylation of the cysteine and subsequent post-translational cleavage of the resulting acetylcysteine. Such an acetylation-dependent pathway has been demonstrated for removal of cysteine from the NH2 terminus of Drosophila actin synthesized in a cell-free translation system ( Rubenstein , P. A., and Martin, D. J. (1983) J. Biol. Chem. 258, 11354-11360). In vivo pulse-chase experiments indicate that the smooth muscle alpha- isoactin intermediate in BC3H1 cells turns over much more slowly than nonmuscle actin intermediates previously identified in mouse L-cells.

The expression of a vascular smooth muscle specific alpha-actin isoform can be induced in mouse BC3H1 smooth muscle cells by treating confluent monolayers with serum-free medium (Strauch, A. R., and Rubenstein, P. A. (1984) J. Biol. Chem. 259, 3152-3159; 7224-7229). Using blot hybridization techniques, two size classes of actin RNA were identified in BC3H1 cells with the relative amount of RNA in each size class varying according to the developmental state of the cells; a 2100-nucleotide actin RNA was most abundant in myoblasts, whereas a smaller 1500-nucleotide actin RNA was found predominantly in fully differentiated myocytes. Results of in vitro translation experiments suggested that the 2100-nucleotide actin RNA on blots of myoblast total RNA corresponded to a mixture of similar size transcripts encoding both beta- and gamma-actin, while the 1500-nucleotide actin RNA in myocytes was an alpha-actin mRNA. Cell-cell contact and serum withdrawal initiated a 6-fold increase in the level of alpha-actin mRNA in BC3H1 cells that was followed by a 3-fold decrease in the amount of beta- and gamma-actin mRNA when confluent cells were exposed to serum-free medium for prolonged periods. Vascular smooth muscle alpha-actin was the major alpha-actin isoform synthesized in L-[35S]cysteine-labeled BC3H1 myocytes, indicating that the 1500-nucleotide actin mRNA size class in these cells may be enriched for vascular smooth muscle alpha-actin transcripts.

The influence of the membrane-stabilizing agents diphenylhydantoin (DPH) and orgotein (a drug with superoxide dismutase activity) on protein synthesis was studied in cultured human muscle cells obtained from 7 patients with Duchenne muscular dystrophy (DMD) and 14 controls. The cultures were obtained by dissociation and subsequent plating of cells from biopsied quadriceps muscle. These cultures were then labeled with tritiated leucine in the presence and absence of the membrane-stabilizing drugs. Total protein synthesis (cpm/mg noncollagen protein) in first-passage muscle cell cultures from DMD patients showed a 35% decrease, but myosin synthesis revealed normal values. DPH and orgotein increased total protein synthesis by 35 and 50%, respectively, in dystrophic cultured cells, but only by 7 and 8%, respectively, in control cultured cells.

It had previously been reported (B. Krzysik, J. P. Vergnes, and I. R. McManus (1971) Arch. Biochem. Biophys. 146, 34-45) that prior to day 11 of embryonic life chick skeletal muscle actin contained little or no 3-methylhistidine, and that between Day 11 and 18, the degree of actin histidine methylation increased until it leveled off at 1 mol of 3-methylhistidine/mol actin. This is the value seen in adult muscle and nonmuscle actins so far analyzed. To determine whether this delayed onset of actin methylation occurred simultaneously throughout the organism or differed from tissue to tissue, the 3-methylhistidine content of cardiac muscle actin from Day 2 of embryonic life to hatching and of brain actin at Days 9, 11, and 14 were analyzed. These results, obtained by analyzing unlabeled actin samples as well as samples labeled in vivo with [3H]histidine, showed that at all stages, 1 mol of 3-methylhistidine was present per mol of actin. When skeletal muscle samples obtained from Day 11 to 18 embryos were analyzed 1 mol of 3-methylhistidine/mol of actin was observed. Thus, in the chick embryo, contrary to those reports published earlier, it was found that actin histidine methylation is not under developmental control.

Class II actin genes usually specify a polypeptide with a Met-Cys-Asp NH2 terminus, whereas the actin itself begins with an acetyl (Ac)-Asp(Glu). Previous studies with Drosophila actin showed that the first detectable intermediate is one with an Ac-Cys NH2 terminus which is subsequently cleaved in a novel reaction to expose the Asp. The initiator Met was probably removed early in translation as a free amino acid. To determine whether the class II actin initiating Met could also be removed in an acetylation-dependent manner, we translated Drosophila mRNA in a rabbit reticulocyte lysate in which protein acetylation was inhibited. After 60 min, three actin intermediates were detected, NH2-Met-Cys-Asp-actin, Ac-Met-Cys-Asp-actin, and NH2-Cys-Asp-actin. During processing in the presence of acetyl-CoA, three additional species were observed with NH2-terminal Ac-Cys-Asp, NH2-Asp, and Ac-Asp segments. In a time- and acetyl-CoA-dependent fashion, Met-Cys-Asp-actin was processed to the mature actin, presumably through an Ac-Met-Cys-Asp intermediate. Thus, two different pathways for removal of the initiator Met of class II actins, acetylation-dependent and independent, are possible. Since no class II actin intermediate containing the initiator Met is seen in vivo, although in class I actins this intermediate is observed, the most probable pathway for class II actins in vivo is the cotranslational removal of the initiator Met as a free amino acid.

An isoactin analysis was performed on L-[35S]cysteine labeled BC3H1 cells to determine if these smooth muscle-like cells synthesize vascular smooth muscle actin. Three different NH2-terminal peptides were identified on thin layer electrophoretograms of DNase I-purified and trypsin-digested BC3H1 cell actin. Results obtained from secondary digestion with thermolysin or Staphylococcus aureus V8 protease showed that the most acidic NH2-terminal peptide was derived from vascular smooth muscle alpha-isoactin. Treatment of cell monolayers with serum-free medium caused a 3-fold increase in the level of alpha-isoactin expression and a concomitant decrease in the level of non-muscle beta- and gamma-isoactin. Cell-cell contact was required for induction of alpha-isoactin, and the effects of serum depletion on isoactin expression and cell growth were reversible. The intensity of about 11 out of 500 polypeptide spots on two-dimensional gels of BC3H1 cell polypeptides also was influenced by the culture conditions. The finding that smooth muscle isoactin expression was coupled to cell growth conditions indicate the potential usefulness of BC3H1 cells in studies of isoactin expression and utilization during vascular smooth muscle development.

the NHz- terminal acetyl and methionyl residues from the poly- peptide chain to generate a polypeptide terminating in aspartic acid. Removal of the methionine apparently requires its prior acetylation. Once the aspartic acid residue is exposed at the NHz terminus, the actin can again be acetylated in an acetyl-CoA-dependent reac- tion to yield a polypeptide probably identical with

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.

We have examined the effects of epidermal growth factor (EGF), platelet-derived growth factor, and insulin on the differentiation of a mouse vascular smooth muscle-like cell line, the BC3H1 cells. On the basis of cell morphology and smooth muscle alpha-isoactin synthesis, we demonstrate that EGF at physiological concentrations prevents the differentiation of these cells, whereas platelet-derived growth factor has no apparent effect. The induction of alpha-isoactin synthesis by serum deprivation is inhibited by EGF in a dose-dependent manner with a half-maximal effect at 3-5 ng/ml and a maximal inhibition at approximately 30 ng/ml. Northern analysis also shows that EGF blocks the accumulation of alpha-isoactin mRNA normally observed during cell differentiation. Addition of EGF to differentiated cells results in a repression of alpha-isoactin synthesis, a stimulation of beta- and gamma-isoactin synthesis, and the stabilization of the nonmuscle isoactins. The synthesis of creatine phosphokinase, a muscle-specific noncontractile protein, is also regulated by EGF in a similar fashion. Modulation by EGF of alpha-isoactin expression is not affected by aphidicolin and is therefore independent of its mitogenic effect on these cells. Insulin is not required for observation of the EGF-dependent effects but instead seems to promote differentiation. Our results show that EGF can replace serum in controlling the differentiation of BC3H1 cells.

An affinity chromotography resin highly specific for rat liver tyrosine aminotransferase (EC 2.6.1.5) has been synthesized and used in the purification of this enzyme. The structure of the resin, N -(5′-phosphopyridoxyl)- l -tyrosyl-aminoocytl-Sepharose 4B, was designed to resemble the tyrosine-pyridoxal phosphate Schiff's base intermediate in the reaction pathway catalyzed by this enzyme. Use of this resin in combination with octyl-agarose chromatography on partially purified enzyme resulted in a tyrosine aminotransferase preparation with a specific activity of about 450 units/mg protein. When analyzed on one-dimensional polyacrylamide-sodium dodecyl sulfate slab gels, the highly purified enzyme was composed of two polypeptides with molecular weights of about 56,000 and 53,000. Radioiodinated tryptic peptides from each of these polypeptides were essentially identical following two-dimensional analysis. Although the two polypeptides could not be separated from each other in an active form, it was found that (i) both polypeptides have pyridoxal phosphate-binding sites, (ii) the coenzyme is probably bound to both polypeptides as a Schiff's base, (iii) both polypeptides have binding sites for l -tyrosine and l -glutamic acid, the two specific substrates for the enzyme, and (iv) both polypeptides can catalyze the formation of the initial amino acid-pyridoxal phosphate Schiff's base adduct in the overall reaction pathway. Since the ratios of these polypeptides differed from preparation to preparation of purified enzyme, the 53,000 M r species probably arises by proteolysis of tyrosine aminotransferase in crude liver extracts. These results imply that if tyrosine aminotransferase isozymes exist, they are not the result of translation products produced by different structural genes.

Acanthamoeba actin is the only actin sequenced to date that has neither an NH2-terminal Ac-Asp nor Ac-Glu residue. The protein begins with an Ac-Gly-Asp and is coded for by a gene that specifies a polypeptide beginning Met-Gly-Asp. Thus, the Acanthamoeba actin gene would appear to specify a class II actin with the usual NH2-terminal Cys replaced with a Gly. Previous studies (Rubenstein, P. A., and Martin, D. J. (1983) J. Biol. Chem. 258, 11354-11360) revealed that for class II actins the Met is probably removed early in translation and the Cys is removed post-translationally as an Ac-Cys residue. Two possibilities might explain why Acanthamoeba actin is not processed in a similar fashion. Either Ac-Gly is not a substrate for the enzyme or the enzyme is absent from the organism. To test these alternatives, Acanthamoeba actin was labeled in vivo with [35S]methionine and incubated with processing enzyme from rat liver, rabbit reticulocytes, and Dictyostelium. In no case did the processing reaction occur, indicating that Ac-Gly is not recognized by the enzyme as a substrate. Furthermore, we could not reproducibly detect the presence of a processing enzyme in Acanthamoeba. We were, however, able to show the presence of such an enzyme in Dictyostelium, the first demonstration of this activity in a lower eukaryote.