Mutational analysis of an actin-binding site of cofilin and characterization of chimeric proteins between cofilin and destrin.

Cofilin and destrin are two related low molecular weight mammalian actin-binding proteins. Cofilin is an F-actin side-binding and pH-dependent actin-depolymerizing protein, and destrin is a pH-independent actin-depolymerizing protein. We have introduced a few point mutations within an actin-binding sequence of cofilin. Biochemical analyses of these mutant proteins have clearly shown that Lys112 and Lys114 of cofilin are crucially but differently involved in its interaction with actin and phosphatidylinositol 4,5-bisphosphate. This is the first example among actin-binding proteins whose point mutations inactivate their interaction with actin in vitro. We have also made and characterized a series of chimeric proteins between cofilin and destrin to identify the regions responsible for the pH dependence and the F-actin side binding activity of cofilin. Our results suggest that a central region consisting of 42 amino acid residues and a carboxyl-terminal quarter of cofilin are both involved in regulation of the pH-dependent actin depolymerizing activity and the activity to bind along F-actin.     

Mitochondrial translocation of cofilin is an early step in apoptosis induction

Increasing evidence suggests that movement of key proteins in or out of mitochondria during apoptosis is essential for the regulation of apoptosis. Here, we report identification of the actin-binding protein cofilin by a proteomic approach, as such a factor translocated from cytosol into mitochondria after induction of apoptosis. We found that after induction of apoptosis, cofilin was translocated to mitochondria before release of cytochrome c. Reduction of cofilin protein levels with small-interfering RNA (siRNA) resulted in inhibition of both cytochrome c release and apoptosis. Only dephosphorylated cofilin was translocated to mitochondria, and the cofilin S3D mutant, which mimicks the phosphorylated form, suppressed mitochondrial translocation and apoptosis. Translocation was achieved through exposure of an amino-terminal mitochondrial targeting signal in combination with carboxy-terminal sequences. When correctly targeted to mitochondria, cofilin induced massive apoptosis. The apoptosis-inducing ability of cofilin, but not its mitochondrial localization, was dependent on the functional actin-binding domain. Thus, domains involved in mitochondrial targeting and actin binding are indispensable for its pro-apoptotic function. Our data suggest that cofilin has an important function during the initiation phase of apoptosis.     

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA

Prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. In many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal RNA, but also to a motif localized within their own mRNA, and thereby regulate expression of the operon. In this study, we computationally predicted an RNA motif present in many bacterial phyla within the 5′ untranslated region of operons encoding ribosomal proteins S6 and S18. We demonstrated that the S6:S18 complex binds to this motif, which we hereafter refer to as the S6:S18 complex-binding motif (S6S18CBM). This motif is a conserved CCG sequence presented in a bulge flanked by a stem and a hairpin structure. A similar structure containing a CCG trinucleotide forms the S6:S18 complex binding site in 16S ribosomal RNA. We have constructed a 3D structural model of a S6:S18 complex with S6S18CBM, which suggests that the CCG trinucleotide in a specific structural context may be specifically recognized by the S18 protein. This prediction was supported by site-directed mutagenesis of both RNA and protein components. These results provide a molecular basis for understanding protein-RNA recognition and suggest that the S6S18CBM is involved in an auto-regulatory mechanism.     

A method of preparing Escherichia coli 16 S RNA possessing previously unobserved 30 S ribosomal protein binding sites

A method of preparing 16 S RNA has been developed which yields RNA capable of binding specifically at least 12, and possibly 13, 30 S ribosomal proteins. This RNA, prepared by precipitation from 30 S subunits using a mixture of acetic acid and urea, is able to form stable complexes with proteins S3, S5, S9, S12, S13, S18 and possibly S11. In addition, this RNA has not been impaired in its capacity to interact with proteins S4, S7, S8, S15, S17 and S20, which are proteins that most other workers have shown to bind RNA prepared by the traditional phenol extraction procedure (Held et al., 1974; Garrett et al., 1971; Schaup et al., 1970,1971).We have applied several criteria of specificity to the binding of proteins to 16 S RNA prepared by the acetic acid-urea method. First, the new set of proteins interacts only with acetic acid-urea 16 S RNA and not with 16 S RNA prepared by the phenol method or with 23 S RNA prepared by the acetic acid-urea procedure. Second, 50 S ribosomal proteins do not interact with acetic acidurea 16 S RNA but do bind to 23 S RNA. Third, in the case of protein S9, we have shown that the bound protein co-sediments with acetic acid-urea 16 S RNA in a sucrose gradient. Additionally, a saturation binding experiment showed that approximately one mole of protein S9 binds acetic acid-urea 16 S RNA at saturation. Thus, we conclude that the method employed for the preparation of 16 S RNA greatly influences the ability of the RNA to form specific protein complexes. The significance of these results is discussed with regard to the in vitro assembly sequence.     

Binding of gelsolin domain 2 to actin. An actin interface distinct from that of gelsolin domain 1 and from ADF/cofilin.

It is generally assumed that of the six domains that comprise gelsolin, domain 2 is primarily responsible for the initial contact with the actin filament that will ultimately result in the filament being severed. Other actin-binding regions within domains 1 and 4 are involved in gelsolin's severing and subsequent capping activity. The overall fold of all gelsolin repeated domains are similar to the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins and it has been proposed that there is a similarity in the actin-binding interface. Gelsolin domains 1 and 4 bind G-actin in a similar manner and compete with each other, whereas domain 2 binds F-actin at physiological salt concentrations, and does not compete with domain 1. Here we investigate the domain 2 : actin interface and compare this to our recent studies of the cofilin : actin interface. We conclude that important differences exist between the interfaces of actin with gelsolin domains 1 and 2, and with ADF/cofilin. We present a model for F-actin binding of domain 2 with respect to the F-actin severing and capping activity of the whole gelsolin molecule.     

A structural basis for the pH-dependence of cofilin. F-actin interactions.

A marked pH-dependent interaction with F-actin is an important property of typical members of the actin depolymerizing factor (ADF)/cofilin family of abundant actin-binding proteins. ADF/cofilins tend to bind to F-actin with a ratio of 1 : 1 at pH values around 6.5, and to G-actin at pH 8.0. We have investigated the mechanism for the pH-sensitivity. We found no evidence for pH-dependent changes in the structure of cofilin itself, nor for the interaction of cofilin with G-actin. None of the actin-derived, cofilin-binding peptides that we had previously identified [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem. 274, 28893-28899] bound cofilin in a pH-sensitive manner. However, we have detected a conformational change in region 75-105 in the actin subdomain 1 by the use of a peptide-directed antibody. A pH-dependent conformational change has also been detected spectroscopically in a similar peptide (84-103) on binding to cofilin. These results are consistent with a model in which pH-dependent motion of subdomain 1 relative to subdomain 2 (through region 75-105) of actin reveals a second cofilin binding site on actin (centered around region 112-125) that allows ADF/cofilin association with the actin filament. This motion requires salt in addition to low pH.     

Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin

Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114. A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins.     

Cofilin is an essential component of the yeast cortical cytoskeleton

We have biochemically identified the Saccharomyces cerevisiae homologue of the mammalian actin binding protein cofilin. Cofilin and related proteins isolated from diverse organisms are low molecular weight proteins (15-20 kD) that possess several activities in vitro. All bind to monomeric actin and sever filaments, and some can stably associate with filaments. In this study, we demonstrate using viscosity, sedimentation, and actin assembly rate assays that yeast cofilin (16 kD) possesses all of these properties. Cloning and sequencing of the S. cerevisiae cofilin gene (COF1) revealed that yeast cofilin is 41% identical in amino acid sequence to mammalian cofilin and, surprisingly, has homology to a protein outside the family of cofilin- like proteins. The NH2-terminal 16kD of Abp1p, a 65-kD yeast protein identified by its ability to bind to actin filaments, is 23% identical to yeast cofilin. Immunofluorescence experiments showed that, like Abp1p, cofilin is associated with the membrane actin cytoskeleton. A complete disruption of the COF1 gene was created in diploid cells. Sporulation and tetrad analysis revealed that yeast cofilin has an essential function in vivo. Although Abp1p shares sequence similarity with cofilin and has the same distribution as cofilin in the cell, multiple copies of the ABP1 gene cannot compensate for the loss of cofilin. Thus, cofilin and Abp1p are structurally related but functionally distinct components of the yeast membrane cytoskeleton.     

Uncoupling actin filament fragmentation by cofilin from increased subunit turnover

The actin depolymerizing factor (ADF)/cofilin family of proteins interact with actin monomers and filaments in a pH-sensitive manner. When ADF/cofilin binds F-actin it induces a change in the helical twist and fragmentation; it also accelerates the dissociation of subunits from the pointed ends of filaments, thereby increasing treadmilling or depolymerization. Using site-directed mutagenesis we characterized the two actin-binding sites on human cofilin. One target site was chosen because we previously showed that the villin head piece competes with ADF for binding to F-actin. Limited sequence homology between ADF/cofilin and the part of the villin headpiece essential for actin binding suggested an actin-binding site on cofilin involving a structural loop at the opposite end of the molecule to the alpha-helix already implicated in actin binding. Binding through the alpha-helix is primarily to monomeric actin, whereas the loop region is specifically involved in filament association. We have characterized the actin binding properties of each site independently of the other. Mutation of a single lysine residue in the loop region abolishes binding to filaments, but not to monomers. Using the mutation analogous to the phosphorylated form of cofilin (S3D), we show that filament binding is inhibited at physiological ionic strength but not under low salt conditions. At low ionic strength, this mutant induces both the twist change and fragmentation characteristic of wild-type cofilin, but does not activate subunit dissociation. The results suggest a two-site binding to filaments, initiated by association through the loop site, followed by interaction with the adjacent subunit through the "helix" site at the opposite end of the molecule. Together, these interactions induce twist and fragmentation of filaments, but the twist change itself is not responsible for the enhanced rate of actin subunit release from filaments.     

In vitro selection of RNA ligands for the ribosomal L22 protein associated with Epstein-Barr virus-expressed RNA by using randomized and cDNA-derived RNA libraries.

The Epstein-Barr virus (EBV)-expressed RNA 1 (EBER1) associates tightly with the ribosomal protein L22. We determined the general requirements for an RNA to bind L22 in a SELEX experiment, selecting RNA ligands for L22 from a randomized pool of RNA sequences by using an L22-glutathione S-transferase fusion protein. The selected sequences all contained a stem-loop motif similar to that of the region of EBER1 previously shown to interact with L22. The nucleotides were highly conserved at three positions within the stem-loop and identical to the corresponding nucleotides in EBER1. Two independent binding sites for L22 could be identified in EBER1, and mobility shift assays indicated that two L22 molecules can interact with EBER1 simultaneously. To search for a cellular L22 ligand, we constructed a SELEX library from cDNA fragments derived from RNA that was coimmunoprecipitated with L22 from an EBV-negative whole-cell lysate. After four rounds of selection and amplification, most of the clones that were obtained overlapped a sequence corresponding to the stem-loop between nucleotides 302 and 317 in human 28S ribosomal RNA. This stem-loop fulfills the criteria for optimal binding to L22 that were defined by SELEX, suggesting that human 28S ribosomal RNA is likely to be a cellular L22 ligand. Additional L22 binding sites were found in 28S ribosomal RNA, as well as within 18S ribosomal RNA and in RNA segments not present in sequence databases. The methodology described for the conversion of a preselected cellular RNA pool into a SELEX library might be generally applicable to other proteins for the identification of cellular RNA ligands.     

Structural conservation between the actin monomer-binding sites of twinfilin and actin-depolymerizing factor (ADF)/cofilin

Twinfilin is an evolutionarily conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. It is composed of two actin-depolymerization factor homology (ADF-H) domains that show approximately 20% sequence identity to ADF/cofilin proteins. In contrast to ADF/cofilins, which bind both G-actin and F-actin and promote filament depolymerization, twinfilin interacts only with G-actin. To elucidate the molecular mechanisms of twinfilin-actin monomer interaction, we determined the crystal structure of the N-terminal ADF-H domain of twinfilin and mapped its actin-binding site by site-directed mutagenesis. This domain has similar overall structure to ADF/cofilins, and the regions important for actin monomer binding in ADF/cofilins are especially well conserved in twinfilin. Mutagenesis studies show that the N-terminal ADF-H domain of twinfilin and ADF/cofilins also interact with actin monomers through similar interfaces, although the binding surface is slightly extended in twinfilin. In contrast, the regions important for actin-filament interactions in ADF/cofilins are structurally different in twinfilin. This explains the differences in actin-interactions (monomer versus filament binding) between twinfilin and ADF/cofilins. Taken together, our data show that the ADF-H domain is a structurally conserved actin-binding motif and that relatively small structural differences at the actin interfaces of this domain are responsible for the functional variation between the different classes of ADF-H domain proteins.     

The amino terminal domain from Mrt4 protein can functionally replace the RNA binding domain of the ribosomal P0 protein

In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein.     

Actin deficiency induces cofilin phosphorylation: proteome analysis of HeLa cells after beta-actin gene silencing

Actin-binding proteins regulate the dynamic structure and function of actin filaments in the cell. Much is known about how manipulation of the actin-binding proteins affects the structure and function of actin filaments; however, little is known about how manipulation of actin in the cell affects actin-binding proteins. We addressed this question by utilizing two technologies: RNA interference and 2-dimensional gel electrophoresis. We knocked down beta-actin expression in HeLa cells using short interfering RNA and applied 2-DGE to examine alterations in the HeLa cell proteome. We revealed a 2-5 fold increases of four protein spots on 2-D gels and identified these proteins by mass spectrometry. Three of the four proteins were actin-binding proteins, including cofilin, which promotes both disassembly and assembly of actin filaments but becomes inactivated when phosphorylated. Further examination revealed that the cofilin total protein level barely increased, but the phosphorylated cofilin level increased dramatically in HeLa cells after beta-actin siRNA treatment. These results suggest that in response to siRNA-induced beta-actin deficiency HeLa cells inactivate cofilin by phosphorylation rather than down-regulate its protein expression level. This study also demonstrates that the combination of RNA interference and 2-dimensional gel electrophoresis technologies provides a valuable method to study protein interactions in a specific cellular pathway.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Isolation of nuclear encoded plastid ribosomal protein cDNAs

Summary A pea leaf cDNA library was constructed in the expression vector gt11 and screened with antisera raised against proteins extracted from 30S and 50S ribosomal subunits and 70S ribosomes prepared from isolated pea chloroplasts. Six recombinant phage were identified that encoded fusion proteins containing plastid ribosomal protein antigenic determinants. Phage-induced cell lysate proteins, containing the fusion proteins, were bound to nitrocellulose membranes and used as affinity matrices to prepare monospecific antibodies. These antibodies were then used to identify by Western blotting which plastid ribosomal protein shared antigenic determinants with the fusion proteins. cDNA inserts from the antigen-producing phage were used to hybrid-select complementary mRNAs. The cell-free translation products of these mRNAs were added to a pea chloroplast in vitro transport system and imported proteins analyzed by two-dimensional gel electrophoresis. The imported proteins comigrated with the plastid ribosomal proteins that were identified as being antigenically related to the fusion proteins produced by the corresponding recombinant phage. The imported proteins were 3,500–5,500 daltons smaller than their precursors.     

Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array: evidence for a second binding site on monomeric actin

The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin regulatory proteins.     

Identification of yeast cofilin residues specific for actin monomer and PIP2 binding.

Cofilin/ADF is a ubiquitous actin-binding protein that is important for rapid actin dynamics in vivo. The long alpha-helix (helix 3 in yeast cofilin) forms the most highly conserved region in cofilin/ADF proteins, and residues in the NH2-terminal half of this alpha-helix have been shown to be essential for actin binding in cofilin/ADF. Recent studies also suggested that the basic residues in the COOH-terminal half of this alpha-helix would play an important role in F-actin binding. In contrast to these studies, we show here that the charged residues in the COOH-terminal half of helix 3 are not important for actin filament binding in yeast cofilin. Mutations in these residues, however, result in a small defect in actin monomer interactions. We also show that yeast cofilin can differentiate between various phosphatidylinositides, and mapped the PI(4,5)P2 binding site by using a collection of cofilin mutants. The PI(4,5)P2 binding site of yeast cofilin is a large positively charged surface that consists of residues in helix 3 as well as residues in other parts of the cofilin molecule. This suggests that cofilin/ADF proteins probably interact simultaneously with more than one PI(4,5)P2 molecule. The PI(4,5)P2-binding site overlaps with areas that are important for F-actin binding, explaining why the actin-related activities of cofilin/ADF are inhibited by PI(4,5)P2. The biological roles of actin and PI(4,5)P2 interactions of cofilin are discussed in light of phenotypes of specific yeast strains carrying mutations in residues that are important for actin and PI(4,5)P2 binding.     

Studies on the interaction between actin and cofilin purified by a new method.

Cofilin is a 21,000-Mr actin-binding protein that widely exists in mammalian tissues. (1) A new purification procedure for porcine brain cofilin has been developed that involves (NH4)2SO4 fractionation and sequential chromatographies on Toyo Pearl and butyl-Toyo Pearl hydrophobic columns, hydroxyapatite, phosphocellulose and Sephadex G-75 gel-filtration columns. The purified cofilin bound to F-actin and increased the amount of G-actin to a limited extent, as previously reported [Nishida, Maekawa & Sakai (1984) Biochemistry 23, 5307-5313]. (2) The binding of cofilin to F-actin was scarcely affected by Mg2+, Ca2+ or by calmodulin. However, the binding was diminished by increasing concentrations of KCl, but was only slightly affected by temperature. (3) Cofilin and either alpha-actinin or filamin could bind to F-actin simultaneously with some competition, but the binding of caldesmon to F-actin was markedly inhibited by cofilin. Phalloidin inhibited the binding of cofilin to F-actin, and protected F-actin from depolymerization by cofilin.     

Ribosomal protein binding with the first intron of the human ribosomal protein S26 pre-mRNA stimulates its interaction with proteins extracted from Hela cells

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, proteins of the 40S ribosome subunit bind to the first intron of the rpS26 pre-mRNA. The binding involved mostly S23, S26 and, to a lesser extent, S13/16. Negligible binding was observed for S2/3a, S6, S8, S10, S11, and S20. Small-subunit proteins did not affect the efficiency of in vitro splicing of a pre-mRNA fragment corresponding to the first intron, second exon, second intron, and a part of the third exon of the rpS26 gene. However, ribosomal proteins substantially increased UV-induced adduction of the pre-mRNA fragments with nuclear extract proteins of HeLa cells. The same set of HeLa proteins was observed with each pre-mRNA fragment. Ribosomal proteins formed adducts only in the absence of HeLa proteins.