Abstract. To define the actin-binding site within the N11 2 -terminal domain (residues 1-245) of chick smooth muscle a-actinin, we expressed a series of a-actinin deletion mutants in monkey Cos cells . Mutant a-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actinbinding site is located within residues 20-195. When a truncated a-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding . The actinbinding site in a-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of a-ACTININ is a rod-shaped F-actin cross-linking protein found in both muscle and nonmuscle cells at sites where actin is attached to a variety of intracellular structures. It is a homodimer with subunits of molecular mass 94-103 kD arranged in an antiparallel orientation . Smooth, skeletal, and nonmuscle isoforms of the protein have been identified, the only clear functional difference being that binding of the nonmuscle isoform to actin is inhibited by calcium, whereas the muscle isoforms are calcium insensitive in this respect (reviewed in Blanchard et al ., 1989) . Analysis of the deduced sequence of the chick smooth muscle isoform of the protein shows that a-actinin can be divided into three distinct domains, an NH2-terminal actin-binding domain spanning Address reprint requests to David R. Critchley, Department of Biochemistry, University of Leicester, University Road, Leicester, UK LEI 7RH .Since this work was submitted for publication, Bresnick et al . (Bresnick, A . R., P. A . Janmey, and J . Condeelis . 1991 . J. Mol. Chem. 268 :12989-12993) have shown that antibodies to the 27 amino acids implicated in binding of ABP120 to actin can immune precipitate ABP120 from cell lysates, suggesting that these residues are on the surface of the protein as expected for an actin-binding site. Fab' fragments of the antibody inhibited ABP120 binding to actin, and the synthetic 27 mer inhibited actin cross-linking by ABP120. Evidence was also presented that the synthetic 27 mer cosediments with actin .
We describe the development of an in vitro library selection system (CIS display) that exploits the ability of a DNA replication initiator protein (RepA) to bind exclusively to the template DNA from which it has been expressed, a property called cis-activity. A diverse peptide library is created by ligation of DNA fragments of random sequence to a DNA fragment that encodes RepA. After in vitro transcription and translation, a pool of protein-DNA complexes is formed where each protein is stably associated with the DNA that encodes it. These complexes are amenable to the affinity selection of ligands to targets of interest. Here we show that RepA is a highly faithful cis-acting DNA-binding protein and demonstrate that libraries encoding >10 12 random 18-mer peptides can be constructed and used to isolate peptides that bind specifically to disparate targets. The use of DNA to encode the displayed peptides offers advantages over in vitro peptide display systems that use mRNA.
Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. In this study we demonstrate a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (Kcap approximately 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH2-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which were previously shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. Our experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells.
CapZ ("capping protein") is a heterodimeric actin capping protein that blocks actin filament assembly and disassembly at the fast growing (barbed) filament ends and is proposed to function in regulating actin filament dynamics as well as in stabilizing actin filament lengths in muscle and nonmuscle cells. We show here that erythrocytes contain a nonmuscle isoform of capZ (EcapZ) that is present exclusively in the cytosol and is not associated with the short actin filaments in the erythrocyte membrane skeleton. This is unlike other cell types where capZ is associated with cytoskeletal actin filaments and suggests that cytosolic EcapZ may be inactive, or alternatively, that the barbed ends are capped by adducin, a membrane skeleton protein that was shown recently to cap actin filament barbed ends in vitro [Kuhlman, P. A., Hughes, C. A., Bennett, V., & Fowler, V. M. (1996) J. Biol. Chem. 271, 7986]. To distinguish between these possibilities, we purified EcapZ from erythrocyte cytosol and characterized its biochemical and functional properties. Two-dimensional gel electrophoresis and western blotting reveals the EcapZ subunit composition to be alpha1beta2, as described for capZ from many other nonmuscle cells, with no evidence for posttranslational modifications. Purified EcapZ is fully functional in blocking actin elongation from barbed filament ends (Kcap approximately 1-5 nM) as well as in nucleating actin polymerization. Furthermore, cytosolic EcapZ binds to actin filament barbed ends, indicating that sequestering of EcapZ by a cytosolic inhibitory factor or insufficient amounts of EcapZ in cytosol also cannot account for its absence from the membrane skeleton. To test directly whether the barbed ends of the erythrocyte actin filaments were already capped, we measured binding of purified EcapZ to isolated membranes. Purified EcapZ does not cosediment with membranes prepared by hypotonic lysis in the presence of magnesium, suggesting that the barbed ends of the erythrocyte actin filaments are capped under these conditions but not by EcapZ. In contrast, purified EcapZ stoichiometrically reassociates with all the actin filament barbed ends in membranes prepared by hypotonic lysis in 5 mM sodium phosphate, pH 8.0 (5P8), conditions in which the barbed filament ends were previously reported to be uncapped. Comparison of the amounts of adducin associated with membranes prepared in the presence and absence of magnesium reveals that 60-80% of the adducin dissociates from the membrane during hemolysis and washing in 5P8 buffer, suggesting that the barbed ends become artifactually uncapped due to loss of adducin. The erythrocyte actin filaments may thus represent a specialized class of membrane-associated actin filaments that are capped by adducin instead of capZ.
We have shown previously that the N-terminal actin-binding domain of cc-actinin retains activity when expressed in E. coil a5 a fusion protein with glutathione-S-transferase. In the present study we have made a series of N-and C-terminal deletions within this domain and show that an actin-binding site is contained within residues 120--134. Amino acid substitutions within this region indicate that several highly ¢on~.ed hydrophobic residues are involved in binding to F-aetin. The hypotlaesis that the interaction between :r-aetinin and F-actin is predominantly hydrophobic in nature is supported by the observation that binding is relatively independent of salt concentration.a-Actinin; Actin-binding site; Dystrophin;/~-Sp~etri~t; ABP-120; Cytoskeleton
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