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Interaction of Actin Monomers with AcanthamoebaActophorin (ADF/Cofilin) and Profilin

1998; Elsevier BV; Volume: 273; Issue: 39 Linguagem: Inglês

10.1074/jbc.273.39.25106

1083-351X

Laurent Blanchoin, Thomas D. Pollard,

Connexins and lens biology

Acanthamoeba actophorin is a member of ADF/cofilin family that binds both actin monomers and filaments. We used fluorescence anisotropy to study the interaction of actin monomers with recombinant actophorin labeled with rhodamine on a cysteine substituted for Serine-88. Labeled actophorin retains its affinity for actin and ability to reduce the low shear viscosity of actin filaments. At physiological ionic strength, actophorin binds Mg-ADP-actin monomers (K d = 0.1 μm) 40 times stronger than Mg-ATP-actin monomers. When bound to actin monomers, actophorin has no effect on elongation at either end of actin filaments by Mg-ATP-actin and slightly increases the rate of elongation at both ends by Mg-ADP-actin. Thus actophorin does not sequester actin monomers. Sedimentation equilibrium ultracentrifugation shows that actophorin and profilin compete for binding actin monomers. Actophorin and profilin have opposite effects on the rate of exchange of nucleotide bound to actin monomers. Despite the high affinity of actophorin for ADP-actin, physiological concentrations of profilin overcome the inhibition of ADP exchange by actophorin. Profilin rapidly recycles ADP-actin back to the profilin-ATP-actin pool ready for elongation of actin filaments. Acanthamoeba actophorin is a member of ADF/cofilin family that binds both actin monomers and filaments. We used fluorescence anisotropy to study the interaction of actin monomers with recombinant actophorin labeled with rhodamine on a cysteine substituted for Serine-88. Labeled actophorin retains its affinity for actin and ability to reduce the low shear viscosity of actin filaments. At physiological ionic strength, actophorin binds Mg-ADP-actin monomers (K d = 0.1 μm) 40 times stronger than Mg-ATP-actin monomers. When bound to actin monomers, actophorin has no effect on elongation at either end of actin filaments by Mg-ATP-actin and slightly increases the rate of elongation at both ends by Mg-ADP-actin. Thus actophorin does not sequester actin monomers. Sedimentation equilibrium ultracentrifugation shows that actophorin and profilin compete for binding actin monomers. Actophorin and profilin have opposite effects on the rate of exchange of nucleotide bound to actin monomers. Despite the high affinity of actophorin for ADP-actin, physiological concentrations of profilin overcome the inhibition of ADP exchange by actophorin. Profilin rapidly recycles ADP-actin back to the profilin-ATP-actin pool ready for elongation of actin filaments. actin depolymerizing factor dithiothreitol. Acanthamoeba actophorin (1Cooper J.A. Blum J.D. Williams Jr., R.C. Pollard T.D. J. Biol. Chem. 1986; 261: 477-485Abstract Full Text PDF PubMed Google Scholar, 2Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Google Scholar) belongs to a family of low molecular weight actin-binding proteins including vertebrate cofilin, destrin, and ADF (3Bamburg J.R. Harris H.E. Weeds A.G. FEBS Lett. 1980; 121: 178-182Crossref PubMed Scopus (157) Google Scholar, 4Adams M.E. Minamide L.S. Duester G. Bamburg J.R. Biochemistry. 1990; 29: 7414-7420Crossref PubMed Scopus (50) Google Scholar, 5Abe H. Endo T. Yamamoto K. Obinata T. Biochemistry. 1990; 29: 7420-7425Crossref PubMed Scopus (69) Google Scholar, 6Nishida E. Biochemistry. 1985; 24: 1160-1164Crossref PubMed Scopus (124) Google Scholar, 7Moriyama K. Nishida E. Yonezawa N. Sakai H. Matsumoto S. Iida K. Yahara I. J. Biol. Chem. 1990; 265: 5768-5773Abstract Full Text PDF PubMed Google Scholar, 8Moriyama K. Matsumoto S. Nishida E. Sakai H. Yahara I. Nucleic Acids Res. 1990; 18: 3053Crossref PubMed Scopus (23) Google Scholar, 9Hatanaka H. Ogura K. Moriyama K. Ichikawa S. Yahara I. Inagaki F. Cell. 1996; 85: 1047-1055Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), echinoderm depactin (10Mabuchi I. J. Biochem. 1981; 89: 1341-1344PubMed Google Scholar, 11Takagi T. Konishi K. Mabuchi I. J. Biol. Chem. 1988; 263: 3097-3102Abstract Full Text PDF PubMed Google Scholar), and yeast cofilin (12Iida K. Moriyama K. Matsumoto S. Kawasaki H. Nishida E. Yahara I. Gene. 1993; 124: 115-120Crossref PubMed Scopus (123) Google Scholar, 13Moon A.L. Janmey P.A. Louie K.A. Drubin D.G. J. Cell Biol. 1993; 120: 421-435Crossref PubMed Scopus (201) Google Scholar). Cofilin is dephosphorylated in response to various agonists (Refs. 14Ohta Y. Nishida E. Sakai H. Miyamoto E. J. Biol. Chem. 1989; 264: 16143-16148Abstract Full Text PDF PubMed Google Scholar, 15Davidson M.M. Haslam R.J. Biochem. J. 1994; 301: 41-47Crossref PubMed Scopus (71) Google Scholar, 16Suzuki K. Yamaguchi T. Tanaka T. Kawanishi T. Nishimaki-Mogami T. Yamamoto K. Tsuji T. Irimura T. Hayakawa T. Takahashi A. J. Biol. Chem. 1995; 270: 19551-19556Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar; and reviewed by Moon and Drubin, Ref.17Moon A. Drubin D.G. Mol. Biol. Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (226) Google Scholar) suggesting that it may be part of a regulated signal transduction mechanism controlling the assembly of the actin cytoskeleton. The original protein of this type, ADF,1 got its name “actin depolymerizing factor” by virtue of its ability to depolymerize actin filaments. Similar evidence led to the name depactin. However, cofilin copolymerizes with actin at pH <7.3, depolymerizing filaments only at higher pH (18Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (239) Google Scholar, 19Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (202) Google Scholar). Subsequent work has not led to a consistent view of the mechanism of action of ADF/cofilin proteins. A variety of evidence suggested that ADF/cofilin proteins sever actin filaments and hasten depolymerization by creating more ends to dissociated subunits (2Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Google Scholar, 18Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (239) Google Scholar, 19Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (202) Google Scholar, 20Moriyama K. Yonezawa N. Sakai H. Yahara I. Nishida E. J. Biol. Chem. 1992; 267: 7240-7244Abstract Full Text PDF PubMed Google Scholar). Carlier et al. (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar) questioned the severing activity of the ADF/cofilin family and proposed a “dynamizing” mechanism for plant cofilin. Their interpretation was that plant cofilin promotes actin filament turnover by increasing the rates of ATP-actin association at the barbed end and ADP-actin dissociation at the pointed end. Either by severing or enhancing dissociation of subunits from the pointed end, ADF/cofilin proteins should release Mg-ADP-actin monomers, which bind actophorin and cofilin with higher affinity than ATP-actin (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar, 22Maciver S.K. Weeds A.G. FEBS Lett. 1994; 347: 251-256Crossref PubMed Scopus (96) Google Scholar). Both porcine brain and yeast cofilin inhibit the exchange of Mg-ATP bound to actin (23Nishida E. Biochemistry. 1985; 24: 1160-1164Crossref PubMed Google Scholar, 24Lappalainen P. Fedorov E.V. Fedorov A.A. Almo S.C. Drubin D.G. EMBO J. 1997; 16: 5520-5530Crossref PubMed Scopus (208) Google Scholar). The effect of ADF/cofilin proteins on ADP exchange is not known, but high affinity binding might trap ADP-actin bound to an ADF/cofilin protein. Using a new fluorescence anisotropy assay, we confirm the higher affinity of actophorin for Mg-ADP-actin than Mg-ATP-actin and characterize in detail the effect of both actophorin and profilin on the exchange of the bound nucleotide. Without profilin, actophorin forms a stable complex with ADP-actin. Profilin competes with actophorin for binding actin monomers. Even with saturating concentrations of actophorin, a low concentration of profilin promotes the rapid exchange of ADP for ATP. Given the higher affinity of Mg-ATP-actin for profilin, these reactions rapidly transfer actin from actophorin to profilin, allowing actophorin to recycle back to actin filaments. We also show that actophorin bound to ADP- or ATP-actin does not inhibit actin filament elongation at either the barbed or pointed end of filaments. Materials came from the following sources: Sigma, dithiothreitol (DTT), EDTA, Tris, sodium azide, Me2SO, hexokinase, ATP, ADP, phalloidin, Sephadex G-25 medium; Molecular Probes (Eugene, OR), 1,N 6-ethenoadenosine-5′-diphosphate (ε-ADP), TCEP (Tris-(2-carboxyethyl)phosphine, 5′-tetramethylrhodamine maleimide isomer; Whatman (Maidstone, United Kingdom), DEAE-cellulose DE-52. Residues Asn-33, Glu-70, and Ser-88 of actophorin were changed to cysteine by reverse polymerase chain reaction mutagenesis (25Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6770) Google Scholar), and the mutations were verified by sequencing. These cysteines were the only reactive sulfhydryls, because neither of the two native cysteines are accessible to solvent (26Leonard S.A. Gittis A.G. Petrella E.C. Pollard T.D. Lattman E.E. Nat. Struct. Biol. 1997; 4: 369-373Crossref PubMed Scopus (61) Google Scholar). Wild type and mutant actophorins in plasmid vector pMW172 were expressed in Escherichia coli strain BL21 (DE3) pLysS and purified according to Quirk et al. (27Quirk S. Maciver S.K. Ampe C. Doberstein S.K. Kaiser D.A. VanDamme J. Vandekerckhove J.S. Pollard T.D. Biochemistry. 1993; 32: 8525-8533Crossref PubMed Scopus (53) Google Scholar), except that 2 mm dithiothreitol was included in all buffers for mutant actophorins to avoid cysteine oxidation. Purified actophorins were stored in 10 mm Tris-Cl, pH 7.5, 1 mm EDTA, 2 mm DTT, 1 mm NaN3. A 4-ml sample of 50 μm actophorin in storage buffer was dialyzed 3 h into labeling buffer (10 mm Tris-Cl, pH 7.5, 1 mm EDTA, 2 mm TCEP, 1 mmNaN3) and then reacted with 7-fold molar excess of 5′-tetramethylrhodamine maleimide isomer from a 20 mm stock in Me2SO for 2 h at 4 °C with gentle stirring. Centrifugation removed precipitated dye. Two steps removed free dye: first, gel filtration on a 60 × 1.5-cm column Sephadex G25 equilibrated with 10 mm Tris-Cl, pH 7.5, 1 mmEDTA, 2 mm DTT, 250 mm NaCl, 1 mmNaN3, at 4 °C; and second, chromatography on a 13 × 1.5-cm column of DEAE-cellulose DE-52 equilibrated with 10 mm Tris-HCl, pH 8.4, 1 mm EDTA, 2 mm DTT, 1 mm NaN3. Labeled protein was eluted with a 0–250 mm gradient of NaCl and dialyzed overnight against 10 mm Tris-Cl, pH 7.5, 1 mmEDTA, 2 mm DTT, 1 mm NaN3. Protein purity was confirmed by SDS-polyacrylamide gel electrophoresis (15% acrylamide), and protein concentration was determined by a solid phase dye binding method (28Minamide L.S. Bamburg J.R. Anal. Biochem. 1990; 190: 66-70Crossref PubMed Scopus (236) Google Scholar). Profilin-II was purified fromAcanthamoeba castellanii (29Kaiser D.A. Goldschmidt-Clermont P.J. Levine B.A. Pollard T.D. Cell Motil. Cytoskeleton. 1989; 14: 251-262Crossref PubMed Scopus (80) Google Scholar). Actin was purified from rabbit muscle acetone powder (30Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) or from Acanthamoeba (31Pollard T.D. J. Cell Biol. 1984; 99: 769-777Crossref PubMed Scopus (187) Google Scholar) and isolated as Ca-ATP-G-actin through Sephacryl S-300 chromatography (32MacLean-Fletcher S. Pollard T.D. Cell. 1980; 20: 329-341Abstract Full Text PDF PubMed Scopus (530) Google Scholar) at 4 °C in G buffer (5 mm Tris-Cl, pH 8.0, 0.2 mm ATP, 0.1 mm CaCl2, 0.5 mm DTT). Actin was labeled on Cys-374 to a stoichiometry of 0.8–1.0 with pyrene iodoacetamide (Ref. 33Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (717) Google Scholar, as modified by Pollard; see Ref. 31Pollard T.D. J. Cell Biol. 1984; 99: 769-777Crossref PubMed Scopus (187) Google Scholar). Mg-ATP G-actin was prepared on ice by addition of 0.2 mm EGTA and an 11-fold molar excess of MgCl2over actin and used within hours. Mg-ADP G-actin was prepared by treatment of Mg-ATP G-actin with soluble hexokinase and glucose (31Pollard T.D. J. Cell Biol. 1984; 99: 769-777Crossref PubMed Scopus (187) Google Scholar). For binding experiments, variable concentrations of actin were mixed with a fixed concentration of rhodamine-S88C-actophorin. Fluorescence measurements were made with a PTI Alpha-scan spectrofluorimeter (Photon Technologies International, South Brunswick, NJ). Fluorescence anisotropy of rhodamine-S88C-actophorin was measured with excitation at 550 nm and emission at 574 nm. 2Vinson, V. K., De La Cruz, E. M., Higgs, H. N., and Pollard, T. D. (1998) in press. Data were analyzed using Kaleidagraph software (Synergy Software, Reading, PA) and fitted to Eq. 1 where r is the observed anisotropy,racf is the anisotropy of free actophorin,racb is the anisotropy of actophorin bound to actin, [C] is the total concentration of actophorin, [A] is the total concentration of actin and K d the dissociation equilibrium constant of the complex.r=racf+(racb−racf)(Kd+[C]+[A])−(Kd+[C]+[A])2−(4[C][A])2(Eq. 1) Seeds were made by polymerizing 20 μmactin for 2 h at 20 °C in F buffer (10 mm Tris-Cl, pH 8.0, 50 mm KCl, 1 mm MgCl2, 1 mm EGTA, 0.2 mm ATP, 0.1 mmCaCl2, 0.5 mm DTT) with a 10-fold molar excess of phalloidin and gelsolin concentrations of 0 or 400 nm. Seeds were diluted 10–50 times into F buffer with 15 μmactophorin and 4 μm pyrenyl-labeled Mg-ADP- or Mg-ATP-actin. The time course of polymerization was measured by fluorescence with excitation at 366 nm and emission at 387 nm. The dissociation Mg-ε-ADP from actin muscle monomers was measured by the decrease in fluorescence with excitation at 360 nm and emission at 410 nm. A Hi-tech SFA-II rapid mixer allowed mixing of equal volumes of 600 μm ATP from one syringe with 2.8 μm Mg-ε-ADP-actin, 6 μm actophorin, and various concentrations of Acanthamoeba profilin-II from the other syringe. Sedimentation equilibrium analytical ultracentrifugation was carried out in a Beckman model XL-I in 6-hole, charcoal-filled centerpieces with quartz windows in a Beckman model An60ti rotor. We determined the molecular weight and the ratio of rhodamine to actophorin after centrifugation to equilibrium at 14,000 rpm. The protein concentration was determinate by Raleigh interferometry assuming 3.3 fringes per mg/ml protein and the rhodamine concentration was measured by absorbance at 541 nm using a molar extinction coefficient of 91,400 cm−1m−1. We analyzed competitive binding of 10 μm rhodamine-S88C-actophorin and various concentrations of profilin-II to 10 μm Mg-ADP-actin monomers by sedimentation equilibrium. We monitored rhodamine absorption at 550 nm. The data was collected and analyzed according to (35Mullins R.D. Kelleher J.F. Xu J. Pollard T.D. Mol. Biol. Cell. 1998; 9: 841-852Crossref PubMed Scopus (70) Google Scholar). Data were fit using Eq. 2ln(absorbance,550 nm)=(ς(r2/2−r2m/2))(Eq. 2) where the logarithm of the absorbance is a function of the effective reduced molecular weightς=Mω2(1−v̄ρ)RT(Eq. 3) M is molecular mass (g/mol), ω is the angular velocity (rad/sec), v̄ is the partial specific volume (cm3/gm), ρ is the solvent density (gm/cm3),R is the gas constant, and T is the absolute temperature (K), and r m and r are the radii of the meniscus and the bottom of the sample cell, respectively. We used KINSIM (36Barshop A. Wrenn R.F. Frieden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (666) Google Scholar) to simulate the time course of actin nucleotide exchange in presence of actophorin and profilin and the redistribution of actophorin/Mg-ADP-actin monomer complex at steady state. Acanthamoeba actophorin has two cysteines, Cys-9 and Cys-60 (27Quirk S. Maciver S.K. Ampe C. Doberstein S.K. Kaiser D.A. VanDamme J. Vandekerckhove J.S. Pollard T.D. Biochemistry. 1993; 32: 8525-8533Crossref PubMed Scopus (53) Google Scholar), but neither reacts with rhodamine maleimide when the protein is folded in its native conformation. To label actophorin with rhodamine, we replaced Asn-33, Glu-70, or Ser-88 individually with cysteine (Fig. 1). We chose these residues for three reasons: (a) each is outside the actin binding site mapped for other members of the ADF/cofilin family (37Muneyuki E. Nishida E. Sutoh K. Sakai H. J. Biochem. 1985; 97: 563-568Crossref PubMed Scopus (25) Google Scholar, 38Sutoh K. Mabuchi I. Biochemistry. 1989; 28: 102-106Crossref PubMed Scopus (35) Google Scholar, 39Yonezawa N. Nishida E. Ohba M. Seki M. Kumagai H. Sakai H. Eur. J. Biochem. 1989; 183: 235-238Crossref PubMed Scopus (44) Google Scholar, 40Yonezawa N. Homma Y. Yahara I. Sakai H. Nishida E. J. Biol. Chem. 1991; 266: 17218-17221Abstract Full Text PDF PubMed Google Scholar); (b) each is a serine in at least one member of the family 26; and (c) each is exposed to solvent in the crystal structure (26Leonard S.A. Gittis A.G. Petrella E.C. Pollard T.D. Lattman E.E. Nat. Struct. Biol. 1997; 4: 369-373Crossref PubMed Scopus (61) Google Scholar). Actophorin labeled with 5′-rhodamine maleimide isomer on any of the new cysteines has the same mobility on SDS-polyacrylamide gel electrophoresis as unlabeled actophorin (Fig. 2 A, lanes 1–6). Rhodamine maleimide labels mutants E70C and S88C (Fig. 2 A, lanes 4 and 5) more efficiently than N33C (Fig. 2 A, lane 6). By sedimentation equilibrium ultracentrifugation rhodamine-S88C-actophorin is a single ideal species with a molecular weight of 15,200 (Fig. 2 B). In this experiment, the ratio of rhodamine (measured byA 550) to protein (measured by Rayleigh interferometry) is constant across the ultracentrifuge cell with 0.44 dyes per protein molecule (Fig. 2 B). If conjugation of the dye with the protein affects its extinction coefficient, the stoichiometry will be different, but in any case, the labeled protein is homogeneous. A second experiment gave similar results. The labeled mutant proteins are indistinguishable from wild type actophorin in their ability to reduce the low shear viscosity of actin filaments (data not shown) using the falling ball assay (1Cooper J.A. Blum J.D. Williams Jr., R.C. Pollard T.D. J. Biol. Chem. 1986; 261: 477-485Abstract Full Text PDF PubMed Google Scholar, 2Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Google Scholar). The fluorescence polarization anisotropy r of rhodamine-labeled S88C and E70C is 0.14 independent of the concentration, as expected for a 14-kDa monomeric protein (41Jameson D.M. Sawyer W.H. Methods Enzymol. 1995; 246: 283-300Crossref PubMed Scopus (152) Google Scholar). Labeling of N33C never exceeded 0.1 dye per protein, so we did not use this mutant. Because actin binding has no effect on the fluorescence excitation or emission of any of our three rhodamine-labeled actophorin mutants, we could use fluorescence anisotropy to determine the affinity of actophorin for monomeric actin. The anisotropy of rhodamine-S88C-actophorin is 0.14 when free and 0.21 when bound to actin monomers. Under all of the conditions tested, a bimolecular binding isotherm with a stoichiometry of 1:1 fit the dependence of the anisotropy on actin concentration (Fig. 3). The nucleotide bound to actin has a strong effect on the affinity of actophorin for actin monomers (Table I). At physiological ionic strength (2 mm MgCl2, 100 mm KCl) actophorin binds Mg-ADP-actin monomers 40 times stronger than Mg-ATP-actin (Fig. 3), as observed with other assays (22Maciver S.K. Weeds A.G. FEBS Lett. 1994; 347: 251-256Crossref PubMed Scopus (96) Google Scholar) and other members of the ADF/cofilin family (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar). In contrast, in low salt concentration actophorin binds Mg-ATP-actin monomers better than Mg-ADP-actin. Rhodamine-S88C-actophorin binds Acanthamoebaand rabbit skeletal muscle actin monomers with similar affinities.Table IDissociation equilibrium constants for actophorin binding to actinSalt concentrationBound nucleotide monomerActophorin +Plant cofilin + muscle actinAmoeba actinMuscle actinμmμmLow saltMgATP0.190.08Low saltMgADP0.660.03High saltMgATP4.55.98High saltMgADP0.150.140.1Carlier et al. (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar). Open table in a new tab Table IIRate and equilibrium constants used for the simulation of nucleotide exchange by actin monomers in the presence of actophorin and profilinConstantValueReferencek +15 μm−1 s−1De La Cruz et al. 1995k −10.08 s−1This studyK 20.066 μmThis studyk +3ndk −30.006 s−1This studyK 4ndk +3 K 45.7 μm−2 s−1K 535 nmVinson et al. 1998k +60.85 μm−1 s−1Vinson et al.1998k −60.3 s−1Vinson et al. 1998K 72 μmVinsonet al. 1998 Open table in a new tab Carlier et al. (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar). To test the ability of actophorin to sequester actin monomers, we measured the rates of elongation from actin filament seeds stabilized with an excess of phalloidin. Phalloidin prevents actophorin binding to the filaments, 3L. Blanchoin and T. D. Pollard, manuscript in preparation. so that the actophorin added to the polymerizing mixture was in a simple equilibrium with 4 μm actin monomers and actophorin did not quench the fluorescence of newly polymerized pyrene-actin (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar). After the addition of the complex of actophorin with Mg-ATP- or Mg-ADP-actin monomers to the end of a filament, phalloidin binds, induces rapid dissociation of actophorin and allows the fluorescence increase associated with pyrenyl-actin polymerization. Elongation is primarily at the barbed end of free seeds and exclusively at the pointed end of seeds capped with gelsolin. Excess actophorin has no effect on the rate or extent of elongation at either end by Mg-ATP-actin (Fig. 4 A) in agreement with the observations of Carlier et al. (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar) but slightly increases the rate of elongation at both ends by Mg-ADP-actin (Fig. 4 B). These experiments show that actophorin does not sequester actin monomers in the sense that bound actophorin does not inhibit subunit addition at either end of actin filaments. We analyzed competition of profilin and actophorin for binding to actin monomers by sedimentation equilibrium ultracentrifugation (Fig. 5). At equilibrium, labeled actophorin distributes with actin. Unlabeled profilin displaces labeled actophorin from actin in a concentration-dependent fashion. This confirms less direct chemical cross-linking experiments (2Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Google Scholar), which suggested that binding of actophorin and profilin to actin monomers is mutually exclusive. Thus, although actophorin does not sequester actin monomers from polymerization, it does inhibit interactions with at least one other ligand, profilin. Saturating concentrations of actophorin inhibit Mg-ADP dissociation from muscle actin by a factor 13 to a rate constant of 0.006 s−1 in buffer with 2 mmMgCl2 (Fig. 6 A, inset). This is similar to the effect of cofilin on ATP dissociation from actin monomers (18Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (239) Google Scholar, 24Lappalainen P. Fedorov E.V. Fedorov A.A. Almo S.C. Drubin D.G. EMBO J. 1997; 16: 5520-5530Crossref PubMed Scopus (208) Google Scholar, 42Nishida E. Biochemistry. 1985; 24: 1160-1164Crossref PubMed Google Scholar). Profilin reverses this effect of actophorin, increasing the rate of nucleotide exchange from actin-actophorin complex in a concentration dependent manner (Fig. 6 A). Scheme FS1 is a minimal mechanism, based on mutually exclusive binding of profilin and actophorin to actin monomers, to explain how the two proteins influence nucleotide exchange. A represents nucleotide-free actin, N nucleotide ATP or ADP, Cactophorin, and P profilin. Remarkably, this simple mechanism and the rate and equilibrium constants measured here and in previous work (43De La Cruz E. Pollard T.D. Biochemistry. 1995; 34: 5452-5461Crossref PubMed Scopus (59) Google Scholar)2 account quantitatively for the time course (Fig. 6 A) and rate (Fig. 6 B) of ADP exchange in the presence of actophorin and a wide range of profilin concentrations. Two constants were unknown: k +3is the rate constant for nucleotide binding the complex of nucleotide-free actin monomer with actophorin, and K 4 is the association equilibrium constant for actophorin binding nucleotide-free actin. Using measured equilibrium constants and detailed balance, we estimated these values to be 1.5 μm−1 s−1 fork +3 and 3.8 μm−1 forK 4. The available data did not constrain these constants further, because variation of the values of these constants ± a factor of 10 does not affect the simulated time course of nucleotide exchange. The present work is the first measurement of the affinity of actophorin for actin. Maciver et al. (44Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Scopus (191) Google Scholar) and Maciver and Weeds (22Maciver S.K. Weeds A.G. FEBS Lett. 1994; 347: 251-256Crossref PubMed Scopus (96) Google Scholar) estimated the affinity of actophorin for actin by a change in critical concentration measured by pyrene fluorescence and light scattering or by gel filtration. However, Carlier et al. (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar) showed (and we confirm3) that ADF-family proteins quench the fluorescence of pyrene-actin when they bind actin filaments. Carlier et al. (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar) used quenching of NBD-actin fluorescence to study the interaction of ADF1 from Arabidopsis thaliana with muscle actin monomers. In our hands, actophorin binding did not affect the fluorescence of NBD-actin monomers, so we used anisotropy to study the interaction of actophorin and actin monomers. This approach has the advantage that the fluorescent dye is placed outside of the binding site where it does not interfere with binding. Assays dependent on a change in fluorescence are more convenient and sensitive, but changes in fluorescence are likely to result from direct or indirect interactions of the fluorophore with the ligand and thus may influence the reaction being studied. Thus fluorescence anisotropy has some advantages, and the labeled protein can also be used for observations in live cells. The fluorescence anisotropy assay confirms that interaction of actophorin with both cytoplasmic and muscle actin monomers depends on the nucleotide bound to actin. At physiological ionic strength, actophorin binds Mg-ADP-actin tightly (K d = 0.15 μm) but Mg-ATP-actin weakly (K d = 5.9 μm). These values are in the range estimated by Maciver and Weeds (22Maciver S.K. Weeds A.G. FEBS Lett. 1994; 347: 251-256Crossref PubMed Scopus (96) Google Scholar) and agree with the affinity of ADF1 for muscle actin monomers (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar). The bound nucleotide also affects the affinity of monomeric actin for other proteins. Some proteins like thymosin-β4 and profilin bind ATP-actin monomers more tightly (45Carlier M.F. Jean C. Rieger K.J. Lenfant M. Pantaloni D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5034-5038Crossref PubMed Scopus (132) Google Scholar,46Pantaloni D. Carlier M.F. Cell. 1993; 75: 1007-1014Abstract Full Text PDF PubMed Scopus (454) Google Scholar),2 and others like ADF1, actophorin, and gelsolin bind ADP actin monomers more tightly (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar, 22Maciver S.K. Weeds A.G. FEBS Lett. 1994; 347: 251-256Crossref PubMed Scopus (96) Google Scholar, 47Laham L.E. Lamb J.A. Allen P.G. Janmey P.A. J. Biol. Chem. 1993; 268: 14202-14207Abstract Full Text PDF PubMed Google Scholar). Because the atomic structures of ATP- and ADP-actin bound to DNase I are nearly identical (48Kabsch W. Mannherz H.G. Suck D. Pai E. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1515) Google Scholar), it is unclear why the nucleotide should have such a large effect on affinity of actophorin and related proteins for actin monomers. The structure of actophorin-G-actin complex will be required to give the answer. Actophorin has the same affinity for muscle and amoeba actin monomers, unlike profilin, which binds better to amoeba actin.2 This is convenient, because muscle actin is somewhat easier to purify. Unfortunately the situation is different with actin filaments where actophorin binds much better to muscle than amoeba actin.3Clearly, every case requires a direct comparison to establish whether muscle and cytoplasmic actins interact the same with test proteins. Actophorin was originally thought to make a nonpolymeric complex with actin (1Cooper J.A. Blum J.D. Williams Jr., R.C. Pollard T.D. J. Biol. Chem. 1986; 261: 477-485Abstract Full Text PDF PubMed Google Scholar, 2Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Google Scholar), but Carlieret al. (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar) called this into question. They showed that multiple effects of ADF/cofilin proteins on actin filaments complicate the design and interpretation of polymerization experiments. Our new assay (Fig. 5) clarifies the effects of actophorin on actin filament elongation. We used phalloidin to inhibit binding of actophorin to actin filaments (2Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Google Scholar),3 so that all added actophorin bound actin monomers and none bound actin filament seeds or elongating pyrene actin filaments. This is essential to avoid quenching of the pyrene fluorescence of the polymerized actin by actophorin. Phalloidin binds actin filaments relatively slowly (49De La Cruz E.M. Pollard T.D. Biochemistry. 1996; 35: 14054-14061Crossref PubMed Scopus (78) Google Scholar), but a concentration of 4 μm will bind with a half-time of 1 s, faster than the rate of phosphate release from ADP-Pi-actin subunits in filaments (50Carlier M.F. Biochem. Biophys. Res. Commun. 1987; 143: 1069-1075Crossref PubMed Scopus (32) Google Scholar). Actophorin does not bind ADP-Pi-actin filaments (2Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Google Scholar). Actin monomers saturated with actophorin still elongate both ends of actin filament seeds. Actophorin has no effect on the rate of elongation at either filament end by Mg-ATP-actin in 100 mmKCl. Carlier et al. (21Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (816) Google Scholar) reported that plant ADF1 increases the rate of muscle actin filament elongation 20-fold at low ionic strength but not in 400 mm KCl. ADP-actin saturated with actophorin consistently elongates both filament ends slightly faster than ADP-actin alone. The pointed end reaction is not diffusion limited (51Drenckhahn D. Pollard T.D. J. Biol. Chem. 1986; 261: 12754-12758Abstract Full Text PDF PubMed Google Scholar), so actophorin may alter the conformation of actin monomers in a way that favors association. Profilin and actophorin compete for binding actin monomers, suggesting that their actin binding sites overlap. This fits well with the proposal of Hatanaka et al. (9Hatanaka H. Ogura K. Moriyama K. Ichikawa S. Yahara I. Inagaki F. Cell. 1996; 85: 1047-1055Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) that the actophorin relative, destrin, binds between subdomains 1 and 3 of actin, directly overlapping the profilin binding site. However, a reconstruction of actin filaments with bound cofilin (34McGough A. Pope B. Chiu W. Weeds A. J. Cell Biol. 1997; 138: 771-781Crossref PubMed Scopus (564) Google Scholar) does not support this model. Instead, cofilin appears to bind along the two start actin filament helix, interacting with two adjacent actin subunits: one at a site between subdomains 1 and 3 and the other at subdomains 2 and 1. More detailed structures of actin bound to ADF/cofilin proteins will be required to understand the competitive interactions of actophorin and profilin. Saturating concentrations of actophorin reduce 13-fold the rate of MgADP exchange for ATP on actin monomers, similar to the effect of brain cofilin (42Nishida E. Biochemistry. 1985; 24: 1160-1164Crossref PubMed Google Scholar) and yeast cofilin (24Lappalainen P. Fedorov E.V. Fedorov A.A. Almo S.C. Drubin D.G. EMBO J. 1997; 16: 5520-5530Crossref PubMed Scopus (208) Google Scholar) on ATP exchange. We assume that the rate of ADP dissociation is rate-limiting, as in other actin nucleotide exchange reactions. Given the 20 μmconcentration of actophorin in Acanthamoeba and its higher affinity for MgADP-actin monomers than filaments,3 a large pool of monomeric ADP-actin might be bound to actophorin if dissociation of ADP were slow. To examine the fate of ADP-actin/actophorin as it is released from depolymerizing filaments, using KINSIM and the measured rate and equilibrium constants, we simulated what happens when 0.4 μm ADP-actin/actophorin is added to a steady-state mixture of cellular concentrations of actin monomers, actin filaments, actophorin, ATP, and ADP (Fig. 7). In the absence of profilin the reaction has two phases. In less than 1 s the actophorin distributes about equally between ADP-actin monomers and ADP-actin filaments. This is followed by slow (t 1/2 = 30 s) exchange of ADP for ATP in the actin monomer pool. Cellular concentrations of profilin accelerate the slow phase (Fig. 7). Profilin promotes the exchange of ADP for ATP on actin monomers, rapidly depleting the pool of high affinity ADP-actin/actophorin complex. This allows actophorin to redistribute rapidly (t 1/2 = 3 s) back to actin filaments and actin monomers to form a high affinity ATP-actin complex with profilin, ready for rapid elongation of actin filament barbed ends. Our new information regarding monomer binding, filament elongation, and nucleotide exchange focuses attention on interactions of actophorin with actin filaments. This is in accord with the effects of mutations in yeast cofilin. Mutations that inhibit binding to actin filaments but not actin monomers are lethal or temperature-sensitive (24Lappalainen P. Fedorov E.V. Fedorov A.A. Almo S.C. Drubin D.G. EMBO J. 1997; 16: 5520-5530Crossref PubMed Scopus (208) Google Scholar). Actophorin binds with high affinity to ADP-actin monomers, the species likely to be released from depolymerizing actin filaments. In a simple system with these two proteins, actophorin would build up a pool of ADP-actin monomers. However, in the presence of profilin, ADP-actin/actophorin released from filaments is only a transitory state. Released actophorin quickly recycles back to actin filaments and the ADP-monomers enter the ATP-actin/profilin pool. Under physiological conditions, actophorin does not appear from its biochemical properties to have the capacity to sequester a large pool of actin monomers in the cell either by blocking elongation or inhibiting nucleotide exchange. More likely its role is to stimulate filament turnover, either by severing filaments or increasing the rate of subunit dissociation or both. ATP hydrolysis within the filament and dissociation of the γ phosphate are essential as a timer for actophorin binding (2Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Google Scholar), singling out older actin filaments for depolymerization. We thank members of the Pollard laboratory for technical assistance, advice, and helpful discussion. We are extremely grateful to R. D. Mullins for advice and suggestions, help in the sedimentation equilibrium experiments, and providingAcanthamoeba actin and profilin II. We thank E. De La Cruz for suggestions during the course of this investigation.