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

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.

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 M) 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.
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 Ͻ7.3, depolymerizing filaments only at higher pH (18,19). 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 depolymeriza-tion by creating more ends to dissociated subunits (2, 18 -20). Carlier et al. (21) 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 ATPactin 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 (21,22). Both porcine brain and yeast cofilin inhibit the exchange of Mg-ATP bound to actin (23,24). 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-ATPactin 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.
Preparation of Mutant Actophorins-Residues Asn-33, Glu-70, and Ser-88 of actophorin were changed to cysteine by reverse polymerase chain reaction mutagenesis (25), 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 (26). 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. (27), 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 NaN 3 .
Other Proteins-Profilin-II was purified from Acanthamoeba castellanii (29). Actin was purified from rabbit muscle acetone powder (30) or from Acanthamoeba (31) and isolated as Ca-ATP-G-actin through Sephacryl S-300 chromatography (32) at 4°C in G buffer (5 mM Tris-Cl, pH 8.0, 0.2 mM ATP, 0.1 mM CaCl 2 , 0.5 mM DTT). Actin was labeled on Cys-374 to a stoichiometry of 0.8 -1.0 with pyrene iodoacetamide (Ref. 33, as modified by Pollard; see Ref. 31). Mg-ATP G-actin was prepared on ice by addition of 0.2 mM EGTA and an 11-fold molar excess of MgCl 2 over actin and used within hours. Mg-ADP G-actin was prepared by treatment of Mg-ATP G-actin with soluble hexokinase and glucose (31).
Binding of Actophorin to Actin Monomers-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. 2 Data were analyzed using Kaleidagraph software (Synergy Software, Reading, PA) and fitted to Eq. 1 where r is the observed anisotropy, r acf is the anisotropy of free actophorin, r acb 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. Effect of Actophorin and Profilin on Nucleotide Exchange from Actin-The dissociation Mg-⑀-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 M ATP from one syringe with 2.8 M Mg-⑀-ADPactin, 6 M actophorin, and various concentrations of Acanthamoeba profilin-II from the other syringe.

Actin Filament Elongation in the Presence of Actophorin-Seeds
Sedimentation Equilibrium-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 Ϫ1 M Ϫ1 . We analyzed competitive binding of 10 M rhodamine-S88C-actophorin and various concentrations of profilin-II to 10 M Mg-ADP-actin monomers by sedimentation equilibrium. We monitored rhodamine absorption at 550 nm. The data was collected and analyzed according to (35). Data were fit using Eq. 2 where the logarithm of the absorbance is a function of the effective reduced molecular weight M is molecular mass (g/mol), is the angular velocity (rad/sec), v is the partial specific volume (cm 3 /gm), is the solvent density (gm/cm 3

RESULTS
Characterization of Rhodamine-Actophorin-Acanthamoeba actophorin has two cysteines, Cys-9 and Cys-60 (27), 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 (37-40); (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 (26).
Actophorin labeled with 5Ј-rhodamine maleimide isomer on any of the new cysteines has the same mobility on SDS-polyacrylamide gel electrophoresis as unlabeled actophorin ( Fig.  2A, lanes 1-6). Rhodamine maleimide labels mutants E70C and S88C ( Fig. 2A, lanes 4 and 5) more efficiently than N33C ( Fig. 2A, lane 6). By sedimentation equilibrium ultracentrifugation rhodamine-S88C-actophorin is a single ideal species with a molecular weight of 15,200 (Fig. 2B). In this experiment, the ratio of rhodamine (measured by A 550 ) to protein (measured by Rayleigh interferometry) is constant across the ultracentrifuge cell with 0.44 dyes per protein molecule (Fig. 2B). 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 (1,2). 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 (41). Labeling of N33C never exceeded 0.1 dye per protein, so we did not use this mutant.
Binding of Rhodamine-S88C-Actophorin to Actin Monomers-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 MgCl 2 , 100 mM KCl) actophorin binds Mg-ADP-actin monomers 40 times stronger than Mg-ATP-actin (Fig. 3), as observed with other assays (22) and other members of the ADF/cofilin family (21). In contrast, in low salt concentration actophorin binds Mg-ATP-actin monomers better than Mg-ADP-actin. Rhodamine-S88C-actophorin binds Acanthamoeba and rabbit skeletal muscle actin monomers with similar affinities.
Actophorin Is Not a Sequestering Protein-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, 3 so that the actophorin added to the polymerizing mixture was in a simple equilibrium with 4 M actin monomers and actophorin did not quench the fluorescence of newly polymerized pyrene-actin (21). 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. 4A) in agreement with the observations of Carlier et al. (21) but slightly increases the rate of elongation at both ends by Mg-ADP-actin (Fig. 4B). 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.
Competitive Binding of Actophorin and Profilin to Mg-ADP Actin-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 (2), 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.
Effect of Actophorin and Profilin on Nucleotide Dissociation from Actin-Saturating concentrations of actophorin inhibit Mg-ADP dissociation from muscle actin by a factor 13 to a rate constant of 0.006 s Ϫ1 in buffer with 2 mM MgCl 2 (Fig. 6A, inset). This is similar to the effect of cofilin on ATP dissociation from actin monomers (18,24,42). Profilin reverses this effect of actophorin, increasing the rate of nucleotide exchange from actin-actophorin complex in a concentration dependent manner (Fig. 6A). 3 L. Blanchoin and T. D. Pollard, manuscript in preparation.  Table I. To avoid polymerization each measurement was made separately just after mixing a fresh sample. Scheme 1 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, C actophorin, and P profilin. Remarkably, this simple mechanism and the rate and equilibrium constants measured here and in previous work (43) 2 account quantitatively for the time course (Fig. 6A) and rate (Fig. 6B) of ADP exchange in the presence of actophorin and a wide range of profilin concentrations. Two constants were unknown: k ϩ3 is 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 M Ϫ1 s Ϫ1 for k ϩ3 and 3.8 M Ϫ1 for K 4 . The available data did not constrain these constants further, because variation of the values of these constants Ϯ a factor of 10 does not affect the simulated time course of nucleotide exchange.

Comparison of Methods to Measure Actophorin Binding to
Actin Monomers-The present work is the first measurement of the affinity of actophorin for actin. Maciver et al. (44) and Maciver and Weeds (22) 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. (21) showed (and we confirm 3 ) that ADF-family proteins quench the fluorescence of pyrene-actin when they bind actin filaments. Carlier et al. (21) used quenching of NBDactin 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.
Effect of Nucleotide on Actophorin Binding to Actin-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 ϭ 0.15 M) but Mg-ATP-actin weakly (K d ϭ 5.9 M). These values are in the range estimated by Maciver and Weeds (22) and agree with the affinity of ADF1 for muscle actin monomers (21). The bound nucleotide also affects the affinity of monomeric actin for other proteins. Some proteins like thymosin-␤4 and profilin bind ATP-actin monomers more tightly (45, 46), 2 and others like ADF1, actophorin, and gelsolin bind ADP actin monomers more tightly (21,22,47). Because the atomic structures of ATPand ADP-actin bound to DNase I are nearly identical (48), 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. 3 Clearly, every case requires a direct comparison to establish whether muscle and cytoplasmic actins interact the same with test proteins.
Effect of Actophorin on Elongation-Actophorin was originally thought to make a nonpolymeric complex with actin (1, 2), but Carlier et al. (21) 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 (2), 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 fila-ments relatively slowly (49), but a concentration of 4 M will bind with a half-time of 1 s, faster than the rate of phosphate release from ADP-P i -actin subunits in filaments (50). Actophorin does not bind ADP-P i -actin filaments (2).
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 mM KCl. Carlier et al. (21) 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 (51), so actophorin may alter the conformation of actin monomers in a way that favors association.
Competition of Actophorin and Profilin for Binding Actin Monomers-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. (9) 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 (34) 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  Table II. The symbols are the observed rates. The inset shows a theoretical curve for nucleotide dissociation rates at higher concentrations of profilin.   1998 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.
Effect of Actophorin and Profilin on Nucleotide Exchange-Saturating concentrations of actophorin reduce 13-fold the rate of MgADP exchange for ATP on actin monomers, similar to the effect of brain cofilin (42) and yeast cofilin (24) on ATP exchange. We assume that the rate of ADP dissociation is ratelimiting, as in other actin nucleotide exchange reactions. Given the 20 M concentration 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 M ADP-actin/actophorin is added to a steadystate 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 ϭ 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 ϭ 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 (24). 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 ␥ phosphate are essential as a timer for actophorin binding (2), singling out older actin filaments for depolymerization.