CARMIL Is a Bona Fide Capping Protein Interactant
2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês
10.1074/jbc.m308829200
ISSN1083-351X
AutoresKirsten Remmert, Thomas E. Olszewski, Meredith Bowers, Mariana N. Dimitrova, Ann Ginsburg, John A. Hammer,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoCARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the α- and β-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is ∼0.4 μm or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, ∼200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer ↔ dimer equilibrium with an association constant of 1.0 × 106m-1. Together, these results indicate that CARMIL self-associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (∼1 and 2 μm for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant. CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the α- and β-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is ∼0.4 μm or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, ∼200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer ↔ dimer equilibrium with an association constant of 1.0 × 106m-1. Together, these results indicate that CARMIL self-associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (∼1 and 2 μm for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant. In 1995, Zot (1.Xu P. Zot A.S. Zot H.G. J. Biol. Chem. 1995; 270: 25316-25319Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and colleagues identified a ∼125-kDa protein from Acanthamoeba on the basis of its ability to bind to the isolated Src homology 3 (SH3) 1The abbreviations used are: SH3, Src homology domain 3; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; CP, capping protein; DSG, disuccinimidyl glutarate; DSP, dithiobis[succinimidyl propionate]; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; GST, glutathione S-transferase; LRR, leucine-rich repeat; MES, 2-(N-morpholino)-ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; RSEM, rotary shadowed electron microscopy; S-NHS, N-hydroxysulfosuccinimide; TBS, Tris-buffered saline; TLCK, Nα-p-tosyl-l-lysine chloromethyl ketone. domain of Acanthamoeba myosin IC (1.Xu P. Zot A.S. Zot H.G. J. Biol. Chem. 1995; 270: 25316-25319Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This protein, which they called Acan 125, coimmunoprecipitated with myosin IC and appeared to colocalize with the myosin in cellular surface projections involved in pinocytosis. The subsequent cloning of the Acanthamoeba gene for Acan 125 (2.Xu P. Mitchelhill K.I. Kobe B. Kemp B.E. Zot H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3685-3690Crossref PubMed Scopus (48) Google Scholar) revealed a multidomain protein dominated by a central, ∼460-residue leucine-rich repeat (LRR) domain. LRRs are ∼29-residue sequences that contain a loose consensus dominated by leucines, form amphipathic α-β-structural units and mediate protein-protein interactions, either by serving as the ligand binding sites themselves or by increasing the affinity and/or specificity of binding at a separate site (3.Kobe B. Deisenhofer J. Curr. Opin. Struct. Biol. 1995; 5: 409-416Crossref PubMed Scopus (322) Google Scholar). The second most striking structural feature of Acan 125 is its ∼200-residue, proline-rich COOH-terminal domain. Consistent with the fact that SH3 domains mediate protein-protein interactions by binding to proline-rich target sequences containing the core element PXXP (4.Kuriyan J. Cowburn D. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 259-288Crossref PubMed Scopus (468) Google Scholar), Xu et al. (2.Xu P. Mitchelhill K.I. Kobe B. Kemp B.E. Zot H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3685-3690Crossref PubMed Scopus (48) Google Scholar) showed that a fusion protein containing the COOH-terminal 344 residues of Acan 125 bound to the isolated SH3 domain of myosin IC and that this interaction was abrogated by an 18-residue deletion spanning two PXXP motifs fitting the consensus for SH3 domain target sequences (2.Xu P. Mitchelhill K.I. Kobe B. Kemp B.E. Zot H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3685-3690Crossref PubMed Scopus (48) Google Scholar, 5.Zot H.G. Bhaskara V. Liu L. Arch. Biochem. Biophys. 2000; 375: 161-164Crossref PubMed Scopus (10) Google Scholar). Subsequent studies have estimated an affinity of 20-150 nm for the interaction between Acan 125 and the SH3 domains of Acanthamoeba myosins IA (6.Lee W.L. Ostap E.M. Zot H.G. Pollard T.D. J. Biol. Chem. 1999; 274: 35159-35171Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and IC (5.Zot H.G. Bhaskara V. Liu L. Arch. Biochem. Biophys. 2000; 375: 161-164Crossref PubMed Scopus (10) Google Scholar). These values, together with the cellular concentrations of myosin I and Acan 125 (∼1 and ∼2 μm, respectively), suggest that type I myosins and Acan 125 may be largely associated in vivo, barring some type of regulation (6.Lee W.L. Ostap E.M. Zot H.G. Pollard T.D. J. Biol. Chem. 1999; 274: 35159-35171Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Parallel efforts in our laboratory to purify and characterize proteins that bind to the SH3 domains of type I myosins from Dictyostelium led to the identification of p116, the Dictyostelium homolog of Acan 125 (7.Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Crossref PubMed Scopus (141) Google Scholar). Importantly, however, the eluates of the Dictyostelium myosin I SH3 domain affinity columns contained on a consistent basis not only p116, but also the seven-member Arp2/3 complex, the central player in the de novo nucleation of actin filament assembly and in the formation of branched filament networks, and capping protein, the central player in the termination of actin filament assembly. Immunoprecipitation reactions and other experiments provided evidence that Dictyostelium myosins IB (myoB) and IC (myoC) form a complex with p116, Arp2/3, and capping protein in vivo and that p116 serves as the scaffold for assembly of the complex, binding myosin I, capping protein, and Arp2/3 at independent sites. Given its central role in complex formation, we proposed the name CARMIL for p116, which stands for Capping protein, ARp2/3, Myosin I Linker. In further work by Jung et al. (7.Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Crossref PubMed Scopus (141) Google Scholar), CARMIL was shown to localize along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration and dorsal, cup-like macropinocytic extensions. Moreover, cells in which the CARMIL gene was rendered nonfunctional by homologous recombination exhibited striking defects in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, and a significant decrease in the efficiency of chemotactic aggregation. Together, these results identified a complex that links key players in the nucleation (Arp2/3) and termination (capping protein) of actin filament assembly with a ubiquitous barbed end-directed motor (myosin I), indicated that the protein responsible for the formation of this complex (CARMIL) is physiologically important, and suggested that previously reported myosin I mutant phenotypes in Dictyostelium might be due, at least in part, to defects in the assembly state of actin. In the present study we sought to purify CARMIL to homogeneity and to begin to characterize its biochemical properties. We chose to purify the protein from Acanthamoeba given our laboratory's previous success in using this organism for large scale preparations of cytoskeletal proteins. We show by a variety of methods that Acanthamoeba CARMIL is a bona fide capping protein (CP) interactant that could bind a significant fraction of cellular CP in vivo. CARMIL Purification—The purification of CARMIL was based on a method described by Xu et al. (2.Xu P. Mitchelhill K.I. Kobe B. Kemp B.E. Zot H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3685-3690Crossref PubMed Scopus (48) Google Scholar) with several modifications. Acanthamoeba castellanii were grown in suspension culture to late-log phase, harvested, washed, and resuspended in 1:2 (w/v) Tris-buffered saline (TBS; 1 mm EDTA, 1 mm dithiothreitol, 150 mm NaCl, 6 mm KCl, 50 mm Tris-Cl, pH 8.0) supplemented with protease inhibitors (5 μg/ml leupeptin, 20 μg/ml soybean trypsin inhibitor, 2 μg/ml aprotinin, 20 μg/ml benzamidine, 20 μg/ml TLCK, 50 μg/ml AEBSF), and broken in a Parr bomb at 375 p.s.i. for 5 min. After clarification of the lysate by centrifugation at 100,000 × g for 3 h ("high speed supernatant"), proteins were fractionated by ammonium sulfate precipitation. The fraction between 25 and 55% (NH4)2SO4 was collected, resuspended in TBS supplemented with 1 m ammonium sulfate and protease inhibitors, and applied to a phenyl-Sepharose 6B column (Amersham Biosciences). Proteins were eluted with a linear gradient from 1.0 to 0 m ammonium sulfate in TBS. Fractions were tested for the presence of CARMIL by Western blotting, and positive fractions were pooled and dialyzed against TBS. CARMIL was bound to a SH3 affinity column and eluted with 5 × TBS. The eluate was dialyzed against buffer A (50 mm KCl, 1 mm dithiothreitol, 1 mm EDTA, 25 mm MOPS, pH 7.2, 5 μg/ml leupeptin, 20 μg/ml TLCK, 50 μg/ml AEBSF), loaded on a Mono Q column (Amersham Biosciences), bound proteins were eluted with a linear 0-500 mm KCl gradient, and fractions were analyzed by SDS-PAGE. CARMIL-containing fractions were loaded on a HiPrep® Sephacryl S300 column (Amersham Biosciences; for analytical gel filtration a Superdex 200 column was used) equilibrated with either buffer L (500 mm KCl, 0.5 mm dithiothreitol, 0.5 mm EDTA, 25 mm MES, pH 5.4, 5 μg/ml leupeptin, 50 μg/ml AEBSF) or buffer P (500 mm KCl, 0.5 mm dithiothreitol, 0.5 mm EDTA, 25 mm MOPS, pH 7.2, 5 μg/ml leupeptin, 50 μg/ml AEBSF). Positive fractions were identified by SDS-PAGE, dialyzed against buffer A, and subjected to a final anion exchange chromatography step on Mono Q to reconcentrate the protein. Other Proteins—Acanthamoeba actin was purified according to Gordon et al. (8.Gordon D.J. Eisenberg E. Korn E.D. J. Biol. Chem. 1976; 251: 4778-4786Abstract Full Text PDF PubMed Google Scholar), with a final gel filtration step on HiPrep® Sephacryl S200. Actin was labeled with N-(1-pyrene)-iodoacetamide (Molecular Probes) according to Kouyama and Mihashi (9.Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (720) Google Scholar) and stored lyophilized at -80 °C. Before use it was resuspended in G buffer (0.1 mm CaCl2, 0.5 mm ATP, 0.75 mm β-mercaptoethanol, 3 mm imidazole, pH 7.5) and dialyzed in the dark against the same buffer. Recombinant Proteins—Generation of the GST fusion protein containing the SH3 domain of Acanthamoeba myosin IC (hereafter referred to as GST-SH3) was described previously (7.Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Crossref PubMed Scopus (141) Google Scholar). Portions of the carboxyl-terminal end of Acanthamoeba CARMIL were amplified by PCR using Pfu polymerase, a full-length CARMIL cDNA clone as template (a kind gift from Henry Zot, Eastern Michigan University, Ypsilanti), and the following primers (5′-oligonucleotides contained an SmaI site, and 3′-oligonucleotides contained an EcoRI site): VAP, 5′-TGC ACC CGG GCG AGG ACT TCT CCC AGC ACA TCT CC-3′ and 5′-TGC AGA ATT CAG CCC ACC TCC AAG CAA GGC GCT-3′; AP, 5′-TGC ACC CGG GCG CCC CGA CCC CGA GGA GCC AG-3′ and 5′-TGC AGA ATT CAG CCC ACC TCC AAG CAA GGC GCT-3′;P,5′-TGC ACC CGG GCG GCG TGG CGC TGC CCT TCG GGG CC-3′ and 5′-TGC AGA ATT CAG CCC ACC TCC AAG CAA GGC GCT-3′; VA, 5′-TGC ACC CGG GCG AGG ACT TCT CCC AGC ACA TCT CC-3′ and 5′-TGC AGA ATT CCT ACA TCG GCA CGC CGG TCG GTC G-3′. PCR products were digested, ligated into pGEX-2T, and transformed into Escherichia coli host strain BL21(DE3) for production of GST fusion proteins. Construction of the GST-LRR construct, which contains the complete LRR domain of Dictyostelium CARMIL, will be described elsewhere. 2G. Jung and J. A. Hammer, manuscript in preparation. Recombinant proteins were prepared as described previously (7.Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Crossref PubMed Scopus (141) Google Scholar). In brief, overnight cultures were diluted 1:10 into 200 ml of fresh LB medium containing 100 μg/ml ampicillin, grown to an A600 nm of 1.0, and induced with isopropyl-1-thio-β-d-galactopyranoside (0.5 mm final concentration) for 3 h at 37 °C. Cells were harvested, resuspended in phosphate-buffered saline supplemented with protease inhibitors (1 mm AEBSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 μg/ml pepstatin A) and 0.1 mm dithiothreitol, and lysed in a French pressure cell. After removal of cell debris by centrifugation at 12,000 × g, fusion proteins were bound to glutathione-Sepharose 4B beads (Amersham Biosciences) for 2 h at 4 °C. Loaded beads were used for CP pull-down experiments after four washes in 1 × TBS. Protein Microsequencing—Peak fractions of the CARMIL-CP complex from the Mono Q eluate (see above) were pooled, resolved on a 10% SDS-PAGE, and the 30- and 32-kDa bands excised. After digestion with trypsin, peptides were separated by high performance liquid chromatography, and sequenced by Edman degradation (Harvard Microchemistry Facility). Peptide sequences were compared with multiple protein data bases (BLASTP 2.0.13, which includes all nonredundant GenBank CDS translations + PDB + SwissProt + PIR + PRF) by BLAST search (10.Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). SDS-PAGE and Western Blotting—SDS-PAGE was performed on minigels using either Tris-acetate- or Tris-glycine-buffered polyacrylamide gels (Invitrogen). Proteins were denatured in SDS-sample buffer (2.5% β-mercaptoethanol, 5% SDS, 0.002% bromphenol blue, 0.75% sucrose, 1 mm EDTA, and 30 mm Tris-HCl, pH 6.8) by boiling for 5 min. After electrophoresis, proteins were either stained with Coomassie Blue or transferred onto nitrocellulose by semidry electroblotting (Bio-Rad). Blots were blocked with 2% bovine serum albumin in TBS and 0.1% Tween 20 (TBST) at 4 °C overnight and incubated with polyclonal antibodies against CARMIL or CP (1:10,000 and 1:1,500, respectively) in 2% bovine serum albumin and TBST for 3 h at room temperature. Specific binding was detected with horseradish peroxidase-conjugated secondary antibodies using an enhanced chemiluminescence system (ECL; Amersham Biosciences). Antibodies—A polyclonal antibody against Acanthamoeba CP was raised using a compound peptide containing two α-subunit sequences that were obtained by microsequencing (see above; DESILNDSAPATFR and FGEVGNGEYLDPR) and three glycine residues as linker. This peptide was conjugated to keyhole limpet hemocyanin and injected into rabbits (Zymed Laboratories Inc.), and the resulting serum was affinity purified using the immunogenic peptide immobilized on nitrocellulose. Polyclonal antibodies against the α- and β-subunits of Dictyostelium CP were a kind gift from John A. Cooper (Washington University, St. Louis). Generation of the polyclonal antibody against a portion of the LRR domain of Acanthamoeba CARMIL was described previously (7.Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Crossref PubMed Scopus (141) Google Scholar). Analytical Ultracentrifugation—Analytical ultracentrifugation was performed in Optima models XL-A and XL-I analytical ultracentrifuges (Beckman Inc.) equipped with a four-place An-Ti rotor. Sedimentation velocity and sedimentation equilibrium experiments were performed at 20 and 4 °C, respectively. The density (ρ) of the dialysate, buffer A (25 mm MOPS, 250 mm KCl, 1 mm EDTA, and 2 mm β-mercaptoethanol, pH 7.0) was determined to be 1.0101 g/ml at 20.00 ± 0.01 °C with the Anton Paar model DMA 58 densitometer, and the relative viscosity was determined to be 1.0139 (11.Shapiro B.M. Ginsburg A. Biochemistry. 1968; 7: 2153-2167Crossref PubMed Scopus (109) Google Scholar). A partial specific volume of 0.720 ml/g for CARMIL was calculated from the amino acid composition and the values of Zamyatnin (12.Zamyatnin A.A. Annu. Rev. Biophys. Bioeng. 1984; 13: 145-165Crossref PubMed Scopus (274) Google Scholar). Specific absorbance coefficients for CARMIL and CP were determined as described previously (13.Nosworthy N.J. Peterkofsky A. Konig S. Seok Y.J. Szczepanowski R.H. Ginsburg A. Biochemistry. 1998; 37: 6718-6726Crossref PubMed Scopus (42) Google Scholar) using a PerkinElmer Life Sciences Lambda 18 spectrophotometer and the model XL-I equipped with interference optics. A capillary synthetic boundary cell centerpiece and sapphire windows were employed with 0.140 ml of dialyzed protein (1.144 mg/ml CARMIL or 0.767 mg/ml CP) loaded in the right side and 0.400 ml of buffer A on the left side. After formation of the boundary by slow acceleration to 15,000 rpm, the speed was decreased to 3,000 rpm for CARMIL and 5,000 rpm for CP, while maintaining the temperature at 20 °C. Repetitive interference scans were taken 20 or 30 s apart for 15 min during which time solvent base line and the protein plateau remained flat. The calibration value of 3.191 ± 005 fringes (mg/ml)-1 (13.Nosworthy N.J. Peterkofsky A. Konig S. Seok Y.J. Szczepanowski R.H. Ginsburg A. Biochemistry. 1998; 37: 6718-6726Crossref PubMed Scopus (42) Google Scholar) was corroborated with a solution of 3.428 mg/ml bovine serum albumin. Specific absorbances (A280 nm, 1 cm) of 0.474 ± 001 (cm2/mg) or ϵ280 nm = 57,640 m-1 cm-1 for CARMIL and 1.140 ± 002 or ϵ280 nm = 70,520 m-1 cm-1 for CP were determined. After determination of the absorbance coefficient for CARMIL, the speed was increased to 44,000 rpm, and 20 scans at 1 min apart were taken to determine the sedimentation coefficient under conditions where >90% of CARMIL was dimeric. For time derivative analysis (14.Stafford W.F. Curr. Opin. Biotechnol. 1997; 8: 14-24Crossref PubMed Scopus (67) Google Scholar), the procedures of Zolkiewski et al. (15.Zolkiewski M. Redowicz M.J. Korn E.D. Hammer III, J.A. Ginsburg A. Biochemistry. 1997; 36: 7876-7883Crossref PubMed Scopus (23) Google Scholar) were used. Observed sedimentation coefficients (sobs) were corrected to the density and viscosity of water at 20 °C, where s20,w = 1.0457 sobs (reported in Svedberg units, S). For sedimentation equilibrium runs in the XL-A, liquid columns of 0.080 or 0.110 ml of CARMIL in buffer A (with 0.015 ml more dialysate buffer A in the reference channel) and a speed of 7,500 rpm were used. Scans at 280 nm were collected at 2-h intervals in step mode (0.001-cm steps) with 11 or 13 averages/scan. Equilibrium was attained in 28 h, as determined by overlaying scans at different times. Global, weighted fits of sedimentation equilibrium data obtained at two concentrations of CARMIL in different runs to a model of reversible monomer-dimer association (with fully competent species present) were made using software provided by Allen P. Minton (NIDDK, NIH). Higher oligomeric forms than dimer were not detected. The monomer molecular weight of CARMIL (calculated from the amino acid composition) and base lines at zero 280 nm absorbance were held constant. Residuals from fits of the data to a monomer ↔ dimer equilibrium were randomly distributed around zero with < 0.01 absorbance deviations. For conversion of the observed association constant (Kobs′) to a true molar concentration-dependent association constant (expressed per mole subunit), Ka′, Equation 1 was used, logKa'=4.539+logKobs'(Eq. 1) where the constant was calculated from log 2/ϵ, where ϵ is the molar extinction coefficient of the monomer for a 1.2-cm path length and the dimer was assumed to equal 2ϵ. Purified CARMIL and CP were mixed together in 0.15 ml of buffer A to give final concentrations of 1.93 and 3.28 μm, respectively (corresponding to a molar ratio of 1.7:1, CP to CARMIL). The mixture was equilibrated versus the same volume of buffer A in a double sector centerpiece in a 12-mm cell with sapphire windows for 48-54 h at 10,000 rpm and 4 °C. Interference optics were used to collect data and later analyzed for the equilibrium components present, where the molecular weights for CP and the CARMIL dimer were 61,860 and 243,200, respectively (see "Results"). Far UV circular dichroism spectra measurements were performed with a Jasco J-710 spectrometer using a water-jacketed cylindrical cell with a path length of 0.01 cm. The temperature of the cell was controlled at 20 °C by an external programmable water bath (Neslab RTE-111). Spectra were corrected for the CD signal of solvent (buffer A without β-mercaptoethanol). For determining secondary structure, far UV spectra were the average of 30 accumulations taken at 200 nm/min. Mean residue molecular weights of 108.5 for CARMIL were used for calculations of [θ]. Secondary structural components were calculated using the SELCON analysis program in Softsec™ applications (Softwood Company). Rotary Shadowing—Purified CARMIL, CP, and mixtures of both proteins at an initial concentration of ∼0.1 mg/ml were diluted with 2 parts of glycerol, sprayed onto freshly cleaved mica chips (16.Tyler J.M. Branton D. J. Ultrastruct. Res. 1980; 71: 95-102Crossref PubMed Scopus (291) Google Scholar), and rotary shadowed at room temperature and at an angle of 9° in a Balzers 301 freeze fracture apparatus, or an RMC RFD 9010 (Boeckeler Instruments Inc., Tucson, AZ). Chemical Cross-linking—DSG, DSP, EDAC, and S-NHS were obtained from Pierce and used at final concentrations of 2.5 mm for DSG and DSP and 5 mm for EDAC and S-NHS. Stock solutions (10×) of DSG and DSP were prepared in dimethyl sulfoxide whereas EDC and S-NHS were dissolved in H2O. The final concentration of dimethyl sulfoxide in cross-linking reactions never exceeded 10%. Cross-linking reactions were incubated for a maximum of 30 min at 24 °C. Products were resolved on 3-8% NuPAGE gels and either stained with Coomassie Blue or processed for Western blotting. Actin Polymerization Assay—The kinetics of actin polymerization were followed at 24 °C by monitoring the change in fluorescence intensity of pyrene-labeled actin upon incorporation into the growing filament (9.Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (720) Google Scholar, 17.Cooper J.A. Walker S.B. Pollard T.D. J. Muscle Res. Cell Motil. 1983; 4: 253-262Crossref PubMed Scopus (370) Google Scholar). In brief, G-actin and pyrene-actin were mixed at a ratio of 20:1 and polymerized at a final actin concentration of 4 μm by adding 10× actin polymerization buffer (500 mm KCl, 10 mm MgCl2, 10 mm EGTA, 100 mm imidazole, pH 7.0). CP was dialyzed into 1× actin polymerization buffer and added to the actin solution immediately before the polymerization reaction was started. Fluorescence intensities (ex, 365 nm; em, 407 nm) were measured at a rate of 0.2 point/sec in a PTI QuantaMaster spectrofluorometer (Photon Technology International, Santa Clara, CA) over 30 min. Capping Protein Copurifies with CARMIL—The procedure we used for the isolation of Acanthamoeba CARMIL was based in part on a protocol devised by Xu et al. (2.Xu P. Mitchelhill K.I. Kobe B. Kemp B.E. Zot H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3685-3690Crossref PubMed Scopus (48) Google Scholar). The key steps in that procedure were hydrophobic-interaction chromatography followed by affinity chromatography that takes advantage of the tight and highly specific association between PXXP motifs located in the proline-rich carboxyl-terminal region of CARMIL and the SH3 domain of Acanthamoeba myosin IC (2.Xu P. Mitchelhill K.I. Kobe B. Kemp B.E. Zot H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3685-3690Crossref PubMed Scopus (48) Google Scholar). Fig. 1 illustrates the enrichment of CARMIL we obtained with a modified procedure that made use of these two steps. In brief, a high speed supernatant of the crude lysate (lane 1) was fractionated with ammonium sulfate, and the 25-55% precipitate (lane 2) was subjected to a hydrophobic interaction chromatography (lane 3). CARMIL-containing fractions were identified by Western blotting with an antibody raised previously against the Acanthamoeba protein (7.Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Crossref PubMed Scopus (141) Google Scholar). Pooled fractions were subjected to SH3 domain affinity chromatography (lane 4). Three predominant polypeptides elute from the GST-SH3 resin: a ∼125-kDa band (filled arrowhead) corresponding to CARMIL and two unknown polypeptides of ∼32 and ∼30-kDa molecular mass (open arrowheads). After a final ion exchange chromatography step using Mono Q (lane 5), we consistently obtained peak fractions containing essentially the three afore-mentioned polypeptides. The fact that the smaller proteins copurified with CARMIL throughout the entire procedure and that their abundance appeared to be roughly equimolar to each other and to CARMIL led us to speculate that they might form a stable complex with CARMIL. In this regard it is interesting to note that these two smaller proteins were not described in the previous study by Xu et al. (2.Xu P. Mitchelhill K.I. Kobe B. Kemp B.E. Zot H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3685-3690Crossref PubMed Scopus (48) Google Scholar). We reported previously that CP is present along with CARMIL in GST-SH3-domain pull-down experiments made using Dictyostelium cell lysates (7.Jung G. Remmert K. Wu X. Volosky J.M. Hammer III, J.A. J. Cell Biol. 2001; 153: 1479-1497Crossref PubMed Scopus (141) Google Scholar). Given those findings, and our observation here that the ∼32-kDa and ∼30-kDa polypeptides are always approximately equimolar, we reasoned that they corresponded to the two chains of heterodimeric CP. Consistent with this, polyclonal antibodies raised against the α- and the β-subunits of CP from Dictyostelium cross-react in a subunit-specific fashion with the ∼32- and ∼30-kDa Acanthamoeba polypeptides, respectively (Fig. 2A). Moreover, using a purified, CARMIL-free fraction of the ∼32/30-kDa proteins (see below) and actin assembly assays, we observed a dose-dependent stimulation of actin nucleation in accordance with the nucleation activity of CPs (Fig. 2B (18.Cooper J.A. Pollard T.D. Biochemistry. 1985; 24: 793-799Crossref PubMed Scopus (83) Google Scholar). To confirm absolutely that the ∼32/30-kDa doublet is CP, both bands were excised from polyacrylamide gels, digested with trypsin, and the resulting fragments subjected to microsequencing by Edman degradation. As shown in Fig. 2C (top), the partial sequences of two peptides derived from the ∼32-kDa band were found to be highly similar to sequences in the α-subunits of CP from Dictyostelium and various vertebrates. Likewise, three peptides obtained from the ∼30-kDa band (Fig. 2C, bottom) exhibited extensive sequence similarity to the β-subunits of CP from various species. Taken together, these results establish unequivocally that the ∼32/30-kDa polypeptides copurifying extensively with CARMIL are the α- and β-subunits of Acanthamoeba CP. CARMIL and CP Can be Separated by Gel Filtration at Low pH—Preparations of CARMIL which are free of CP are required to investigate the biochemical properties of the protein. Given the large difference in the molecular masses of CARMIL (∼125 kDa) and CP (∼60 kDa for the obligate heterodimer), we chose size exclusion chromatography as an appropriate method for their separation. Chromat
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