Article |
Address correspondence to Pekka Lappalainen, Institute of Biotechnology, P.O. Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Helsinki, Finland. Tel.: 358-9-1915-9499. Fax: 358-9-1915-9366. E-mail: pekka.lappalainen{at}helsinki.fi
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Abstract |
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Key Words: actin; twinfilin; capping protein; PI(4,5)P2; budding yeast
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Introduction |
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A number of actin monomerbinding proteins regulate the size and dynamics of the actin monomer pool in cells. However, only three classes of small actin monomerbinding proteins are found in organisms as diverse as yeast and mammals (Lappalainen et al., 1998): cofilin/actin-depolymerizing factors (ADFs),* profilins, and twinfilins.
Cofilin/ADF proteins promote the addition of actin monomers to the cytoplasmic pool by depolymerizing (and severing) preexisting actin filaments (Carlier et al., 1997; Lappalainen and Drubin, 1997; Rosenblatt et al., 1997) and interacting with actin monomers. Under physiological conditions, cofilin/ADFs have an 100-fold higher affinity for ADP- than for ATP-actin monomers (Maciver and Weeds, 1994; Carlier et al., 1997), and they inhibit the nucleotide exchange of the actin monomers (Hawkins et al., 1993).
In contrast to cofilin/ADF proteins, profilins have a higher affinity for ATP- than for ADP-actin monomers and promote nucleotide exchange on actin monomers (Goldschmidt-Cleremont et al., 1991; Vinson et al., 1998). In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, profilin's ability to enhance nucleotide exchange on actin monomers in vivo is important (Wolven et al., 2000; Lu and Pollard, 2001). Profilin can also promote filament assembly because profilinactin complexes can add to barbed ends (Pantaloni and Carlier, 1993). Furthermore, profilin binding to monomers suppresses the spontaneous nucleation of actin filaments, functioning as an actin monomersequestering protein in the absence of free filament ends (Vinson et al., 1998).
Twinfilin is a 3540-kD actin monomerbinding protein that was originally identified from the budding yeast S. cerevisiae (Goode et al., 1998). Homologues of twinfilin have been found in S. pombe, Caenorhabditis elegans, mice, and humans, suggesting that twinfilins are present across the entire spectrum of eukaryotic organisms (Vartiainen et al., 2000). Twinfilins are composed of two domains homologous to cofilin/ADF proteins (ADF-H domain) that are separated by a short linker. Unlike cofilin/ADF proteins, twinfilin only interacts with actin monomers and inhibits the assembly of actin filaments in a stoichiometric manner in vitro (Goode et al., 1998; Vartiainen et al., 2000). Furthermore, twinfilin inhibits the nucleotide exchange on actin monomers (Goode et al., 1998). Deletion of the twinfilin gene in yeast results in abnormal cortical actin patches, defects in bipolar bud site selection pattern, and synthetic lethality with certain cofilin and profilin mutations (Goode et al., 1998; Wolven et al., 2000). Overexpression of twinfilin in yeast and mouse cells results in the formation of abnormal actin structures (Goode et al., 1998; Vartiainen et al., 2000). These findings suggest that twinfilin, together with cofilin/ADF and profilin, is involved in the regulation of the dynamics of the actin cytoskeleton. However, the mechanism by which twinfilin contributes to actin filament turnover is not understood.
Here we show that twinfilin is an abundant protein, that it binds ADP-actin preferentially, and that it localizes to cortical actin patches in yeast. The localization of twinfilin to actin patches is disrupted by mutations at the actin monomerbinding site of twinfilin. Furthermore, localization of twinfilin to actin patches depends on the presence of the actin filament barbed-end capping protein, Cap1/2p. We suggest that twinfilin localizes ADP-actin monomers at sites of rapid actin filament assembly in cells; therefore, it may serve as a link between actin filament depolymerization and actin filament assembly.
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Results |
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We studied the localization of twinfilin in yeast cells by using the affinity-purified polyclonal antibodies described in the legend to Fig. 2. These antibodies are also specific for twinfilin in immunofluorescence because a twinfilin deletion strain shows no specific staining (Fig. 3 C). In a wild-type yeast strain, twinfilin shows cytoplasmic staining but it is also strongly colocalized with the cortical actin patches (Fig. 3, A and B). No twinfilin staining could be detected on the cytoplasmic actin cables.
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Mutations in the actin-binding site affect the localization of twinfilin
To understand whether twinfilin's interaction with an actin monomer is required for localization, we designed a mutant twinfilin that did not interact with actin monomers. In designing such a mutant, we took advantage of the sequence homology between the ADF-H domains of twinfilin and cofilin/ADF proteins. Based on our multiple sequence alignments, residues R88 and 90 in the NH2-terminal, and residues K254 and R256 in the COOH-terminal ADF-H domain of yeast twinfilin, correspond to residues that are crucial for actin binding in cofilin/ADFs (Moriyama et al., 1992; Lappalainen et al., 1997). Therefore, we chose to replace these residues by alanines either in the NH2-terminal ADF-H domain (Twf1-1p), the COOH-terminal ADF-H domain (Twf1-2p), or in both ADF-H domains (Twf1-3p).
Purified wild-type and mutant twinfilins were analyzed for actin monomer interactions by actin filament sedimentation and native gel electrophoresis assays. In actin filament sedimentation assays, we compared the ability of wild-type and mutant twinfilins to shift actin from filaments (pellet) to the monomer pool (supernatant). Wild-type twinfilin efficiently shifts actin from filaments to the monomer pool with the monomer-sequestering activity saturated at 4 µM twinfilin. Twf1-1p, in which only residues in the first ADF-H domain are replaced by alanines, is somewhat less efficient in sequestering actin monomers in this assay. Also, Twf1-2p shows detectable actin monomer sequestering activity, but it is significantly less efficient than Twf1-1p in shifting actin into the monomeric fraction. Twf1-3p, in which residues in both ADF-H domains are mutated, can no longer shift detectable amounts of actin into the supernatant fraction with the protein concentration range probed in this study (Fig. 4 A). The defect in actin monomer interactions was also seen in native gel electrophoresis assays. Whereas wild-type twinfilin forms a stable complex with ADP-actin monomers on native gels, no detectable complex formation between Twf1-3p and ADP-actin monomers could be observed (unpublished data). The far UV CD spectra of purified wild-type and Twf1-3p twinfilins are almost identical to each other, suggesting that the composition of secondary structure elements in these proteins is very similar (Fig. 4 B). Additionally, the mutations in Twf1-3p do not decrease the protein stability because wild-type twinfilin has a Tm value of 55°C, whereas the Tm value of Twf1-3p is 59°C.
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Two other yeast strains in which twinfilin showed an abnormal localization are cap1 and cap2
. These carry deletions of the
and ß subunits of actin filament barbed-end capping protein, Cap1/2p. In these cells, twinfilin localized diffusely in the cytoplasm, and stronger staining was only occasionally observed at and/or near cortical actin patches (Fig. 6, CF). Whereas virtually all actin patches were positive for twinfilin staining in the wild-type parent strain, detectable patch-like twinfilin staining could be observed in <3% of the actin patches in the cap2
strain. Based on a quantitative Western blot analysis, the cap2
cells have the same level of twinfilin as wild-type cells, demonstrating that the lack of twinfilin localization to the cortical actin patches in the cap2
strain is not a result of decreased twinfilin levels (unpublished data). The defect in actin patch localization in the cap2
strain is also specific for twinfilin because other actin patch components, such as cofilin and Abp1p, localize normally to cortical actin patches in the cap2
strain (unpublished data). These results show that Cap1/2p is required for efficient localization of twinfilin to actin patches. Twinfilin also localizes to the cortical actin patches in the cof1-22 strain (Fig. 6, G and H). In this yeast strain, the actin monomer pool is depleted due to a mutation in the actin filament depolymerizing protein cofilin (Lappalainen and Drubin, 1997). Therefore, it is unlikely that the localization defect observed in cap1
and cap2
strains results from a decrease in the cytoplasmic actin monomer pool in the absence of Cap1/2p.
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Discussion |
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Twinfilin is an abundant protein in yeast, found in an 1:10 ratio to actin and a 1:2.5 ratio to cofilin. Because twinfilin appears to have a high affinity for ADP-actin monomers, our results suggest that it can bind and/or sequester a significantly large proportion of actin monomers in yeast cells at any given time. The abundance of profilin in yeast cells has not been reported, but in Acanthamoeba cells, profilin is found in a 1:2 ratio to actin (Tseng et al., 1984). However, it is important to note that the actin monomer pool in yeast S. cerevisiae cells has been reported to be very small compared with the one in more motile organisms and their cell types (Karpova et al., 1995). In contrast to profilin, twinfilin prefers to interact with ADP-actin monomers and inhibits the nucleotide change upon binding (Goode et al., 1998); therefore, it may have a significant effect on the size and nucleotide status of the actin monomer pool in yeast cells.
The actin cytoskeleton in yeast is composed of two types of filamentous structures: cortical actin patches and cytoplasmic actin cables. These two cytoskeletal structures have a different, but somewhat overlapping, composition of actin-binding proteins. Furthermore, the dynamics of these two cytoskeletal domains are different; patches are more dynamic and composed of shorter filaments than cables (Karpova et al., 1998). Twinfilin shows diffuse cytoplasmic staining, but is also concentrated to cortical actin patches. We did not observe any twinfilin staining in actin cables, although it is possible that twinfilin staining on faint actin cables would not be visible above the background (Fig. 3). Localization of twinfilin to actin filament structures is unexpected, because twinfilin binds actin monomers in vitro.
To study whether twinfilin needs to be associated with an actin monomer to localize to patches, we constructed a mutant twinfilin that was unable to bind actin monomers. A twinfilin mutant (Twf1-3p) in which two key charged residues in both ADF-H domains are replaced by alanines is no longer able to bind and sequester actin monomers. These residues correspond to the most critical actin monomerbinding residues (R96 and K98) in yeast cofilin. Therefore, cofilin/ADF proteins and the ADF-H domains in twinfilin appear to interact with actin through overlapping interfaces. Interestingly, mutations in the COOH-terminal ADF-H domain (Twf1-2p) have a stronger effect on actin monomer sequestering activity than the mutations in the NH2-terminal ADF-H domain (Twf1-1p) (Fig. 4 A), suggesting that the former domain may be more important for actin monomer interactions. The mutant twinfilin (Twf1-3p) interacts with Cap1/2p and PI(4,5)P2, suggesting that these binding sites do not overlap with the actin-binding site (Figs. 7 and 8). When expressed in yeast cells under a GPD promoter or as a GFP fusion protein under a Gal promoter, wild-type twinfilin localized at the cortical actin patches, whereas Twf1-3p showed diffuse cytoplasmic localization. These results suggest that, in order to localize at the cortical actin cytoskeleton, twinfilin may require the ability to associate with an actin monomer. It is possible that the interaction with an actin monomer promotes a conformational change in the three-dimensional structure of twinfilin or in the orientation of the two ADF-H domains with respect to each other, increasing the affinity of the twinfilinactin monomer complex for its ligand at the cortical actin patches.
The localization of twinfilin to the cortical actin cytoskeleton is dependent on actin filaments (Fig. 3, E and F). Therefore, we speculated that the localization of twinfilin's binding partner at the cortical actin patches would also be actin dependent. From the currently known yeast actinbinding proteins, this criterion narrows the number of proteins to seven (for review see Pruyne and Bretscher, 2000). Twinfilin showed normal cortical actin patch localization in all but two of the mutant strains carrying deletions or mutations in these and some other known actin patch associated proteins (Table II.). These exceptions are cap1 and cap2
strains, which carry deletions of the subunits of capping protein, Cap1/2p. It has been shown that a deletion of the gene for either of the capping protein subunits leads to a loss of also the other subunit (Amatruda et al., 1992). This implies that twinfilin requires the presence of intact capping protein, Cap1/2p, to localize to the cortical actin filament structures in yeast cells. Twinfilin and capping protein also interact with each other in native gel assays, suggesting that a direct interaction between these two proteins may promote the localization of twinfilin to cortical actin cytoskeleton. However, the interaction between twinfilin and Cap1/2p can be also detected in the absence of actin monomers in vitro, whereas a mutation (Twf1-3p) that disrupts the actin-binding site of twinfilin no longer localizes at the cortical actin patches in vivo. It is possible that Twf1-3p mutation also has some other, currently unidentified, defects that prevent its correct localization in cells. Alternatively, interaction with an actin monomer may increase the affinity of twinfilin for capping protein, and may therefore be essential for the correct localization of twinfilin in vivo. Because there are currently no quantitative methods available for determining the affinities of twinfilin for capping protein in presence and absence of actin, we cannot distinguish between these two alternatives.
The activity of several actin-binding proteins is regulated by PIPs in vitro. As shown in Fig. 8, A and B, yeast twinfilin binds PI(4,5)P2 and this interaction downregulates its actin monomersequestering activity. Therefore, the interaction with PIP2 may serve as a mechanism to prevent twinfilin from sequestering actin monomers at the regions of rapid actin filament nucleation and assembly in cells.
Fig. 9 shows a hypothetical model for the function of twinfilin in yeast cells. ADP-actin monomers dissociate from the minus end of the filament either spontaneously or by a cofilin/ADF-stimulated mechanism. Because twinfilin and cofilin interact with actin monomers through overlapping interfaces (Fig. 4), we speculate that twinfilin can sequester the actin monomer from cofilin. Twinfilin inhibits the spontaneous nucleotide exchange on the actin monomer (Goode et al., 1998). Therefore, it is probably able to keep the actin monomer in ADP form and prevent the assembly of this monomer into a filament. The function of twinfilin may be to transport actin monomers, in their inactive ADP form, to cortical actin patches. The possible function of twinfilin as an actin monomerlocalizing protein is supported by the synthetic lethality between twf1-null mutation and specific cofilin (cof1-22) and profilin (pfy1-4) mutations. In cof1-22 cells, the actin monomer pool is depleted due to defects in actin filament depolymerization (Lappalainen and Drubin, 1997). In pfy1-4 cells, the exchange of actin nucleotide form ADP to ATP is defective due to mutations in profilin (Wolven et al., 2000). In combination with a possible defect in actin monomer localization in twf1cells, either one of these mutations would be expected to result in a dramatic decrease in the amount of ATP-actin monomers at the sites of rapid actin filament assembly in cells.
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In conclusion, yeast twinfilin is an abundant ADP-actin monomerbinding protein that localizes to the cortical actin cytoskeleton. The localization of twinfilin at the cortical actin filament structures appears to be dependent on the interaction with an actin monomer. Twinfilin also interacts with Cap1/2p, and the presence of intact capping protein is required for its localization to the cortical actin cytoskeleton. Therefore, twinfilin may function as a protein that links the actin filament depolymerization to filament assembly by localizing actin monomers to the sites of rapid actin filament assembly.
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Materials and methods |
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Protein expression and purification
Wild-type and mutant yeast twinfilins were expressed and purified as described (Goode et al., 1998). Actin was purified from yeast cell extracts by DNaseI affinity chromatography as described by Rodal et al. (1999). Yeast cofilin and mouse twinfilin were purified as described by Lappalainen et al. (1997) and Vartiainen et al. (2000), respectively. Yeast Cap1/2p protein was expressed and purified as described by Amatruda et al. (1992). Skeletal muscle actin was purified from chicken pectoral muscle as described by Spudich and Watt (1971), and pyrene actin was prepared as described by Cooper et al. (1983).
The plasmid for the expression of mouse capping protein 1 and ß2 subunits was constructed in a pET3d vector (pET3d[m-
1/ß2]) using the strategy described by Soeno et al. (1998) for chicken capping protein (
1ß1). Mouse
1ß2 capping protein was expressed and purified from BL21(DE3) Escherichia coli. One liter of LB media containing carbenicillin (50 µg/ml-1) was grown shaking at 37°C until the A600 nm was between 0.6 and 1.0. Expression was induced by the addition of IPTG to 1 mM and by growth for 3 h. Cells were harvested by centrifugation (3000 g for 15 min), resuspended, and washed once in 100 ml of 40 mM TrisCl, pH 8.0, 40 mM EDTA, and 140 mM NaCl. The cell pellet was resuspended in 100 ml of ice-cold 50 mM Tris, pH 8.0, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF, and sonicated on ice for 12 x 10-s bursts. The cell lysate was subjected to centrifugation in a Beckman Coulter Ti45 rotor at 35,000 rpm for 1 h at 4°C. A 5070% ammonium sulphate fraction was obtained from the high-speed supernatant, and the precipitate obtained by centrifugation at 32,000 g for 20 min at 4°C. The pellet was resuspended in 40 ml of ice-cold HA buffer (10 mM KH2PO4, pH 7.0, 500 mM KCl, 1 mM DTT, 1 mM PMSF, 0.01% NaN3), dialyzed overnight against HA buffer, and applied to a hydroxyapatite column (2.5 x 10 cm) equilibrated in HA buffer. Proteins were eluted with a 10250-mM KH2PO4 gradient in HA buffer and analyzed by SDS-PAGE. Fractions containing capping protein were pooled and dialyzed against Q buffer (10 mM TrisCl, pH 8.0, 10 mM KCl, 0.5 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.01% NaN3). The dialyzed sample was loaded onto a Mono Q column (2.6 x 10 cm) equilibrated in Q buffer. Proteins were eluted with a 10400-mM KCl gradient in Q buffer. Fractions containing capping protein were pooled and dialyzed against SA buffer (10 mM MES, pH 6.0, 0.5 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.01% NaN3) followed by dialysis for 1.5 h against SB buffer (10 mM MES, pH 5.8, 0.5 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.01% NaN3). The sample was loaded onto a Mono-S column (2.6 x 10 cm) equilibrated in SB buffer. The column was eluted with a 0350-mM NaCl gradient in SB buffer, and the fractions containing capping protein were pooled, concentrated, and dialyzed against 10 mM TrisCl, pH 8.0, 40 mM KCl, 0.5 mM DTT, and 50% glycerol for storage at 70°C.
Native gel electrophoresis assays
Native PAGE for studying the twinfilinactin monomer interaction was performed as described by Safer (1989) and as modified by Maciver and Weeds (1994). ADP-actin was prepared by incubating ATP-actin with agarose-linked yeast hexokinase (Sigma-Aldrich) for 3 h at 4°C in the presence of 1 mM glucose and 0.2 mM EGTA (Pollard, 1986). Native PAGE to study proteinlipid interactions was performed as described by Gungabissoon et al. (1998). The tissue-extracted lipids with heterogeneous fatty acid composition were purchased from Sigma-Aldrich, with the exception of PI(3,4)P2, PI(3,4,5)P3, polar lipid mix, cardiolipin, and phosphatidic acid that were from Matreya, Inc.
TwinfilinCap1/2p interaction was studied on 10% native polyacrylamide gels. Purified yeast and mouse capping protein and twinfilin (either alone or in a mixture with each other), was diluted to the desired concentrations in 10 mM Tris, pH 7.5, 50 mM NaCl, and 0.5 mM DTT, and incubated for 60 min at room temperature. A 20-µl aliquot was then mixed at a ratio of 4:1 with loading buffer (125 mM Tris, pH 9.0, 250 mM NaCl, 2.5 mM DTT, 50% glycerol) and then loaded onto the gel. The gel was run at 120 V for 100 min with native running buffer (25 mM Tris, 194 mM glycine, pH 9.0, 0.5 mM DTT). For analysis in a second dimension with SDS-PAGE, an entire lane from the first dimension native gel was excised using a razor blade. The edges of the lane were trimmed to remove the "smile" section of the gel. The gel piece was then placed horizontally 5 mm from the top of a clean glass plate. The gel lane was then incubated in
23 ml of SDS-PAGE running buffer and
1 ml of 10% SDS for 15 min at room temperature. The excess liquid was carefully removed, the gel piece clamped between two glass plates, and a 12% (wt/vol) SDSpolyacrylamide separating gel carefully poured underneath. After this had set, a 4% (wt/vol) SDSpolyacrylamide stacking gel was poured around the gel piece, completely covering it. Protein standard lanes were also placed in the stacking gel.
Actin filament sedimentation and depolymerization assays
Actin filament sedimentation assays were carried out as described by Goode et al. (1998). The concentration of actin was constant (4 µM) and twinfilin was used in final concentrations of 0, 2, 4, and 8 µM. Kinetics of actin filament disassembly was monitored by pyrene fluorescence with excitation at 365 nm and emission at 407 nm. 6 µM actin (5:1, yeast actin pyrene-labeled rabbit skeletal muscle actin; Cytoskeleton, Inc.) was polymerized in F buffer for 30 min. Disassembly of F-actin was induced by mixing 40 µl of F-actin with 10 µl of 25-µM yeast twinfilin, and monitored by the decrease in fluorescence at 407 nm for 10 min in Hitachi F-4010 fluorescence spectrophotometer. Twinfilin was pre-incubated for 5 min with PI(4,5)P2 before mixing with F-actin. The final concentrations of PI(4,5)P2 in this assay were 0, 25, and 50 µM.
Immunofluorescence microscopy
Cells were grown in appropriate medium at 30°C to an optical density of 0.5 at 600 nm, and prepared for immunofluorescence as described by Ayscough and Drubin (1998). The antibody against yeast twinfilin was raised by immunizing rabbits with the purified recombinant first ADF-H domain (residues 1162). The antibodies were then affinity purified from the rabbit antiserum with the same protein. The anti-actin antiserum was generated by immunizing guinea pigs with purified yeast actin. The guinea pig antiyeast actin serum was used at a dilution of 1:1,000, and the rabbit antiyeast twinfilin antibody was used at a dilution of 1:50. Latrunculin-A (Molecular Probes) was used at a final concentration of 500 µM for 20 min at 37oC.
Western blotting and coimmunoprecipitation
Cells were grown to confluence overnight in YEPD medium (1% [wt/vol] Bacto yeast extract, 2% Bacto peptone, 2% glucose) at 30oC, diluted (1:10), and allowed to grow 3 h, after which cells from a 3-ml cell culture were spun down and resuspended in 100 µl 20 mM Tris-HCl, pH 7.5, 0.6 mM PMSF, and protease-inhibitor cocktail (1:1,000) (500 µg each of antipain, leupeptin, pepstatin, chymostatin, and aprotin per ml; Sigma-Aldrich). Cells were lysed by adding glass beads (1:1) and vortexing for 5 min at room temperature. Western blotting was carried out as described by Vartiainen et al. (2000) with the following primary antibodies: rabbit antiyeast actin (1:500), cofilin (1:500), or twinfilin (1:1,000). Coimmunoprecipitation experiment using 108 cells of DDY1102, DDY1436, YJC0388, and YJC0391 yeast strains was carried out as described (Paunola et al., 1998). The primary antibodies (rabbit anti-Twf1p and guinea pig anti-Cap2p) were covalently coupled to protein A-Sepharose beads. Twinfilin and capping protein were visualized from Western blots with rabbit anti-Twf1p (1:1,000) and guinea pig anti-Cap2p (1:10,000) antibodies.
CD spectroscopy
CD measurements were recorded with a Jasco J-700 spectropolarimeter equipped with a microcomputer and a Jasco PTC-348WI thermostat. Spectra were collected with a scan speed of 50 nm/min, step resolution of 0.2 nm, bandwidth of 2.0 nm, sensitivity of 20 millidegrees, and with a response time of 1 s. Each spectrum was the average of at least 20 scans. Far UV CD spectra were recorded at a protein concentration of 3 µM in 2 mM NaPO4, pH 7.4, and 25 mM UV-free NaCl (Sigma-Aldrich) with a 2-mm pathlength optical cell. For temperature transition studies, six scans at the desired temperature were recorded after an incubation time of 4 min in a given temperature, and the distortion of helixes (Yang et al., 1986) was plotted at 222 nm.
Miscellaneous
PAGE was carried out by using the buffer system described by Laemmli (1970). Protein concentrations were determined with Hewlett Packard 8452A Diode Array Spectrophotometer by using calculated extinction coefficients for yeast twinfilin (at 280 nm = 11.6 mM-1 cm-1), yeast actin (at 290 nm -320 nm
= 26.6 mM-1 cm-1), cofilin (at 280 nm
= 14.7 mM-1cm-1), and mouse capping protein (
1ß2) (at 280 nm
= 76.3 mM-1 cm-1). The concentration of yeast Cap1/2p was quantified from Coomassie bluestained SDS gels compared with purified yeast twinfilin and actin. Protein distributions in SDS-PAGE gels were quantified by Fluor-STM MultiImager with Quantity One (v. 4.1.0; Bio-Rad Laboratories) or TINA (v. 2.09c) software.
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Footnotes |
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Acknowledgments |
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This study was supported by Academy of Finland and Biocentrum Helsinki (to P. Lappalainen). P.J. Ojala was supported by a fellowship from the Viikki Graduate School in Biosciences.
Submitted: 29 June 2001
Revised: 30 August 2001
Accepted: 5 September 2001
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