Identification of a Novel Actin-binding Protein in Yeast
In a search of the yeast Saccharomyces cerevisiae
genome database for proteins with sequence homology to the actin filament depolymerizing protein cofilin, we discovered a previously uncharacterized open reading frame (YGR080W) encoding a novel cofilin-like protein. This gene, predicted to encode a 332–amino acid protein, is composed of not one, but two cofilin-like sequences with 21% and 19% amino acid sequence identity to yeast cofilin (Lappalainen et al., 1998
). These cofilin-like repeats also show ~15% sequence identity to each other, and therefore we named this protein twinfilin (TWF1
). The protein encoded by TWF1
also shows homology to the human and mouse A6 proteins, previously identified by a screen of an embryonic cDNA expression library using a anti-phospho-tyrosine antibody (Beeler et al.,
1994) and to a sequence in the C. elegans
genome (these data are available from GenBank/EMBL/ DDBJ under accession number U46668
). Biochemical analyses in this earlier report suggested that human A6 protein might be a protein tyrosine kinase. However, A6 protein lacks any sequence homology to known protein kinases. Furthermore, we did not detect any kinase activity for purified yeast twinfilin using identical substrates and conditions to those described by Beeler et al. (1994; data not shown; see Materials and Methods).
A sequence alignment of yeast cofilin and twinfilin repeats 1 and 2 from yeast, human, and mouse (Fig. ) shows that the positions of cofilin secondary structure elements identified from the yeast cofilin crystal structure (Fedorov et al.,
1997) are relatively well conserved between cofilin and twinfilins. Furthermore, the sequence insertions in the twinfilins are located in regions predicted to form loops, suggesting that each twinfilin repeat has a tertiary structure similar to the ADF/cofilin proteins. Each twinfilin repeat also has an ~20–amino acid extension at its COOH-terminal region not found in any of the known ADF/cofilin proteins (Lappalainen et al., 1998
Figure 1 Sequence alignment of yeast cofilin and repeats 1 and 2 of yeast, mouse and human twinfilins. The secondary structure elements identified from the yeast cofilin crystal structure are shown above the sequences. The twinfilin sequences that are either (more ...)
The residues of cofilin that have been shown to be essential for interactions with actin monomers and actin filaments (indicated by asterisks above the sequences in Fig. ) also are relatively well conserved in each repeat of twinfilins (see Fig. ). However, the residues in yeast cofilin that have been shown to be essential for binding to actin filaments are less conserved in twinfilins than residues implicated in monomer binding. The overall structural conservation as well as the conservation of the actin-binding residues between cofilin and twinfilins suggests that twinfilins might bind directly to actin, and that their interactions with actin may be similar to ADF/cofilin proteins. In support of the hypothesis that twinfilin is an actin-binding protein, we also have identified twinfilin as a protein enriched from yeast extracts on an actin affinity column (Goode, B.L., D. Shieltz, J. Yates, and D.G. Drubin, unpublished observations).
Twinfilin Sequesters Actin by Forming a 1:1 Complex with Actin Monomers
To test directly whether twinfilin binds to actin filaments and/or actin monomers, we expressed full-length twinfilin in E. coli as a glutathione-S-transferase fusion protein. The twinfilin GST–fusion proteins were purified from E.coli extracts using glutathione-agarose beads. Twinfilin was subsequently cleaved from GST by digestion with thrombin. Purification was finalized by gel-filtration chromatography on a Superdex-75 column. The majority (70– 80%) of the full-length twinfilin eluted from the column as a single peak at the expected position for a monomer (at ~ 54 ml). However, a small fraction of twinfilin (20–25%) eluted in the void volume, suggesting some aggregation under these conditions. After freezing and thawing of the monomeric fraction of twinfilin, we again observed that 20–25% of the twinfilin sedimented on its own upon ultracentrifugation for 20 min at 90,000 rpm in a TLA-100 rotor (Fig. , lane 2). In the pelleting assays described below, the presence of the insoluble twinfilin fraction that pellets on its own is subtracted from the results. In monomer binding gel shift assays (see Fig. ), the insoluble twinfilin fraction appears to not enter the gel and therefore should not effect on the results.
Figure 4 Analysis of twinfilin–actin monomer complexes on native gels. Lane 1, actin alone; lane 2, twinfilin; lane 3, actin + twinfilin; lane 4, repeat-1; and lane 5, repeat-1 + actin. Each protein was mixed in solution and then loaded (more ...)
To study the interactions of the purified twinfilin with actin filaments, we first carried out actin filament cosedimentation assays using a constant concentration (2 μM) of twinfilin and variable concentrations (0–8 μM) of purified yeast F-actin. As shown in Fig. , addition of twinfilin to the actin filaments leads to a significant increase in the amount of actin present in the supernatant. Whereas ~90% of the actin is normally found in the pellet fraction under these conditions, addition of equimolar amounts of twinfilin to F-actin decreases the amount of actin in the pellet to ~25% (compare Fig. , lane 1 with 3). At higher actin concentrations twinfilin appears to shift actin to the supernatant in an ~1:1 molar ratio (i.e., for every molecule of twinfilin added to the reaction, one molecule of actin shifts to the supernatant). Several different mechanisms of action by twinfilin could underlie these observations, including actin monomer sequestration and/or capping of the barbed end of the filaments to prevent new subunit addition; however, the 1:1 stoichiometry of actin and twinfilin in the supernatant strongly suggests monomer sequestration. Fig. also shows that only a small increase in the amount of twinfilin in the pellet occurs upon addition of increasing concentrations of F-actin, suggesting that twinfilin does not bind tightly to actin filaments.
To further examine the ability of twinfilin to depolymerize actin filaments, we carried out a cosedimentation assay using a constant (2 μM) concentration of actin and variable twinfilin concentrations (0 and 6 μM). As shown in Fig. , a and c, addition of twinfilin causes an equimolar amount of actin to redistribute from the pellet fraction to the supernatant. Because ~25% of twinfilin pellets on its own, the addition of 2 μM twinfilin to actin filaments can shift a maximum of 1.5 μM actin to the supernatant. The observation that twinfilin–actin monomer binding saturates at 2 μM twinfilin and 2 μM actin (see Fig. ) strongly suggests that twinfilin forms a tight 1:1 complex with actin monomers.
To further test the ability of twinfilin to form a complex with actin monomers, we used native gel electrophoresis. Reactions containing a final concentration of 15 μM twinfilin and 15 μM yeast actin in G-buffer (see Materials and Methods) were fractionated on a 7.5% native polyacrylamide gel run at 100 V for 120 min. The motilities of yeast actin and recombinant twinfilin alone are shown in Fig. (lanes 1
, respectively). Lane 3
in Fig. shows that addition of twinfilin to actin at a 1:1 molar ratio causes in a dramatic shift in the motility of actin, resulting in the formation of a smear between the twinfilin and actin bands. These results suggest that the complex that forms between twinfilin and actin is relatively stable in vitro. As an independent test of monomer binding, we also investigated whether addition of twinfilin could inhibit nucleotide exchange that normally occurs for actin monomers in solution. Inhibition of nucleotide exchange has been demonstrated previously for ADF/cofilin proteins (Nishida, 1985
). As shown in Fig. , twinfilin inhibits nucleotide exchange of ATP-actin monomers in a concentration-dependent manner similar to that described for cofilin (Nishida, 1985
). Taken together, these data (the shift in F-actin steady state towards monomer in a 1:1 molar ratio, the detection of twinfilin/actin monomer complexes on native gels, and the inhibition of nucleotide exchange by actin when twinfilin is bound) demonstrate that twinfilin functions in vitro like a bona fide actin monomer-sequestering protein.
Figure 5 Effects of twinfilin and repeat-1 on the nucleotide exchange of yeast actin monomers. The reaction-rates are indicated on the y axis as the inverse of the reaction half-life (t1/2). Both full-length twinfilin and repeat-1 inhibit the nucleotide exchange (more ...)
ADF/cofilin proteins have been shown to promote the rapid depolymerization of actin filaments from their pointed ends (Carlier et al.,
1997). Because twinfilin has sequence homology to ADF/cofilin proteins, we tested whether it also effects actin filament depolymerization kinetics in a similar manner to ADF/cofilin proteins. To specifically measure the filament depolymerization from the pointed end, actin filaments capped at their barbed ends were prepared by polymerizing 6 μM yeast actin in the presence of 5 nM gelsolin as described by Carlier et al. (1997)
. Depolymerization was then induced by the addition of twinfilin, cofilin or the actin monomer-sequestering drug latrunculin-A. As shown in Fig. , twinfilin and latrunculin-A have similar effects in this assay, consistent with the conclusion that twinfilin sequesters actin monomers without stimulating filament depolymerization. In contrast, cofilin stimulates rapid filament depolymerization. Because the stoichiometric cofilin-induced depolymerization was too rapid to monitor using our experimental system (Fig. D
), we also performed this assay using substoichiometric cofilin and a sufficient concentration of latrunculin A to sequester depolymerized actin monomers (Fig. C
). These conditions allowed detection of the cofilin-induced rapid actin filament depolymerization. It is important to note that the rapid decrease in signal may be in part due to fluorescence quenching of the pyrene actin upon cofilin binding (Carlier et al., 1997
). However, it is clear from our results that, despite its sequence homology with ADF/cofilin proteins, twinfilin has little or no effect on filament depolymerization rate at the pointed end. One potential caveat of these experiments is that the recombinant twinfilin used in these assays could be improperly folded. However, the recombinant twinfilin eluted as a single peak on a gel filtration column and showed strong activities on actin, suggesting that these preparations are homogeneous and active. On the other hand, these results do not rule out the possibility that native yeast twinfilin might have additional activities not detected here for the recombinant protein.
Figure 6 Depolymerization assay using yeast actin filaments capped at their barbed-ends with gelsolin. Actin filaments (6 μM, 1:5 pyrene rabbit actin/yeast actin) were polymerized in the presence of 5 nM gelsolin. Depolymerization was induced by mixing (more ...)
Both Cofilin-like Repeats in Twinfilin Are Required for Strong Actin Monomer Binding
Because twinfilin is composed of two ADF homology domains (repeats 1 and 2), we investigated whether individual repeat domains might have activities similar to those of full-length twinfilin. Each repeat was expressed in E. coli as a GST–fusion protein and purified as described above for full-length twinfilin. As shown in Table , both repeats were expressed at high levels in E. coli, but only repeat-1 was soluble after cleavage from GST with thrombin. All of the repeat-2 eluted in void volume from the Superdex-75 gel-filtration column after cleavage from glutathione- S-transferase and sedimented on its own upon ultracentrifugation for 20 min at 90,000 rpm in TLA-100 rotor. Therefore, only repeat-1 was used for the assays described below.
Bacterial Expression and Solubility of Yeast Twinfilin and Its Two Cofilin Homology Domains Expressed Individually
In cosedimentation assays using a range of F-actin concentrations (0–8 μM) and a constant repeat-1 concentration (2 μM), repeat-1 did not exhibit copelleting with actin filaments (data not shown). However, repeat-1 was able to increase the amount of actin in the supernatant in a concentration-dependent manner (Fig. , b and c). This activity was significantly weaker than that observed for full-length twinfilin, which suggests that both repeats are necessary for strong actin monomer sequestering. We also tested directly the interaction of repeat-1 with actin monomers by native gel electrophoresis and by the inhibition of nucleotide exchange on actin monomers. As shown in Fig. (lanes 4 and 5) addition of repeat-1 to G-actin results in the formation of a weak smear between the repeat-1 and the actin bands, suggesting that these two proteins form a low affinity molecular complex with each other. As shown in Fig. , Repeat-1 also inhibits nucleotide exchange by actin monomers in a concentration dependent manner. Together, these results suggest that repeat-1 forms a complex with actin monomers similar in nature, but weaker in strength to the one formed by cofilin and full-length twinfilin.
A GFP–Twinfilin Fusion Protein Localizes to the Cytoplasm and the Cortical Actin Cytoskeleton in Yeast Cells
To investigate the localization of twinfilin protein in yeast, a GFP–twinfilin fusion protein was expressed and its localization observed in living cells. As shown in Fig. a
, the majority of cells expressing the GFP–twinfilin fusion protein showed strong cytoplasmic staining with additional cortical punctate staining. The cortical spot structures moved in real time, with some of the patches holding a stable position and others dramatically translocating over the span of seconds (data not shown). Since these movements are very similar to those of cortical actin patches described in previous reports (Doyle and Botstein, 1996
; Waddle et al.,
1996), we used double immunofluorescence with anti-actin and anti-GFP antibodies to address whether the GFP–twinfilin patches correspond to actin patches. In the majority of cells examined, the anti-GFP staining localized primarily to the cytoplasm, but many cells also showed patch-like staining. Examples of cells with clear patch staining are shown in Fig. b
. In these cells, the GFP-staining patches overlapped with a subset of the cortical actin patches. Taken together, these results suggest that twinfilin localizes primarily to the cytoplasm, but also to the cortical actin cytoskeleton. However, it is important to remember that this localization was carried out in cell overexpressing GFP–twinfilin fusion protein and may therefore not fully represent the localization of twinfilin in wild-type cells. While the cytoplasmic localization of twinfilin is consistent with its activities as an actin monomer-sequestering protein, the patch-like staining raises intriguing possibilities about the regulation of twinfilin function. One possibility is that a fraction of twinfilin is associated with cortical actin patches through binding interactions with patch components other than actin. This also could explain the above mentioned isolation of twinfilin from yeast extracts on actin filament affinity columns.
Figure 7 Localization of GFP–twinfilin fusion protein in yeast cells. DDY759 cells were transformed with a plasmid encoding a GFP–twinfilin fusion protein under regulation of the galactose promoter. To induce expression of the GFP fusion protein, (more ...)
Deletion of the TWF1 Gene Results in Synthetic Lethality with a Cofilin Mutant
To investigate the in vivo functions of twinfilin, we generated a strain in which the TWF1 gene is deleted and replaced by the URA3 gene. Haploid twf1Δ cells exhibit normal growth and have normal morphologies over a temperature range of 20–37°C (data not shown). The growth of twf1Δ cells also was indistinguishable from wild-type cells on a variety of stressful media, including media produced with low and high pH, high NaCl, KCl, MgCl2, CaCl2, and formamide (data not shown). Furthermore, twf1Δ cells have no detectable defects in fluid phase endocytosis (data not shown; see Materials and Methods) and their actin cytoskeletons appear normal by immunofluorescence except for consistently brighter actin patch staining (Fig. ).
Figure 8 Organization of the actin cytoskeleton in wild-type (A), twf1Δ (B), cof1-22 (C) and twf1Δ × cof1-22 (D) cells. The cells were grown at 20°C to an OD600 of ~0.3, fixed with formaldehyde, and then the actin was (more ...)
The absence of a strong detectable phenotype in twf1Δ
cells suggests that there may be functional redundancy between twinfilin and other proteins in yeast. Because the actin cytoskeleton is characterized by a high complexity of protein components, and by many examples of genetic redundancy, gene disruption of one actin binding protein often has no significant effects on growth rate of cells or the overall appearance of the actin cytoskeleton by immunofluorescence. However, such gene disruptions can lead to strong synergistic defects in combination with other mutations in genes that encode actin-binding proteins (e.g., Holtzman et al.,
1993). To test the possibility that functional redundancy explains the lack of pronounced defects in twf1Δ
mutants, we crossed twf1Δ
mutants with mutants of other genes encoding actin binding proteins, concentrating on genes that encode proteins with ADF-homology domains (COF1
; Lappalainen et al., 1998
) and on genes that encode known actin monomer binding proteins (profilin/PFY1
, and SRV2
). As shown in Table , twf1Δ
demonstrates a strong and specific synthetic phenotype with the cofilin allele cof1-22
. This cofilin mutant has been shown to have significant defects in F-actin binding and depolymerization both in vivo and in vitro, and it results in lethality at the temperatures >30°C. However, at 20°C cof1-22
cells show normal morphology and exhibit growth rates similar to wild-type cells (Lappalainen and Drubin, 1997
). After 3 d at 20°C, none of the twf1Δ cof1-22
double mutants formed visible colonies. However, after prolonged incubation (5–7 d at 20°C), tiny twf1Δ cof1-22
colonies appeared. The cells in these colonies were abnormally large. To visualize the actin cytoskeletons in such twf1Δ cof1-22
cells, the segregants were inoculated into a small volume of YPD and grown at 20°C for 48 h. Fig. shows a comparison of the morphologies of the actin cytoskeletons in wild-type, twf1Δ
, and twf1Δ cof1-22
double mutant cells grown at 20°C. Whereas twf1Δ
cells show some increase in the brightness (= size) of the cortical actin structures compared with wild-type cells, most twf1Δ cof1-22
double mutant cells have completely depolarized cortical actin cytoskeletons and abnormally large and chunky actin patches. These results suggest that TWF1
genes may share a function required for the regulation of actin-based processes.
Genetic Interactions between Δtwf1 and Actin-binding Protein Mutants
Deletion of the TWF1 Gene Causes Random Budding Pattern and Bumpy Surface Morphology in Diploid Yeast Cells
We also examined the morphology of diploid yeast cells homozygous for the twf1Δ
gene deletion (DDY1436). Fig. a
shows that twf1Δ/twf1Δ
cells appear to form normal buds, but the cells have large bumps on their surfaces. Similar phenotypes have been reported previously for a subset of actin alleles that have defects in bipolar bud patterning (Drubin et al.,
1993; Yang et al.,
1997). Calcofluor staining of the twf1Δ/twf1Δ
cells revealed that each bump is marked by a bud scar, suggesting that the bumps represent sites of past bud formation and cytokinesis. In normal diploid yeast, the first bud to emerge from a daughter cell is usually formed at the pole opposite to the birth scar, and subsequent buds form at sites that are either at the same pole as the birth scar or the opposite pole (Chant and Pringle, 1995
). In wild-type cells, this leads to a bipolar budding pattern (the accumulation of multiple bud scars positioned at either pole). It has been shown that disruption of the actin cytoskeleton does not affect the position of the first bud to emerge from the daughter cell, but subsequently results in a random budding pattern in diploid cells (Yang et al.,
1997). Diploid-specific bud pattern defects also have been observed in actin-binding protein mutants, including sla2Δ, rvs167Δ
, and sac6Δ
(Drubin et al.,
1993; Yang et al.,
1997). We found that 56% of twf1Δ/ twf1Δ
cells exhibit random bud scar patterning (examples are shown in Fig. b
), compared with only 2% of wild-type cells. Such frequencies of random budding are similar to those reported previously for actin and actin-binding protein mutants, and support the model that TWF1
is involved in actin cytoskeletal functions in vivo.
Figure 9 Budding pattern defects and bumpy surface abnormalities in twf1Δ/twf1Δ cells. (A) DIC imaging of living twf1Δ/twf1Δ cells reveals abnormal bumpy surface projections not found in wild-type diploid cells. (B) Calcofluor (more ...)
Overexpression of Twinfilin Causes Depolarization of the Cortical Actin Cytoskeleton
Finally, we examined the effects of overexpressing twinfilin in yeast cells. One might predict that increased levels of an actin monomer-sequestering protein would lead to reduced polymer levels, and the build up of a larger pool of sequestered actin monomers in the cytoplasm. Recently it has been shown that the overexpression of previously identified actin monomer-binding proteins in yeast have different effects on the actin cytoskeleton depending on the actin-binding protein (Hofmann, C., and D.G.Drubin, unpublished results). While overexpression of Srv2p has no detectable effects, overexpression of profilin and cofilin both lead to a partial depolarization of the cortical actin cytoskeleton and the formation of cytoplasmic actin bars (aberrant structures that are not likely to be composed of filamentous actin since they do not stain with rhodamine-phalloidin). As shown in Fig. , overexpression of twinfilin leads to a complete depolarization of the actin cytoskeleton and the accumulation of cytoplasmic actin bars. Both of these effects support the conclusion that twinfilin functions as an actin monomer-sequestering protein in vivo.
Figure 10 Effects of overexpression of twinfilin in yeast cells. Wild-type diploid cells (DDY759) were transformed with either a control plasmid (pRB1438) or a plasmid carrying the TWF1 gene under the regulation of the galactose promoter (pGAL-TWF1). To induce (more ...)