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(1998) 16, 121 ± 130  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00

Isolation and characterization of a novel ®lament-binding from

Takeshi Asakura1, Takuya Sasaki1, Fumiko Nagano1, Ayako Satoh2, Hiroshi Obaishi2, Hideo Nishioka2, Hiroshi Imamura1, Kazuhiko Hotta1, Kazuma Tanaka1, Hiroyuki Nakanishi2 and Yoshimi Takai1,2

1Department of Molecular and , Osaka University Medical School, Suita 565, and 2Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-22, Japan

We puri®ed a novel actin ®lament (F-actin)-binding downstream target molecules have been isolated, but protein from the soluble fraction of Saccharomyces the modes of and action of the Rho family cerevisiae by successive column chromatographies by members have not been fully understood. use of the 125I-labeled F-actin overlay method. The In the Saccharomyces cerevisiae, the actin puri®ed protein showed a minimum Mr of about 140 kDa is involved in the establishment and on SDS-polyacrylamide gel and we maintenance of polarized and growth named it ABP140. A search with the partial amino (Madden et al., 1992; Welch et al., 1994). Yeast sequences of ABP140 against the Saccharomyces possesses the homologs of mammalian Rho family, Database revealed that the including RHO1, RHO2 (Madaule et al., 1987), RHO3, of the ABP140 (ABP140) corresponded to and RHO4 (Matsui and Toh-e, 1992). RHO1 is a YOR239W fused with YOR240W by the +1 transla- counterpart of the mammalian RhoA gene and we have tional frame shift. The encoded protein consisted of 628 shown that the rho1 mutants are de®cient in the amino with a calculated Mr of 71,484. The budding process (Yamochi et al., 1994). We have recombinant protein interacted with F-actin and showed recently identi®ed the downstream targets of Rho1p to the activity to crosslink F-actin into a bundle. Indirect be Pkc1p, 1,3-b-glucan synthase, and Bni1p (Nonaka et immuno¯uorescence study demonstrated that ABP140 al., 1995; Drgonova et al., 1996; Kohno et al., 1996). was colocalized with both cortical actin patches and Pkc1p is a homolog of mammalian protein C cytoplasmic actin cables in intact cells. However, and regulates integrity through activation of elimination of ABP140 by gene disruption did not show the MAP kinase cascade (Levin and Errede, 1995). a deleterious e€ect on or a€ect the Glucan synthase is involved in cell wall synthesis organization of F-actin. These results indicate that (Shematek and Cabib, 1980). Bni1p is known to be ABP140 is not required for cell growth but may be implicated in and establishment of cell involved in the reorganization of F-actin in the budding polarity (Jansen et al., 1996; Zahner et al., 1996). We yeast. have moreover found that Bni1p directly interacts with pro®lin, an actin -binding protein, which Keywords: F-actin; F-actin-binding protein; yeast; accelerates the polymerization of actin (Imamura et ABP140 al., 1997). It is likely that Rho1p regulates the reorganization of the actin cytoskeleton through the Bni1p-pro®lin system in the budding yeast. In yeast, a single (ACT1) encodes the Introduction sole source of `conventional' actin (Gallwitz and Sures, 1980; Ng and Abelson, 1980). Yeast actin is about Reorganization of the actin cytoskeleton is essential for 90% identical to muscle actin at the cell shape change, cell , and regulation of cell- sequence level, and the biochemical properties of yeast to-cell and cell-to-matrix adhesion (Pollard and Cooper, actin are similar to those of rabbit muscle actin. The 1986; Gumbiner, 1996; Lau€enburger and Horwitz, product of this single gene in yeast assembles into two 1996). Many actin-binding and many factors di€erent : cortical patches and cytoplasmic which regulate reorganization of the actin cytoskeleton cables (Adams and Pringle, 1984). The distribution of have been identi®ed (Pollard and Cooper, 1986; both patches and cables changes dramatically over time Vandekerckhove, 1990; Gips et al., 1994). Recent during the cell (Kilmartin and Adams, 1984). studies have clari®ed that the Rho family members, During vegetative growth, an asymmetric distribution which belong to the Ras-related small of these structures is apparent, with the cortical patches superfamily, are a novel type of the factors which localizing to sites of cell surface growth and the cables regulate cell shape change, cell motility, and cell-to-cell often oriented toward these regions. Following entry adhesion through reorganization of the actin cytoske- into the from G0 or after , when the leton (Takaishi et al., 1997; for reviews, see Hall, 1994; cortical patches are delocalized over the entire cell Takai et al., 1995). Many upstream regulators and surface, the bud starts to grow from the cells. As the bud develops, most of the patches appear in the growing tip of the daughter cell, with the cables Correspondence: Y Takai appearing to be oriented along the long axis of the Received 5 July 1997; revised 8 August 1997; accepted 8 August 1997 mother cell. As the daughter cell matures, the patches A novel F-actin-binding protein TAsakuraet al 122 are localized to the septum, with the cables appearing to radiate from this region. These dynamic changes of Mr (kDa) the actin structures require the actin-binding proteins which interact directly with actin and regulate the 205 reorganization of the actin cytoskeleton (Madden et al., 1992; Welch et al., 1994). Several encoding actin-binding proteins have been identi®ed and characterized in yeast: these include MYO1 ( 120 I), MYO2 (myosin II), COF1 (co®lin), PFY1 (pro®lin), and TPM1 () (Madden et al., 1992; Welch et al., 1994). Genetic analyses have demonstrated that 85 the activities of these actin-binding proteins are important for proper actin in yeast. Specifi- cally, in the genes that actin-binding proteins often result in similar to those of actin mutations. We have recently found that the Rho subfamily members regulate the of ERM (/ 47 /) with the plasma membrane and thereby regulate the reorganization of the actin cytoskeleton in (Takaishi et al., 1995; Hirao 1 2 3 et al., 1996). In general, actin ®laments are associated Figure 1 Detection of an F-actin-binding protein by the 125I- with the plasma membrane through peripheral labeled F-actin blot overlay method. The homogenate, the cytosol membrane proteins which directly interact with actin fraction, and the membrane fraction of yeast cells (BJ5457) (50 mg ®lament (F-actin), such as ERM, , and each of protein) were subjected to SDS ± PAGE, followed by the (Luna and Hitt, 1992; Tsukita et al., 1993; Cowin and 125I-labeled F-actin blot overlay. The protein markers used were Burke, 1996). However, this type of F-actin-binding myosin (Mr=205 000), b-galactosidase (Mr=120 000), bovine (Mr=85 000), and (Mr=47 000). protein has not been identi®ed in yeast. Moreover, a Lane 1, the homogenate; lane 2, the cytosol fraction; lane 3, the computer search against Saccharomyces membrane fraction Genome Database has revealed that there is no counterpart of these F-actin-binding proteins in the budding yeast. We have therefore attempted here to 140 kDa which was identi®ed by protein with isolate F-actin-binding proteins which may serve as a (Figure 2C and D). linker of F-actin to the plasma membrane. We have isolated a novel actin-binding protein from Sacchar- and amino acid sequences of ABP140 omyces cerevisiae. We have named this protein

ABP140, because it has a minimum Mr of about The TSKgel phenyl-5PW RP sample of ABP140 (30 mg 140 kDa on SDS-polyacrylamide of protein) was subjected to SDS ± PAGE. The protein (PAGE). band corresponding to ABP140 was cut out from the gel and digested with lysyl endopeptidase, and the digested were separated by TSKgel ODS-80Ts reverse phase high-performance liquid column chroma- Results tography. Over 20 peptides were observed, and the amino acid sequences of the ®ve peptides (-10, Puri®cation of ABP140 from the yeast cytosol fraction -14, -15, -18, and -19) were determined. Furthermore, To identify novel F-actin-binding proteins from the the N-terminal amino acid sequence of ABP140 was budding yeast, we attempted to detect F-actin-binding determined. A search with these amino acid sequences proteins by use of the 125I-labeled F-actin blot overlay against the Saccharomyces Genome Database revealed method. The homogenate was prepared from a yeast that the N-terminal peptide, peptide-14, and peptide-15 strain BJ5457, which contained less , and were included in the amino acid sequence of a putative subjected to SDS ± PAGE, followed by the 125I-labeled open reading frame, YOR239w, in XV. F-actin blot overlay (Figure 1). An F-actin-binding On the contrary, peptide-10 and peptide-19 were

protein band with a Mr of about 140 kDa was included in YOR240w, which is the next open reading detected. This F-actin-binding protein band was frame downstream of YOR239w. The amino acid detected in the cytosol fraction, whereas no F-actin- sequence of peptide-18 consisted of the parts of the binding protein was detected in the membrane fraction. C-terminal region of YOR239w and the N-terminal This protein was named ABP140. region of YOR240w, suggesting that YOR239w and ABP140 was highly puri®ed from the cytosol YOR240w constitute the ABP140 gene (ABP140). We fraction by successive column chromatographies, determined DNA sequences encompassing the C- including Mono S HR 16/10, hydroxyapatite, and terminal region of YOR239w and the N-terminal TSKgel phenyl-5PW RP column chromatographies. In region of YOR240w and con®rmed the DNA each column , the 125I-labeled F-actin- sequence reported in the Saccharomyces Genome binding protein band appeared in a single peak (Figure Database to be correct. A +1 translational frame 2A ± C). On the ®nal TSKgel phenyl-5PW RP column shift has been reported in seven sequence of chromatography, the 125I-labeled F-actin-binding pro- the TYB gene of yeast Ty retrotransposons (Belcourt

tein band well coincided with a protein with a Mr of and Farabaugh, 1990). Surprisingly, YOR239w pos- A novel F-actin-binding protein TAsakuraet al 123

D F-Actin overlay

Coomassie brilliant blue staining 205

120 85

Mr (kDa) 41 42 43 44 45 46 47 48 49 50 51 52 Fraction number Figure 2 Elution pro®les of ABP140 on various column chromatographies. An aliquot of each fraction of Mono S column chromatography (20 ml), hydroxyapatite column chromatography (5 ml), or TSKgel phenyl-5PW column chromatography (5 ml) was subjected to SDS ± PAGE, followed by the 125I-labeled F-actin blot overlay. (A) Mono S column chromatography. (B) hydroxyapatite column chromatography. (C) TSKgel phenyl-5PW column chromatography. (*), the radioactivity; (- - -), 125 absorbance at 280 nm; (Ð), NaCl or KH2PO4/K2HPO4 concentration. (D) the protein staining and the I-labeled F-actin blot overlay of the phenyl-5PW column chromatography. An aliquot (5 ml) of each fraction was subjected to SDS ± PAGE, followed by the 125I-labeled F-actin blot overlay. Another aliquot (80 ml) of each fraction was subjected to SDS ± PAGE, followed by protein staining with Coomassie brilliant blue. The protein markers used were the same as those used in Figure 1. Arrows indicate the positions of ABP140 sessed exactly the same sequence, CTTAGGC, in expressed the HA-tagged protein in the yeast cells which CTT corresponded to the codon 277 for (BJ5457). The cytosol fraction was prepared from the . If the +1 translational frame shift would HA-tagged ABP140-overexpressing cells and subjected occur at the codon 277, YOR239W would be fused to SDS ± PAGE, followed by the 125I-labeled F-actin with YOR240W and the amino acid sequence of blot overlay. An F-actin-binding protein band was peptide-18 would perfectly match the predicted amino detected in the cytosol fraction (1 mg of protein), acid sequence of the resulting open reading frame. We although native ABP140 was not detected in the same concluded that this amino acid sequence is most amount of the cytosol fraction prepared from the wild- probable for ABP140 (Figure 3). The encoded protein type cells (Figure 4A). Recombinant HA-tagged consisted of 628 amino acids and showed a calculated ABP140 showed the mobility similar to that of native

Mr of 71 484, which was much lower than the observed ABP140 on SDS ± PAGE. Therefore, we concluded

Mr of about 140 kDa on SDS ± PAGE. This discre- that this DNA fragment encoded full-length ABP140. pancy might be due to an abnormal tertiary Recombinant HA-tagged ABP140 was puri®ed from of ABP140 or a dimer formation of ABP140 which is the cytosol fraction of the yeast cells overexpressing this resistant to a reducing reagent. protein by successive column chromatographies, includ- A computer homology search revealed that the ing Mono S HR10/10, hydroxyapatite, and Superdex 200 deduced amino acid sequence of expressed sequence HR10/30 column chromatographies. On the ®nal Super- tag from (accession number R63104) is 54% dex 200 HR10/30 column chromatography, ABP140 identical to a limited region (amino acid positions appeared in a single peak at a position with a Mr of about 529 ± 615) of the deduced amino acid sequence of 240 kDa (data not shown). Recombinant ABP140 was ABP140, suggesting that there may be a counterpart of highly puri®ed as estimated by SDS ± PAGE and showed ABP140 in mammal. the 125I-labeled F-actin-binding activity (Figure 4B). Therefore, this recombinant protein was used to study the properties of ABP140 as described. Recombinant ABP140 To con®rm whether the DNA fragment encoding full- Properties of ABP140 length ABP140, we isolated this DNA fragment from the total yeast genome by PCR, constructed the In addition to the 125I-labeled F-actin blot overlay, the with this DNA fragment, and binding of ABP140 to F-actin was examined by A novel F-actin-binding protein TAsakuraet al 124 cosedimentation with F-actin. When recombinant al., 1993) (data not shown). This result indicated that ABP140 was incubated with F-actin followed by ABP140 bound along the sides of F-actin. ultracentrifugation, ABP140 was recovered with F- The e€ect of recombinant ABP140 on the viscosity actin in the pellet (Figure 5). ABP140 bound to F-actin of F-actin was next examined by the falling-ball at a molar ratio of one ABP140 molecule to about 30 method. This e€ect was compared to that of a- actin molecules (data not shown). It was next examined , a well-characterized protein which shows the by a competition experiment whether ABP140 bound F-actin-cross-linking activity (Burridge and Feramisco, along the sides of F-actin or its ends. The binding of 1981). ABP140 increased the viscosity in time- and 125I-labeled F-actin to recombinant ABP140 was dose-dependent manners, but the viscosity became inhibited by an excessive amount of myosin subfrag- maximally about ®vefold (Figure 6A). In contrast, a- ment 1, a well-characterized protein which binds along actinin increased the viscosity, so that the steel ball the sides of F-actin (Rayment et al., 1993; Schroder et became lodged in the gel (data not shown). These

Figure 3 Nucleotide and deduced amino acid sequences of ABP140. The amino acid sequences determined from the TSKgel phenyl-5PW RP sample of ABP140 are indicated by solid underlines; numbers below these sequences correspond to the peptide numbers. Nucleotide sequence is referred to the data from the Saccharomyces Genome Database. Nucleotide sequences used for designing PCR primers are indicated by dashed underlines. The possible slip site for the +1 translational frame shift is indicated by bold letters A novel F-actin-binding protein TAsakuraet al 125 results suggest that ABP140 has weak F-actin-cross- intensity with actin polymerization (Cooper and linking activity. This activity of ABP140 was Pollard, 1982). ABP140 did not a€ect actin - con®rmed by transmission electron of ization (Figure 7). negatively stained specimens. ABP140 caused F-actin to associate into thin bundles (Figure 6B), under the Localization of ABP140 in yeast cells conditions where a-actinin caused F-actin to associate into thick bundles (data not shown). We ®nally To examine the function of ABP140, we established the examined the e€ect of recombinant ABP140 on actin ABP140-null mutant yeast cells by gene disruption. polymerization using pyrene-conjugated actin. Pyrene- The ABP140-null mutant cells were normal in conjugated actin is known to increase its ¯uorescent phenotypes so far tested, including growth at 248Cor at 378C, mating potency with the wild-type cells of an opposite mating type, and budding pattern (data not A B shown). To assess the localization of ABP140, the ABP140-null mutant cells expressing HA-tagged Mr (kDa) Mr (kDa) ABP140 were next prepared. These mutant cells were also morphologically indistinguishable from the wild- 201 201 type cells and used for the indirect immuno¯uorescence microscopy with the anti-HA monoclonal 120 120 (mAb). The same preparation was doubly stained with rhodamine-phalloidin to visualize F-actin. The staining 85 with the anti-HA mAb was observed in both cortical 85 actin patches (Figure 8) and actin cables (data not shown).

1 2 1 2 Discussion Figure 4 Expression of recombinant ABP140. (A) The 125I- labeled F-actin blot overlay of the cytosol fractions of the wild We have puri®ed here a novel F-actin-binding protein type cells and the HA-tagged ABP140-overexpressing cells. The from the cytosol fraction of S. cerevisiae and cytosol fractions of the wild type cells (50 mg of protein) and of characterized it. Because this novel F-actin-binding the HA-tagged ABP140-overexpressing cells (1 mg of protein) protein shows a minimum M of about 140 kDa on were subjected to SDS ± PAGE, followed by the 125I-labeled F- r actin blot overlay. Lane 1, the wild type cells; lane 2, the ABP140- SDS ± PAGE, we have named it ABP140. Many actin- overexpressing cells. (B) the protein staining and the 125I-labeled binding proteins in yeast show amino acid sequences F-actin blot overlay of puri®ed recombinant HA-tagged ABP140. similar to those of mammalian proteins. However, The puri®ed sample of recombinant HA-tagged ABP140 was ABP140 does not have any homologous regions to subjected to SDS ± PAGE, followed by protein staining with any known actin-binding proteins in mammal or Coomassie brilliant blue or the 125I-labeled F-actin blot overlay. Lane 1, protein staining; lane 2, the 125I-labeled F-actin blot yeast. Our biochemical study has revealed that overlay ABP140 directly interacts with F-actin and binds

ABP140 Mr (kDa) ABP140 + F-Actin

ABP140

120

66

45 Actin

1 2 1 2 Figure 5 Cosedimentation of ABP140 with F-actin. Recombinant HA-tagged ABP140 was incubated in the presence or absence of F-actin, and the mixture was then centrifuged. The supernatant and the precipitate were subjected to SDS ± PAGE (8 ± 13% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. Lane 1, the supernatant; lane 2, the precipitate A novel F-actin-binding protein TAsakuraet al 126 A

B

Figure 7 E€ect of ABP140 on actin polymerization. E€ect of recombinant HA-tagged ABP140 or on actin polymeriza- tion was examined using the pyrene-conjugated actin (*), with ABP140; (D), with gelsolin; (*), without ABP140 and gelsolin

Figure 6 F-Actin cross-linking activity of ABP140. (A) E€ect of actin and to associate F-actin into bundles than a- ABP140 on the viscosity of F-actin. (a) dose-dependent e€ect. G- Actin was mixed with the indicated amounts of recombinant HA- actinin. ABP140 may belong to a family of cross- tagged ABP140 and incubated for 45 min, followed by low shear linking proteins di€erent from those thus far reported. viscometry. (b) time-dependent e€ect. G-Actin was mixed with Actin-binding proteins are located at either cortical recombinant HA-tagged ABP140 (20 nM) and incubated for the actin patches or cytoplasmic actin cables, or both in indicated periods of time, followed by low shear viscometry. (*), yeast. Our immuno¯uorescence study has revealed with ABP140; (*), without ABP140. (B) e€ect of ABP140 on the arrangement of F-Actin. F-Actin was incubated in the presence or that ABP140 is colocalized with both the F-actin absence of recombinant HA-tagged ABP140 and observed by structures. This result has provided additional transmission electron microscopy. (a) F-actin alone; (b) F-actin evidence that ABP140 is an F-actin-binding protein. and ABP140. The bar represents 1 mm. All photographs were However, the ABP140-null mutant cells show no taken with the same magni®cation defects in these two structures or morphology of the cells. Mutations in the genes that encode actin-binding proteins often a€ect the organization of the actin along the sides of F-actin. Moreover, ABP140 has cytoskeleton. For example, of CAP1, encod- weak F-actin-cross-linking activity. Most actin-binding ing a homolog of mammalian capping protein, CapZ, proteins in mammal and yeast belong to one of causes aberrant cell morphology, disorganization of several families: monomer-binding proteins, capping cortical actin structures, and partial disappearance of proteins, severing proteins, and cross-linking proteins. cytoplasmic cables (Amatruda et al., 1992). In According to this classi®cation, ABP140 belongs to contrast, in some cases, only speci®c combinations of the family of cross-linking proteins. Most cross-linking mutations of two or more actin-binding proteins a€ect proteins share a common actin-, and the reorganization of the actin cytoskeleton. For this domain is combined with spacer domains example, ABP1, encoding one of the actin-binding consisting of a variable number of repeated a-helical proteins, has been reported to show no e€ect on the and b-sheet motifs, which generate proteins that di€er organization of F-actin through its elimination by in their ability to form actin bundles or networks gene disruption (Drubin et al., 1988; Adams et al., (Matsudaira, 1991). However, structural analysis has 1989). By the synthetic lethal screening of ABP1, revealed that ABP140 does not have such a common mutations that create a requirement for ABP1 have actin-binding domain with spacer domains. Moreover, been identi®ed in three genes; SAC6, SLA1, and SLA2 although the stoichiometry of the binding of most (Adams et al., 1989; Holtzman et al., 1993). One of cross-linking proteins to actin molecules is 1:4 ± 1:6, these genes, SAC6, encodes the yeast counterpart of that of ABP140 is about 1:30. Consistently, ABP140 mammalian ®mbrin, an F-actin cross-linking protein has weaker activities to increase the viscosity of F- (Adams et al., 1989; Holtzman et al., 1993). Therefore, A novel F-actin-binding protein TAsakuraet al 127 A Phase Actin HA

B

Figure 8 Localization of ABP140 in yeast cells. The ABP140-null mutant cells carrying pRS316-GAL-HA or pRS316-GAL-HA- ABP140 were used for the indirect immuno¯uorescence microscopy. The cells were ®xed and processed for costaining with rhodamine-phalloidin (F-actin) and with the anti-HA mAb (HA-ABP140). (A) pRS316-GAL-HA-ABP140. (B) pRS316-GAL-HA. All photographs were taken with the same magni®cation

our results together with these earlier observations for 125I-labeled F-actin blot overlay suggest that there might be another (s) 125I-Labeled F-actin blot overlay was done as described that cooperates with ABP140 in regulating the (Chia et al., 1991; Pestonjamasp et al., 1995). Actin reorganization of the actin cytoskeleton. Genetic monomer (G-actin) was prepared from rabbit muscle as screening to identify such proteins is necessary to described (Pardee and Spudich, 1982). Puri®ed G-actin was clarify the exact function of ABP140. labeled with 125I-Bolton Hunter reagent (Amersham). 125I- Rho1p regulates the reorganization of the actin Labeled G-actin (1 mg/ml, average speci®c activity of cytoskeleton at least through the Bni1p-pro®lin system 63.3 mCi/mg) was polymerized with 18 mg/ml of gelsolin in the budding yeast (Kohno et al., 1996; Imamura et (Sigma) (molar ratio, 100:1) by incubation for 10 min at 48C in a solution containing 20 mM piperazine-N,N'-bis(2- al., 1997). We have examined a possible interaction of ethanesulfonic acid)/NaOH at pH 7.0, 50 mM KCl, and ABP140 with Rho1p, Bni1p, and pro®lin by use of the 2mMMgCl2. Phalloidin was then added to give a ®nal yeast two-hybrid system. However, ABP140 does not concentration of 40 mM. The mixture was then incubated interact with these proteins (data not shown). There- for another 15 min at room and stored at 48C fore, it is unlikely that ABP140 is involved in at least as 125I-labeled F-actin. The sample to be tested was the Rho1p-regulated reorganization of the actin subjected to SDS ± PAGE and transferred to a nitrocellu- cytoskeleton. However, it is possible that the Rho lose membrane sheet (0.45 mmporesize,Schleicher& family members other than Rho1p and their related Schuell). The sheet was blocked in TBST (20 mM Tris/HCl proteins interact with ABP140. Further studies are at pH 7.5 containing 140 mM NaCl and 0.05% (w/v) necessary for our understanding of the functions and Tween 20) containing 5% (w/v) defatted powder . The sheet was then incubated for 1 h at room temperature modes of action of the Rho family members and with 10 mg/ml of 125I-labeled F-actin in TBST containing ABP140 in the budding yeast. 5% defatted powder milk and 5 mM phalloidin. After the incubation, the sheet was washed three times (1 min/wash) with TBST, followed by autoradiography using an image Materials and methods analyser (Fujix BAS2000). For competition experiments, 125I-labeled F-actin was Growth conditions for yeast cells prepared as described above except that the concentration of gelsolin was reduced to 7.2 mg/ml (molar ratio, 250:1). For puri®cation of ABP140, a Saccharomyces cerevisiae Twenty mg/ml of 125I-labeled F-actin was incubated for strain (BJ5457, aUra3-52 trp1 lys2-801 leu2D1his3D200 30 min at room temperature with 0.42 mg/ml of myosin pep4: :HIS3 prb1D1.6R can1) was cultured in YPDAU subfragment 1 (Sigma) in a solution containing 20 mM medium (1% Bacto-, 2% Bacto-peptone, 2% piperazine-N,N'-bis(2-ethanesulfonic acid)/NaOH at pH 7.0, , 0.04% and 0.02% ) with continuous 58 mM KCl, 2 mM MgCl2, 130 mM CaCl2 and 0.8 mM phalloi- agitation at 308C unti late logarithmic phase, and cells din in the presence or absence of 2 mM ATP. After the were harvested and stored at 7808Cuntiluse. incubation, the mixture was diluted with an equal volume of A novel F-actin-binding protein TAsakuraet al 128 TBST containing 10% defatted powder milk, and the constructions resulting mixture was added to the blot membrane sheet, Standard molecular biological techniques were used to followed by incubation for 1 h at room temperature. construct (Sambrook et al., 1989). A 1.9 kilobase pairs (kbp) DNA fragment encoding the entire open reading frame of ABP140 with the BamHI and KpnI sites Puri®cation of ABP140 from yeast cells upstream of the codon and the BamHI and All manipulations were carried out at 0 ± 48C. Yeast cells of PvuII sites downstream of the termination codon was a strain BJ5457 (30 g wet weight) were suspended in 60 ml isolated from the total yeast genome by polymerase chain of Bu€er A (25 mM Tris/HCl at pH 7.5, 5 mM EGTA, reaction (PCR), which was performed using the 5' primer 0.5 mM EDTA, 1 mM DTT, 0.3 M sorbitol, 10 mg/ml of 5'-GCGCGGATCCGGTACCATGGGTGTCGCAGATT- leupeptin, 20 mg/ml of aprotinin, 10 mM (p-amidino-phenyl) TGATC-3' and 3' primer 5'-GCGCGGATCCCAGCTG- methanesulfonyl ¯uoride and 1 mg/ml of pepstatin A). The GGTACCTTATGATGAGAGAGGAGGTGG-3' corre- cell suspension was homogenized with 30 ml of glass beads sponding to the N and C terminus, respectively. The (0.45 mm diameter). The homogenate was centrifuged at 1.9 kbp BamHI ± PvuII PCR fragment containing ABP140 1700 g for 10 min. The resulting supernatant was further was cloned into the BamHI-PvuII site of URA3-bearing centrifuged at 100 000 g for 1 h. The supernatant (60 ml, multicopy vector pKT10-GAL-HA, which had GAL1 700 mg of protein) was used as the cytosol fraction and and two copies of the HA , to construct stored at 7808C until use (Masuda et al., 1994). The pellet pKT10-GAL-HA-ABP140. The 1.9 kbp BamHI ± BamHI was resuspended with the same volume of Bu€er A and PCR fragment containing ABP140 was cloned into the used as the crude membrane fraction (60 ml, 600 mg of BamHI site of pTrcHis vector. A plasmid pTrcHis- protein). abp140::LEU2 was made from pTrcHis-ABP140 by The cytosol fraction of the yeast cells was pooled to insertion of the 2.2 kbp SalI±XhoI LEU2 fragment into 300 ml. One-tenth of the cytosol fraction (30 ml, 350 mg of the SalI site of ABP140.The1.9kbpBamHI ± PvuII PCR protein) was applied to a Mono S HR16/10 column fragment of ABP140 wasclonedintotheBamHI ± SmaI (1.6610 cm, Pharmacia) equilibrated with Bu€er B (20 mM site of URA3-bearing single copy vector pRS316-GAL- HEPES/NaOH at pH 7.4, 1 mM DTT and 10 mM (p- HA, which had GAL1 promoter and two copies of the HA amidino-phenyl)methanesulfonyl ¯uoride). The column was epitope, to construct pRS316-GAL-HA-ABP140. washed with 100 ml of Bu€er B and elution was performed with a 200 ml-linear gradient of NaCl (0 ± 500 mM) in Bu€er Expression and puri®cation of recombinant ABP140 B. Fractions of 10 ml each were collected. ABP140 appeared in Fractions 11 ± 14 (see Figure 2A). The same procedures The plasmid pKT10-GAL-HA-ABP140 was introduced were repeated ten times and the active fractions of the ten into a yeast strain BJ5457 by the acetate method column chromatographies were pooled. A half of these (Gietz et al., 1992). The transformants were screened for samples (200 ml, 40 mg of protein) was applied to a growth on SD-ura plate medium (2% glucose, 0.7% yeast hydroxyapatite column (1.0610 cm, Koken) equilibrated base without amino acids, and required amino with Bu€er C (20 mM phosphate at pH 7.5 and acids and bases) and cultured in SGATA medium (3% 1mMDTT). Elution was performed with a 120 ml-linear galactose, 0.2% sucrose, 0.5% casamino acids, 0.7% yeast gradient of potassium phosphate (20 ± 500 mM) in Bu€er C. nitrogen base without amino acids, 0.025% L-, Fractions of 4.0 ml each were collected. ABP140 appeared in and 0.025% adenine sulfate dehydrate) with continuous Fractions 10 ± 13 (see Figure 2B). The same procedures were agitation at 308C. The cells were harvested and the cytosol repeated twice and the active fractions of the two column fraction of the cells was prepared as described above. chromatographies were pooled. One-sixth of these samples To purify recombinant HA-tagged ABP140, the cytosol (5.2 ml, 3.9 mg of protein) was applied to a TSKgel phenyl- fraction (10 ml, 200 mg of protein) was applied to a Mono S 5PW RP column (0.7567.5 cm, Tosoh) equilibrated with HR10/10 column (1.0610 cm) equilibrated with Bu€er B. 0.05% tri¯uoroacetic acid. Elution was performed with a The column was washed with 50 ml of Bu€er B, and elution 100 ml-linear gradient of acetonitrile (0 ± 80%) in 0.05% was performed with an 80 ml-linear gradient of NaCl (0 ± tri¯uoroacetic acid. Fractions of 1.0 ml each were collected. 500 mM) in Bu€er B. Fractions of 4.0 ml each were ABP140 appeared in Fractions 42 ± 46 (see Figure 2C). The collected. ABP140 appeared in Fractions 11 ± 14. The active same procedures were repeated six times and each peak fractions (16 ml, 4.8 mg of protein) were applied to a fraction (Fraction 43) of the six column chromatographies hydroxyapatite column (0.565 cm) equilibrated with Bu€er (6.0 ml, 0.6 mg of protein) was combined and stored at 48C C. Elution was performed with a 15 ml-linear gradient of until use. potassium phosphate (20 ± 500 mM) in Bu€er C. Fractions of 0.5 ml each were collected. ABP140 appeared in Fractions 13 ± 16. The active fractions (2.0 ml, 2.0 mg of protein) were applied to a Superdex 200 HR10/30 column (1.0630 cm, Peptide mapping of ABP140 Pharmacia) equilibrated with Bu€er D (20 mM HEPES/ The TSKgel phenyl-5PW RP sample of ABP140 (30 mgof NaOH at pH 7.4, 1 mM DTT and 250 mM KCl). Elution protein) was subjected to SDS ± PAGE. The protein band was performed with Bu€er D. Fractions of 0.5 ml each were corresponding to ABP140 was cut out from the gel and collected. ABP140 appeared in Fractions 23 ± 26. The active digested with lysyl endopeptidase, and the digested fractions (2 ml, 0.5 mg of protein) were collected and stored peptides were separated by TSKgel ODS-80Ts at 48C until use. (0.46615 cm, Tosoh) reverse phase high-performance liquid column chromatography as described (Imazumi et Assay for of F-actin al., 1994). The amino acid sequences of the ®ve peptides were determined with a peptide sequencer (PSQ-1-gas G-actin was polymerized by incubation for 30 min at room phase sequencer, Shimazu; HP G1005A protein sequen- temperature in a polymerization bu€er (20 mM /

cing system, HP). To determine the N-terminal amino acid HClatpH7.0,2mMMgCl2,1mMATP and 90 mM KCl). sequence, the phenyl-5PW RP sample of ABP140 (1.4 mgof Puri®ed recombinant HA-tagged ABP140 (300 nM)was protein) was subjected to SDS ± PAGE and transferred to a mixed with F-actin (0.5 mg/ml) and incubated for 45 min polyvinylidene di¯uoride membrane. The protein band was at 258C in 100 ml of a solution containing 20 mM HEPES/ cut out from the membrane and directly subjected to the NaOH at pH 7.4, 10 mM imidazole/HCl at pH 7.4,

peptide sequencer. 120 mM KCl, 2 mM MgCl2,and1mMATP. The mixture A novel F-actin-binding protein TAsakuraet al 129 was then centrifuged at 130 000 g for 20 min at 48C. The Disruption and localization of ABP140 supernatant was removed and the precipitate was carefully ABP140 was disrupted as follows. The pTrcHis- washed with the same solution. The supernatant and abp140::LEU2 was cut with BamHI and the digested precipitate were solubilized and subjected to SDS ± DNA was introduced into a wild type haploid strain PAGE, followed by protein staining with Coomassie OHNY1 (MATa ura3 leu2 trp1 his3 ade2). The genomic brilliant blue. DNA was isolated from each transformant and the proper disruption of ABP140 was veri®ed by PCR. Low shear viscometry The pRS316-GAL-HA-ABP140 was introduced into these ABP140-null mutant cells, and cultured in SG-ura medium Low shear viscosity was measured as described (Pollard (3% galactose, 0.2% sucrose, 0.7% yeast nitrogen base and Cooper, 1982; Kato et al., 1996). Brie¯y, recombinant without amino acids, and required amino acids and bases) at HA-tagged ABP140 (5 ± 40 nM) was mixed with G-actin 248C. To visualize the localization of ABP140, the ABP140- (0.3 mg/ml) in 100 ml of a solution containing 20 mM null mutant cells expressing HA-tagged ABP140 were HEPES/NaOH at pH 7.4, 140 mM KCl, 0.1 mM ATP, processed for indirect immuno¯uorescence microscopy by and 1.0 mM EGTA, and the mixture was sucked into a the method as described (Yamochi et al., 1994). Brie¯y, the 0.1 ml micropipette. After the incubation at 258C, the time cells were ®xed with 4% formaldehyde for 2 h at 248C, and for a stainless steel ball to fall a ®xed distance in the the anti-HA mAb (25 mg/ml) was used as a primary antibody pipette was measured. and the FITC-conjugated anti- IgG (10 mM/ml, Cappel Labo.) was used as a secondary antibody. F-Actin Electron microscopy of F-actin was stained with rhodamine-phalloidin (0.5 mM, Molecular Probes Inc.) as described (Yamochi et al., 1994). Stained cells Recombinant HA-tagged ABP140 (150 nM) was mixed with were observed and photographed using a Zeiss Axiophot F-actin (0.15 mg/ml) in 100 ml of a solution containing microscope (Carl Zeiss). 20 mM HEPES/NaOH at pH 7.4, 3 mM imidazole/HCl at pH 7.4, 90 mM KCl, 2 mM MgCl2,and1mMATP, and the mixture was incubated for 45 min at 258C.Thesamplewas Other procedures negatively stained with 2% uranyl acetate and viewed with SDS ± PAGE (8% polyacrylamide gel) was performed as a transmission (model H-7100, described (Laemmli, 1970). Protein concentrations were Hitachi) (Pollard and Cooper, 1982). determined with bovine as a reference protein (Bradford, 1976). Assay for pyrene-conjugated actin assembly Pyrene-conjugated actin polymerization was measured as described (Cooper and Pollard, 1982). Brie¯y, G-actin (0.125 mg/ml, containing 20% pyrene-conjugated actin) Acknowledgements was mixed with recombinant HA-tagged ABP140 The investigation at Osaka University Medical School was (200 nM) or gelsolin (200 nM)in100ml of a solution supported by grants-in-aid for Scienti®c Research and for containing 5 mM HEPES/NaOH at pH 7.4, 1.3 mM Tris/ Research from the Ministry of Education, Science,

HCl, 0.25 mM DTT, 0.13 mM CaCl2,150mMKCl and Sports, and Culture, Japan (1996), by grants-in-aid for

2mMMgCl2. The actin polymerization reaction was Abnormalities in Mechanisms and for initiated by the addition of KCl and MgCl2,andthe Aging and Health from the Ministry of Health and mixture was incubated in a cuvette at 258C. Pyrene Welfare, Japan (1996), and by grants from the Human ¯uorescence was monitored continuously by a spectro- Frontier Science Program (1996) and the Uehara Memorial ¯uorimeter (excitation 365 nm, emission 386 nm). Foundation (1996).

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