©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Actin Monomer Binding Activity Localizes to the Carboxyl-terminal Half of the Saccharomyces cerevisiae Cyclase-associated Protein (*)

(Received for publication, November 30, 1994)

Nancy L. Freeman (§) Zunxuan Chen (§) Jeffrey Horenstein (2) Annemarie Weber (1) Jeffrey Field (¶)

From the  (1)Department of Pharmacology and theDepartment of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and the (2)Department of Biological Sciences, Columbia University, New York, New York 10027

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Saccharomyces cerevisiae adenylyl cyclase complex contains at least two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-associated protein, CAP (also called Srv2p). Genetic studies suggested two roles for CAP, one as a positive regulator of cAMP levels in yeast and a second role as a cytoskeletal regulator. We present evidence showing that CAP sequesters monomeric actin (K in the range of 0.5-5 µM), decreasing actin incorporation into actin filaments. Anti-CAP monoclonal antibodies co-immunoprecipitate a protein with a molecular size of about 46 kDa. When CAP was purified from yeast using an anti-CAP monoclonal antibody column, the 46-kDa protein co-purified with a stoichiometry of about 1:1 with CAP. Western blots identified the 46-kDa protein as yeast actin. CAP also bound to muscle actin in vitro in immunoprecipitation assays and falling ball viscometry assays. Experiments with pyrene-labeled actin demonstrated that CAP sequesters actin monomers. The actin monomer binding activity is localized to the carboxyl-terminal half of CAP. Together, these data suggest that yeast CAP regulates the yeast cytoskeleton by sequestering actin monomers.


INTRODUCTION

The Saccharomyces cerevisiae adenylyl cyclase enzyme is a multisubunit protein containing at least two subunits, a 200-kDa catalytic subunit, the product of the cyr1 gene (Kataoka et al., 1985), and a 70-kDa subunit, the product of the CAP gene (Field et al., 1988, 1990). In this yeast, adenylyl cyclase is activated by the two RAS gene products, RAS1 and RAS2 (Toda et al., 1985). Genetic studies suggested that CAP (^1)too is a positive regulator of cAMP levels in yeast, because the elevated cAMP levels, heat shock sensitivity, and nitrogen starvation sensitivity characteristic of the hyperactive RAS2 allele were suppressed by some loss of function mutations in the CAP gene. In fact, CAP is allelic to supC and srv2, two genes identified independently through genetic screens for suppressors of RAS2 (Fedor-Chaiken et al., 1990; Field et al., 1990). Biochemical tests were unable to establish how CAP regulates cAMP levels, but demonstrated that CAP is not essential for RAS activation of cyclase in vitro (Wang et al., 1992; Mintzer and Field 1994).

After CAP was cloned, yeast knockout technology was used to construct strains with disruptions in the CAP gene. Studies with such cap strains led to the discovery of another function for CAP, characterized by a second group of phenotypes. These, which we will refer to as the cytoskeletal phenotypes, included failure to grow on rich media, swelling, aberrant actin and tubulin staining, and abnormal bud scar patterns. The cytoskeletal phenotypes were distinct from any reported for the cAMP pathway in yeast and could not be suppressed by any of the known suppressors of the cAMP pathway, such as plasmids that overexpress the cAMP-dependent protein kinases or cAMP phosphodiesterases (Fedor-Chaiken et al., 1990; Field et al., 1990). Further evidence supporting this as a function distinct from cAMP regulation came from deletion studies of the CAP gene. The cAMP regulatory region and adenylyl cyclase binding site mapped to the amino-terminal third of CAP and the cytoskeletal phenotypes mapped to the carboxyl-terminal third of CAP (Gerst et al., 1991; Mintzer and Field 1994).

Genetic suppression studies in yeast provided some insight into the nature of the second function of yeast CAP. In these studies it was found that the cytoskeletal phenotypes, but not the cAMP phenotypes, of cap strains could be suppressed by overexpression of profilin (Vojtek et al., 1991). Profilin is an actin monomer-binding protein (Carlson et al., 1977; Haarer et al., 1990) with several important biological functions: it binds actin monomers, increases the nucleotide exchange rate of the monomers (Goldschmidt-Clermont et al., 1992), and delivers actin monomers to the barbed filament ends (Pollard and Cooper 1984; Pring et al., 1992), causing actin polymerization even at subcritical actin concentrations (Pantaloni and Carlier, 1993). In addition, profilin binds polyproline (Tanaka and Shibata, 1985) and phospholipids (Lassing and Lindberg, 1985). Although studies with mutant profilins suggested that phospholipid binding was most important for cap suppression, this was not established biochemically (Vojtek et al., 1991). Here we demonstrate that CAP from S. cerevisiae associates with actin in yeast and binds actin monomers in vitro.


EXPERIMENTAL PROCEDURES

Strains and Plasmids

Strain T158 5H1 is MATa leu2 trp1 his3 cyr1:: URA3 ade 8 pSCH9; strain 17-14d is MATa leu2 trp1 his3 cap::HIS3 ura3 ade 8 containing pEF-CYR1 integrated into the chromosome (this plasmid expresses an epitope fusion cyclase and contains the TRP1 gene) (Field et al., 1988); strain SKN34 is MATa leu2 trp1 his3 cap::HIS3 ura3 ade 8. Escherichia coli expression plasmids for yeast CAP have been described elsewhere (Field et al., 1990). pGST ABP expresses amino acids 535-592 of ABP1 in the plasmid pGEX3X (a generous gift from Tom Lila and David Drubin).

Growth of Bacteria and Yeast

Yeast was grown on either YPD media (1% yeast extract, 2% peptone, and 2% glucose) or, when cap- strains were used, synthetic media (Rose et al., 1990). Bacterial expression of CAP using the T7 promoter system (Field et al., 1990) has been described elsewhere. Expression and purification of the GST-SH3 fusion proteins has been described (Cicchetti et al., 1992).

Antibodies

The CAP monoclonal antibodies have been described elsewhere (Mintzer and Field, 1994). Monoclonal antibody 12CA5, used for purification of an epitope-tagged adenylyl cyclase, has been described elsewhere (Field et al., 1988). The rabbit anti-actin serum was a generous gift from Susan Brown and Brian Haarer.

S Labeling

Yeast cells were grown in 100 ml of synthetic media lacking methionine to A of 0.5 to 1.0. They were quickly harvested and suspended in 1.0 ml of media containing 0.5-1.0 mCi of S-labeled methionine and cysteine (ICN Biomedicals, Irvine CA). After incubation for 1 h at 30 °C, cells were harvested by centrifugation, suspended in 0.5-1.0 ml buffer C (20 mM MES, pH 6.2, 0.1 mM MgCl 0.1 mM EGTA, 1.0 mM beta-mercaptoethanol, 1.0 mM phenylmethylsulfonyl fluoride (PMSF)) and lysed by vigorous vortexing with glass beads (Sigma). The lysate was centrifuged 15 min in a Microfuge at 12,000 times g, and the supernatant was used for immunoprecipitations. A typical extract was labeled to a specific activity of 10^5 to 10^6 cpm/µl.

Immunoprecipitations

Typically, immunoprecipitations were carried out by incubating about 10^7 cpm of S-labeled extracts with 2 µg of the indicated antibodies (Mintzer and Field, 1994) and 20 µl of a 50% suspension of protein A-agarose beads (Pharmacia Biotech Inc.) in a reaction volume of 100 µl for 60 min at 4 °C. The beads were washed three times with 1.0 ml of buffer C containing 1% Lubrol and 0.5 M NaCl, followed by a wash with buffer C alone. The beads were transferred to a new Microfuge tube before the last wash. After washing, the beads were boiled in sample buffer and analyzed on SDS gels.

Protein Purifications

CAP was purified by monoclonal antibody affinity chromatography either from yeast or from E. coli expressing CAP. The column was made by cross-linking about 1 mg of protein A-purified antibody JF2 (a mouse anti-CAP monoclonal antibody) to CNBr activated beads (Pharmacia). Typically, one liter of cells were grown, harvested, and suspended in buffer C. At this point all subsequent procedures were carried out at 0-4 °C with frequent additions of fresh PMSF. Cells were lysed either by sonication (when E. coli expression systems were used) or by passage through a french press at 20,000 p.s.i. (when yeast were used). After disruption, the extracts were centrifuged at 15,000 rpm in a Sorval SS34 rotor for 1 h, and the supernatant was collected and stored at -80 °C until needed. Typically, extracts had a protein concentration of 10-20 mg/ml. Extracts were applied to the monoclonal antibody column, which was then washed with buffer C and eluted with 100 mM glycine, pH 1.9. Fractions (1 ml) were collected and immediately neutralized with 100 µl of 1.0 M Tris-HCl, pH 7.0. The carboxyl terminus of CAP and one preparation of full-length CAP were purified using an SH3 affinity column made with the SH3 domain from ABP1 expressed as a glutathione S-transferase (GST) fusion protein. The GST ABP1 SH3 column was prepared by cross-linking glutathione affinity-purified fusion protein to CNBr activated beads. An extract from E. coli harboring the plasmid pT7.CAPDelta29 (which expresses amino acids 248-526) was passed over the column and protein was eluted with a 0.0-1.0 M NaCl gradient. The peak of CAP or its carboxyl-terminal fragment, as determined by electrophoresis, eluted at about 0.2 M NaCl. The pooled protein peak was concentrated by ultrafiltration against 50 mM Tris, pH 7.5, and then stored at -80 °C. The actin binding properties of full-length CAP were about the same after purification by SH3 affinity chromatography and by antibody affinity chromatography. Yeast actin was purified by DNase I affinity chromatography (Worthington) using the method of Zechel as described (Zechel, 1980; Haarer et al., 1990). For falling ball assays, actin was purified from an acetone powder prepared from rabbit muscle (Sigma), using the method of Spudich (Spudich and Watt, 1971). For pyrene labeling experiments actin was prepared according to Murray et al. (1981) and pyrene labeled according to Kouyama and Mihashi(1981) with the modifications described previously (Northrop et al., 1986). Gelsolin was generously donated by J. Bryan (Bryan, 1988). Spectrin-actin complexes were prepared according to Cohen and Branton(1979) with the modifications previously described (Weber et al., 1994).

Falling Ball Viscometry Assays

The effect of CAP on actin polymerization was checked by falling ball viscometry (Pollard and Cooper, 1982). Actin samples were incubated in a 50-µl reaction volume, and polymerization was induced by addition of KCl to a concentration of 100 mM and MgCl(2) to a concentration of 2 mM. Samples were quickly transferred to 100-µl capillary pipettes. The pipettes were placed at an angle of 20 °C and, after a 40-min incubation at room temperature, the time it took a 0.025-inch steel ball (New England Miniature Ball Company, Norfolk, CN) to fall 3.7 cm was measured.

Fluorescence and Kinetic Measurements

Changes in the rate and extent of actin polymerization were determined from fluorescence measurements of pyrenyl-actin (excitation 366.5 and emission 407 nm, usually about 10% pyrenyl-actin) (Weber et al., 1987), using a Photon Technology International photon counting fluorimeter. Measurements were standardized against a Raman excitation peak (357 nm; emission 407 nm). The absolute fluorescence readings from different experiments shown here are not directly comparable because varying slit widths were used. All experiments were carried out at 20 °C with Mg-actin.

Measurements of elongation rates at the pointed and the barbed filament ends and data analysis were carried out as described previously (Young et al., 1990; Weber et al., 1994) using a mixture of 5 or 10% pyrenyl-labeled and native actin in a medium containing 10 mM imidazole buffer, pH 7.4, 0.1 M KCl, 2 mM MgCl 1 mM azide, 1 mM dithiothreitol, 0.1 mM leupeptin, ATP as indicated in the legends, 5.0 mM EGTA. Short gelsolin-actin filaments (20 actin monomers/filament) were used as nuclei for elongation from the pointed filament end. Gelsolin-actin dimers were present (10 nM) during the elongation assay to insure full capping after dilution of the stock solution. Spectrin-actin complexes of known number concentration of barbed filament ends (Weber et al., 1994) were used as nuclei for elongation from the barbed filament ends. End points of polymerization were measured in some cases at the end of the day and in other cases after overnight incubation. We determined that CAP binds with similar affinity to pyrenyl-actin and to native actin by comparing its effect on the polymerization from the pointed filament end of 100% and of 5%-pyrene-labeled actin.

Protein Concentrations

Protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard (Bradford, 1976) (reagents for protein assays were purchased from Bio-Rad). However, when during the polymerization assays it became apparent that the CAP concentration was significantly underestimated by this application of the Bradford test, the extinction coefficient was calculated from the amino acid sequence (E = 47.7 mM cm for CAP and E = 10.8 mM cm for the COOH-terminal half of CAP), and the protein concentrations were obtained by light absorption; concentrations obtained by the Bradford test were multiplied by a factor of 2.5. Protein concentrations for actin and gelsolin were obtained by light absorption, using E = 24.9 mM cm and E = 150 mM cm.


RESULTS

CAP Isolated from Yeast Is Associated with Actin

In order to search for CAP-binding proteins, five monoclonal antibodies against CAP were used to immunoprecipitate CAP from S-labeled yeast extracts. The proteins removed from the extracts were then analyzed by autoradiography of SDS gels. Three of the antibodies immunoprecipitated a protein of relative molecular size of 46 kDa in addition to the 70-kDa CAP protein (Fig. 1, lanes 3-5). The other two monoclonal antibodies (Fig. 1, lanes 1 and 2) and six polyclonal antisera (data not shown) against CAP all extracted CAP from the lysates, but failed to extract the 46-kDa protein. These antibodies may have interfered with binding of CAP to the 46-kDa protein. The 46-kDa band could not have been derived from adenylyl cyclase, because this experiment was carried out in an adenylyl cyclase (cyr1) disruption strain, which lacks any coding sequences for adenylyl cyclase. An experiment with an extract from a cap strain established that the 46-kDa band was binding to CAP and not to the antibodies (Fig. 2). When antibody alone was used with this extract, neither the 46-kDa nor the 70-kDa bands were seen (Fig. 2, lane 2). However, the 46-kDa band was clearly visible when recombinant CAP was included in the immunoprecipitations (Fig. 2, lanes 3-5). Together, these experiments demonstrate that a 46-kDa yeast protein binds CAP.


Figure 1: Immunoprecipitation of CAPfrom S-labeled cells. A, an extract was prepared from T158-5H1 cells labeled with S and immunoprecipitated with the indicated antibodies as described under ``Experimental Procedures.'' Samples were run on an SDS gel. Then the gel was treated with Entensify (DuPont NEN), dried, and then exposed to x-ray film for 17 h at -80 °C. The control antibody was monoclonal antibody 12CA5, which does not bind CAP (Field et al., 1988). B, regions of CAP where the monoclonal antibodies bind and the major functional regions of CAP.




Figure 2: Immunoprecipitation of CAP in a cap strain. An extract was prepared from cells from a cap strain (17-14d) labeled with S and immunoprecipitated with monoclonal antibody JF2 as described in the legend to Fig. 1. Extracts from E. coli (E. coli extract) or E. coli expressing CAP (CAP extract) were included in immunoprecipitations. Delta indicates that the extract was heated to 90 °C for 10 min prior to addition to the immunoprecipitation. Extracts are expressed as volume, in microliters, added per immunoprecipitation. Similar results were obtained when purified CAP was substituted for the E. coli extract (data not shown). The 70-kDa CAP band was not seen in this experiment because it was unlabeled. However, CAP was readily visualized when the same gels were stained for protein with Coomassie Blue (not shown). Immunoprecipitations were carried out and analyzed by autoradiography of an SDS gel as described in the legend to Fig. 1.



Earlier studies suggested that actin may bind CAP so we purified CAP from S. cerevisiae using a monoclonal antibody affinity column and tested for the presence of actin (Fig. 3). The strain used contained a disruption of the CYR1 gene so the 200-kDa catalytic subunit was not seen. In Fig. 3A the purified CAP is shown along with a preparation of yeast actin on an SDS-polyacrylamide gel. The purified CAP was seen as three closely spaced bands on the gel, all of which were recognized by CAP antibodies on a Western blot (Western blot data not shown). The three bands may either be isoforms of CAP or the result of degradation during purification. The CAP preparation contained a 46-kDa band that co-migrated with actin. We estimate from the intensity of staining of the bands that as much as 1 actin/CAP was isolated. In order to identify the 46-kDa unequivocally as actin, we carried out a Western blot on the purified CAP and found that the 46-kDa band was recognized by an actin antiserum (Fig. 3B). These data indicate that CAP associates with actin in yeast and that, in all likelihood, the 46-kDa protein visualized by S labeling experiments was actin.


Figure 3: Co-purification of actin with CAP from yeast. CAP was purified on a monoclonal antibody column as described under ``Experimental Procedures'' from strain T158 5H1. Actin was purified on a DNase I column as described under ``Experimental Procedures'' from strain T158 5H1. A, about 1 µg of each protein was run on a 10% SDS-polyacrylamide gel and stained with Coomassie Blue. B, Western blot of purified CAP probed with anti-actin antisera. The CAP preparation shown in Fig. 3A was run on a 10% SDS gel, blotted onto nitrocellulose paper, and then probed with an actin antisera. Visualization was by a horseradish peroxidase-linked goat anti-rabbit detection system purchased from Bio-Rad.



CAP Binds Actin in Vitro

To characterize the interaction of CAP and actin, we used in vitro binding assays. Full-length CAP was purified from an E. coli expression system using an anti-CAP monoclonal antibody column and the carboxyl terminus of CAP was purified by SH3 affinity chromatography using the SH3 domain of ABP1 as the ligand (Fig. 4A). We used yeast, rabbit muscle, and platelets (data not shown for platelet actin) as sources of actin. Yeast and mammalian actin are about 90% identical (Gallwitz and Sures, 1980; Ng and Abelson, 1980) and possess many of the same biochemical properties (Kron et al., 1992; Nefsky and Bretscher, 1992). In immunoprecipitations, recombinant yeast CAP bound to purified rabbit actin (Fig. 4B).


Figure 4: Interaction of yeast CAP with rabbit skeletal actin. A, SDS gel of purified CAP and carboxyl-terminal fragment of CAP stained with Coomassie Blue. Lane 1, full-length CAP; lane 2, carboxylterminal half of CAP. Proteins were purified from an E. coli expression system as described under ``Experimental Procedures.'' B, immunoprecipitation of yeast CAP incubated with rabbit actin. 50 µl of an extract from E. coli expressing CAP was incubated with 10 µg of actin purified from rabbit skeletal muscle, 3.0 µg of monoclonal antibody JF2, and 25 µl of a suspension of 50% protein A-agarose in a reaction volume of 100 µl. Samples were rocked gently for 60 min at 4 °C, washed three times with buffer C containing 0.5 M NaCl and 1.0% lubrol buffer with the final wash with buffer C alone. Samples were separated in a 10% SDS gel stained with Coomassie Blue.



Falling ball viscometry was used to measure the effect of CAP on actin filament networks (Pollard and Cooper, 1982). In this assay a steel ball is dropped through a capillary tube containing the actin solution, and the time it takes the ball to fall a fixed distance is determined. The rate that the ball falls through the solution is reduced when the actin polymerizes and forms networks. Several types of actin-binding proteins will reduce the viscosity of the actin solutions in this assay. These include proteins that shorten the length of actin filaments and proteins that sequester actin monomers. Fig. 5A shows a decrease in viscosity with increasing CAP concentrations using yeast actin, and Fig. 5B shows similar results using rabbit muscle actin. The effect disappeared after heating CAP as would be expected for a protein (data not shown).


Figure 5: Falling ball viscometry of yeast and rabbit muscle actin incubated with yeast CAP. Where indicated, varying amounts of CAP purified from an E. coli expression system and actin purified from rabbit skeletal muscle or yeast were mixed together in 50-µl reaction volumes, and polymerization was measured as described under ``Experimental Procedures.'' Measurements were repeated three times. An average of the three data points were plotted. The error bars indicate one standard deviation from this average. The data are plotted as the ratio of CAP: actin using a molecular weight of 58,000 daltons for CAP (this is calculated from the deduced amino acid sequence; on SDS gels CAP has a M(r) of 70,000) and 42,000 for actin. A, yeast actin. B, rabbit muscle actin.



CAP Sequesters Actin Monomers in Vitro

The experiments described so far suggest very strongly that CAP binds to actin, but they do not indicate whether CAP binds to G-actin (globular actin) or to F-actin (filamentous actin). The next series of experiments with purified recombinant CAP and muscle actin indicates that CAP sequesters actin monomers, preventing their incorporation into actin filaments. We evaluated the action of CAP by kinetic assays using pyrenyl-actin to measure the effect of CAP on polymerization from either end of actin filaments. We first measured the effect of CAP on the elongation of short gelsolin-capped actin filaments, i.e. on the elongation from the pointed, slowly reacting, actin filament ends (Fig. 6A). CAP inhibited the rate of elongation, and it decreased the final extent of polymerization. At low concentrations of added G-actin, CAP caused the depolymerization of the gelsolin-capped actin filaments used as nuclei for elongation.


Figure 6: A, inhibition of polymerization from the pointed filament end by CAP. Polymerization was initiated by the simultaneous addition of short gelsolin-capped actin filaments (80 nM F-actin + 4 nM gelsolin) and polymerizing salt to actin monomers which had been converted to Mg-G-actin in the absence or presence of CAP (4 µM); the medium contained 2 mM creatine phosphate + 0.2 mg/ml creatine phosphokinase and 10 µM ATP. K = 3.6 µM for CAP binding to actin monomers, calculated on the basis of the concentration of CAP, the decrease in the concentration of polymerized actin (measured at the end of the day), and the pointed end critical concentration of 0.55 µM (K= {critical concentration times (total CAP - bound CAP)}/(control F-actin - F-actin )). B, inhibition of polymerization from the barbed filament end by CAP. Polymerization was initiated as before except that the short gelsolin-actin filaments were replaced by spectrin-actin complexes containing 0.4 nM free barbed filament ends. Samples contained 3 µM G-actin and 3 µM G-actin + 5 µM CAP, as indicated, and 0.5 mM ATP. K = 1.1 µM, calculated as before, with a value for the critical concentration of 0.1 µM. C, inhibition of polymerization from the pointed filament end by the COOH-terminal half of CAP. The experiment was carried out as in A using 3 µM actin and, where indicated, 5 µM of the COOH-terminal half of CAP. The medium contained 0.5 mM ATP and no creatine phosphate + creatine phosphokinase. K = 0.55 µM.



These effects of CAP could have been caused by either one of two actions: actin monomer sequestration or a tropomodulin-like action at the pointed-filament-end, which consists of partial capping together with an increase in the pointed end critical concentration (Weber et al., 1994). To distinguish between the two possibilities we measured the effect of CAP on the polymerization from the barbed filament end using spectrin-actin complexes as nuclei for elongation (both filament ends uncapped). CAP lowered the final extent of polymerization and also the rate of elongation when the filament ends were free (Fig. 6B). (When the barbed filament ends are not capped they are the preferential site for actin elongation because they are the fast-reacting ends with a 10 times higher rate constant of elongation than the rate constant at the pointed filament ends). The results of the experiment rule out a tropomodulin-like action of CAP, since tropomodulin has no effect on polymerization when the barbed filament ends are free (Weber et al., 1994). However, all of these findings can be produced by monomer sequestration by CAP. The COOH-terminal end of CAP had a similar effect on polymerization from both filament ends (Fig. 6C, data not shown for barbed end). The K(d) values for CAP binding to actin monomers varied (the lowest was 0.6 µM and the highest was 6 µM); however the means, calculated separately in the absence (three determinations) or presence of gelsolin (four determinations), with values of 2.2 and 3.6 µm respectively, were relatively close. The reasons for the variability of the data appears to be the instability of purified CAP and variability between different preparations.


DISCUSSION

Genetic studies indicated that CAP is a multifunctional protein. The first function known was regulation of adenylyl cyclase, and a second function was predicted based on phenotypes observed when the coding region of CAP was deleted from yeast to create cap- stains (Field et al., 1988, 1990; Fedor-Chaiken et al., 1990). Many of the genetic observations suggested the second function involved regulation of the cytoskeleton. Here we present biochemical evidence that the second function of S. cerevisiae CAP is binding actin monomers. CAP associates with actin in yeast; in vitro CAP was shown to bind actin from yeast, rabbit skeletal muscle, and platelets. Also consistent with the genetic studies on the second function of CAP, we localized the actin binding region to the carboxyl-terminal half of the protein.

The kinetics show clearly that yeast CAP sequesters monomers of muscle and platelet (data not shown) actin. The extent of actin polymerization is decreased when the barbed filament ends are free and when they are capped and the rate of elongation is inhibited at either filament end as expected for a decrease in the free actin monomer concentration. The similarity of the effect of CAP on falling ball viscometry with yeast and muscle actin strongly suggest that the results of the kinetic study may be extended to yeast actin. The lowest K(d) value for monomer binding by CAP of 0.4 µM is the same as that of thymosin-beta(4) for platelet actin (0.4 µM) (Weber et al., 1992) and higher than that of spleen profilin for muscle actin (0.1 µM) (Pantaloni and Carlier, 1993). The highest values are about three times higher than those of thymosin-beta(4) for muscle actin (2 µM). These K(d) values are higher than expected on the basis of the experiments showing that the complex of CAP with yeast actin persisted after purification by column chromatography or after washing of protein A beads containing the complex. However, in view of the variability of different CAP preparations and in view of the difference in the actin species, muscle versus yeast actin, this may not be surprising. The kinetics do not give information about the stoichiometry of the CAP actin monomer complex.

It remains to be determined how CAP binding to actin is related to regulation of cAMP in yeast. One possibility is that CAP allows two different pathways to coordinate their actions, one pathway regulating cAMP and the other pathway regulating the cytoskeleton. However, although CAP is readily detected in adenylyl cyclase preparations, we have been unable to detect the presence of actin in these preparations (Field et al., 1988). Therefore, it is not easy to see how the two pathways can be coordinately regulated. Also, the cytoskeletal and cAMP functions can be mapped to different regions of CAP, suggesting that the two functions of CAP can act independently. Further work is necessary to determine how the actions of the two pathways may be coordinated.

Little is known about the way CAP regulates actin. However, the interaction of profilin and actin has been studied in a number of laboratories. Initially, it was shown by in vitro assays that profilin, like CAP, sequesters actin monomers and inhibits actin polymerization (Carlson et al., 1977). However, later studies with platelets demonstrated that the bulk of the unpolymerized actin is bound to thymosin-beta another actin monomer-binding protein (Safer et al., 1991). Recently, it was demonstrated that profilin can deliver monomers to the barbed filament ends (but not the pointed ends) (Pollard and Cooper, 1984) even in the presence of thymosin-beta(4) and at subcritical monomer concentrations (Pantaloni and Carlier, 1993). Our kinetic data so far have not produced any evidence for a similar action of CAP, although in view of the unusual variability of the preparations, such activity cannot be unequivocally ruled out. A different way that CAP may serve as a monomer delivery protein would be through its SH3 binding domain. We recently demonstrated that CAP binds SH3 proteins through a proline rich domain and that, in yeast, this domain is required for co-localization with a number of SH3 proteins to cortical actin patches. (^2)Thus CAP may participate in SH3-mediated cytoskeletal signaling.

Homologs of CAP have been identified and cloned in other species, including another yeast (Schizosaccharomyces pombe) and mammals (Gieselmann and Mann, 1992; Kawamukai et al., 1992; Matviw et al., 1992; Zelicof et al., 1993). Only the S. pombe CAP binds its own cyclase (Kawamukai et al., 1992). Where tested, all CAP homologs were able to suppress the cytoskeletal phenotypes of cap strains. In fact the pig homolog, ASP-56, was first identified, because it associated with actin in platelets, probably by sequestering actin monomers. These results demonstrate the wide conservation of CAP as an actin sequestering protein.


FOOTNOTES

*
This work was supported by National Institutes of Health R01-GM-48241. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to this manuscript.

Beckman Young investigator. To whom correspondence should be addressed.

(^1)
The abbreviations used are: CAP, cyclase-associated protein; MES, 2-(N-morpholino)ethanesulfonic acid.

(^2)
N. L. Freeman, T. Lila, K. Mintzer, Z. Chen, A. J. Pahk, R. Ren, D. G. Drubin, and J. Field, unpublished observations.


ACKNOWLEDGEMENTS

We thank Susan Brown and Brian Haarer for providing us with actin antisera and also for helpful discussions. We also thank Amita Sehgal and Vivianne Nachmias for helpful discussions and Cynthia Pennise for helping us with the kinetic studies.


REFERENCES

  1. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  2. Bryan, J. (1988) J. Cell Biol. 106, 1553-1562 [Abstract]
  3. Carlson, L., Nystrom, L.-E., Sundkvist, I., Markey, F., and Lindberg, U. (1977) J. Mol. Biol. 115, 465-483 [Medline] [Order article via Infotrieve]
  4. Cicchetti, P., Mayer, B. J., Thiel, G., and Baltimore, D. (1992) Science 257, 803-806 [Medline] [Order article via Infotrieve]
  5. Cohen, C. M., and Branton, D. (1979) Nature 279, 163-165 [Medline] [Order article via Infotrieve]
  6. Fedor-Chaiken, M., Deschenes, R. J., and Broach, J. R. (1990) Cell 61, 329-340 [Medline] [Order article via Infotrieve]
  7. Field, J., Nikawa, J., Broek, D., MacDonald, B., Rodgers, L., Wilson, I. A., Lerner, R. A., and Wigler, M. (1988) Mol. Cell. Biol. 8, 2159-2165 [Medline] [Order article via Infotrieve]
  8. Field, J., Vojtek, A., Ballester, R., Bolger, G., Colicelli, J., Ferguson, K., Gerst, J., Kataoka, T., Michaeli, T., Powers, S., Riggs, M., Rodgers, L., Wieland, I., Wheland, B., and Wigler, M. (1990) Cell 61, 319-327 [Medline] [Order article via Infotrieve]
  9. Gallwitz, D., and Sures, I. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2546-2550 [Abstract]
  10. Gerst, J. E., Ferguson, K., Vojtek, A., Wigler, M., and Field, J. (1991) Mol. Cell. Biol. 11, 1248-1257 [Medline] [Order article via Infotrieve]
  11. Gieselmann, R., and Mann, K. (1992) FEBS Lett. 298, 149-153 [CrossRef][Medline] [Order article via Infotrieve]
  12. Goldschmidt-Clermont, P. J., Furman, M. I., Wachsstock, D., Safer, D., Nachmias, V. T., and Pollard, T. D. (1992) Mol. Biol. Cell 3, 1015-1024 [Abstract]
  13. Haarer, B. K., Lillie, S. H., Adams, A. E. M., Magdolen, V., Bandlow, W., and Brown, S. S. (1990) J. Cell Biol. 110, 105-114 [Abstract]
  14. Kataoka, T., Broek, D., and Wigler, M. (1985) Cell 43, 493-505 [Medline] [Order article via Infotrieve]
  15. Kawamukai, M., Gerst, J., Field, J., Riggs, M., Rodgers, L., Wigler, M., and Young, D. (1992) Mol. Biol. Cell 3, 167-180 [Abstract]
  16. Kouyama, T., and Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38 [Abstract]
  17. Kron, S. J., Drubin, D. G., Botstein, D., and Spudich, J. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4466-4470 [Abstract]
  18. Lassing, I., and Lindberg, U. (1985) Nature 314, 472-474 [Medline] [Order article via Infotrieve]
  19. Matviw, H., Yu, G., and Young, D. (1992) Mol. Cell. Biol. 12, 5033-5040 [Abstract]
  20. Mintzer, K. A., and Field, J. (1994) Cell. Signalling 6, 681-694 [CrossRef][Medline] [Order article via Infotrieve]
  21. Murray, J. M., Weber, A., and Knox, M. K. (1981) Biochemistry 20, 641-649 [Medline] [Order article via Infotrieve]
  22. Nefsky, B., and Bretscher, A. (1992) Eur. J. Biochem. 206, 949-955 [Abstract]
  23. Ng, R., and Abelson, J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3912-3916 [Abstract]
  24. Northrop, J. A., Weber, A., Mooseker, M. S., Franzini-Armstrong, C., Bishop, M. F., Dubyak, G. R., Tucker, M., and Walsh, T. P. (1986) J. Biol. Chem. 261, 9274-9281 [Abstract/Free Full Text]
  25. Pantaloni, D., and Carlier, M.-F. (1993) Cell 75, 1007-1014 [Medline] [Order article via Infotrieve]
  26. Pollard, T. D., and Cooper, J. A. (1982) Methods Enzymol. 85, 211-233 [Medline] [Order article via Infotrieve]
  27. Pollard, T. D., and Cooper, J. A. (1984) Biochemistry 23, 6631-6641 [Medline] [Order article via Infotrieve]
  28. Pring, M., Weber, A., and Bubb, M. R. (1992) Biochemistry 31, 1827-1836 [Medline] [Order article via Infotrieve]
  29. Rose, M. D., Winston, F., and Hieter, P. (1990) Methods in Yeast Genetics: A Laboratory Course Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  30. Safer, D., Elzinga, M., and Nachmias, V. T. (1991) J. Biol. Chem. 266, 4029-4032 [Abstract/Free Full Text]
  31. Spudich, J. A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871 [Abstract/Free Full Text]
  32. Tanaka, M., and Shibata, H. (1985) Eur. J. Biochem. 151, 291-297 [Abstract]
  33. Toda, T., Uno, I., Ishikawa, T., Powers, S., Kataoka, T., Broek, D., Cameron, S., Broach, J., Matsumoto, K., and Wigler, M. (1985) Cell 40, 27-36 [Medline] [Order article via Infotrieve]
  34. Vojtek, A., Haarer, B., Field, J., Gerst, J., Pollard, T. D., Brown, S., and Wigler, M. (1991) Cell 66, 497-505 [Medline] [Order article via Infotrieve]
  35. Wang, J., Suzuki, N., and Kataoka, T. (1992) Mol. Cell. Biol. 12, 4937-4945 [Abstract]
  36. Weber, A., Northrop, J., Bishop, M. F., Bishop, M. F., Ferrone, F. A., and Mooseker, M. S. (1987) Biochemistry 26, 2528-2536 [Medline] [Order article via Infotrieve]
  37. Weber, A., Nachmias, V. T., Pennise, C. R., Pring, M., and Safer, D. (1992) Biochemistry 31, 6179-6185 [Medline] [Order article via Infotrieve]
  38. Weber, A., Pennise, C. R., Babcock, G. G., and Fowler, V. M. (1994) J. Cell Biol. 127, 1627-1635 [Abstract]
  39. Young, C., Southwick, F. S., and Weber, A. (1990) Biochemistry 29, 2232-2240 [Medline] [Order article via Infotrieve]
  40. Zechel, K. (1980) Eur. J. Biochem. 110, 343-348 [Abstract]
  41. Zelicof, A., Gatica, J., and Gerst, J. E. (1993) J. Biol. Chem. 268, 13448-13453 [Abstract/Free Full Text]

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