(Received for publication, November 30, 1994)
From the
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.
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 ()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.
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.
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.
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.
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 of 70,000) and 42,000 for actin. A, yeast actin. B, rabbit muscle actin.
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
(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 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.
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 value
for monomer binding by CAP of 0.4 µM is the same as that
of thymosin-
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-
for muscle actin (2 µM). These K
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- 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-
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. (
)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.