![Profilin increases the turnover rate of actin filaments. A solution of 10 M [ 3 H]ADP-F-actin prepared as described under " Materials and Methods " was split in two samples supplemented or not with the indicated amount of profilin. A chase amount of ATP (0.2 mM) was applied to each sample at time 0. Aliquots were centrifuged at different times over a period of 8 h. The linear increase in [ 3 H]ADP in the supernatant was measured and normalized to 1 for the control run in the absence of profilin. The increase in turnover rate in the presence of profilin is plotted as percentage increase of the rate measured in the control. Different symbols refer to independent experiments. The inset represents typical time courses of [ 3 H]ADP increase in the supernatant in the absence (q) or in the presence (E) of 5 M profilin.](https://www.researchgate.net/publication/232285835/figure/fig2/AS:349285930553364@1460287663513/Profilin-increases-the-turnover-rate-of-actin-filaments-A-solution-of-10-M-3_Q320.jpg)
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Role of Nucleotide Exchange and Hydrolysis in the Function of
Profilin in Actin Assembly*
(Received for publication, January 31, 1996, and in revised form, March 18, 1996)
Irina Perelroizen‡, Dominique Didry§, Hans Christensen§, Nam-Hai Chua
¶
,
and Marie-France Carlier‡
i
From the ‡Laboratoire d’Enzymologie, CNRS, 91198 Gif-sur-Yvette, France, the §Institute of Molecular Agrobiology,
Singapore, and the
¶
Laboratory of Plant Molecular Biology, Rockefeller University, New York, New York 10021
Profilin, an essential G-actin-binding protein, has two
opposite regulatory functions in actin filament assem-
bly. It facilitates assembly at the barbed ends by lower-
ing the critical concentration (Pantaloni, D., and Car-
lier, M.-F. (1993) Cell 75, 1007–1014); in contrast it
contributes to the pool of unassembled actin when
barbed ends are capped. We proposed that the first of
these functions required an input of energy. How profi-
lin uses the ATP hydrolysis that accompanies actin po-
lymerization and whether the acceleration of nucleotide
exchange on G-actin by profilin participates in its func-
tion in filament assembly are the issues addressed here.
We show that 1) profilin increases the treadmilling rate
of actin filaments in the presence of Mg
21
ions; 2) when
filamentsareassembledfromCaATP-actin,whichpolym-
erizes in a quasireversible fashion, profilin does not pro-
mote assembly at the barbed ends and has only a G-
actin-sequestering function; 3) plant profilins do not
accelerate nucleotide exchange on G-actin, yet they pro-
mote assembly at the barbed end. The enhancement of
nucleotide exchange by profilin is therefore not in-
volved in its promotion of actin assembly, and the pro-
ductive growth of filaments from profilin-actin complex
requires the coupling of ATP hydrolysis to profilin-actin
assembly, a condition fulfilled by Mg-actin, and not by
Ca-actin.
Living cellsundergochangesin shape and motile behavior by
spatially and temporally controlled rearrangements of the ac-
tin cytoskeleton. In the physiological ionic conditions, F-actin is
assembled at steady state in the cell medium. Changes in the
F-actin/G-actin ratio, which occur in response to stimuli, are
made possible by shifts in steady state, i.e. changes in the
critical concentration for filament assembly. These changes are
elicited by capping proteins and profilin (1, 2) and amplified by
G-actin-binding proteins (3). A high level of capping of barbed
ends maintains the high critical concentration of pointed ends
in the cytoplasm. A steep energetic gradient is therefore cre-
ated between the cell medium and the loci where uncapped
barbed ends are nucleated at the plasma membrane. We un-
derstand that in this way capping of barbed ends in the cyto-
plasm is required for a more efficient local actin assembly. In
support of this view, recent evidence indeed indicates that the
level of motility in fibroblasts (4) and Dictyostelium (5) corre-
lates with the level of barbed end capping. Similarly, the actin-
based propulsive movement of Listeria results from the local
creation and maintenance of new uncapped barbed ends at the
bacterium surface, while filaments are capped in the bulk
cytoplasm (6).
Profilin has unique properties among G-actin-binding pro-
teins. Under physiological ionic conditions (Mg-actin, 0.1
M
KCl), it binds G-actin tightly (K 5 10
7
M
21
for vertebrate
profilin I) and participates in the establishment of the pool of
unassembled actin when barbed ends are capped. In contrast,
when barbed ends are uncapped, the participation of profilin-
actin complex in filament assembly (7, 8) results in a decrease
in critical concentration (2), i.e. profilin then synergizes with
the uncapping effect to promote more efficient barbed end actin
assembly. We demonstrated that the profilin-induced decrease
in critical concentration at the barbed ends could not be ac-
counted for in the context of reversible actin assembly, and we
proposed that the free energy of ATP hydrolysis associated
with actin polymerization was used by profilin. However, no
evidence was provided in that work (2) on the nature of the
reactions that could support profilin function in barbed end
assembly and the detailed pathways in which ATP binding and
hydrolysis could be used. The property of profilin to increase
the rate of nucleotide exchange on G-actin (9–12) might be
involved. For instance, ATP hydrolysis and/or P
i
release might
be accelerated by profilin upon association of the profilin-actin
complex to a filament barbed end. It was also suggested in a
short review (13) that the enhancement of nucleotide exchange
on G-actin itself might also be part of its function in the pro-
motion of actin assembly at the barbed end.
The above issues are raised in the present work. We show
that when the polymerization of ATP-actin is quasireversible
(CaATP-actin), profilin does not promote barbed end assembly;
hence, the coupling of ATP hydrolysis to actin polymerization is
required in the function of profilin. Profilins from Arabidopsis
thaliana promote assembly of MgATP-actin at the barbed ends,
like vertebrate profilins, while being unable to enhance the
rate of nucleotide exchange on G-actin, which demonstrates
that the latter property is not used in the main biological
function of profilin in living cells.
MATERIALS AND METHODS
Proteins—Actin was purified from rabbit muscle and isolated in the
CaATP-G-actin form as described (14). Profilin was isolated from bovine
spleen by poly-
L-proline affinity chromatography as described (14). Ac-
tin was pyrenyl-labeled as described (15). Gelsolin was a generous gift
from Dr Yukio Doi. Thymosin
b
4
(T
b
4
)
1
was purified from bovine spleen
as described (2). Recombinant A. thaliana profilin 1 (vegetative form)
and profilin 3 (floral form) were expressed in Escherichia coli and
* This work was supported in part by the Ligue Nationale Franc¸aise
contre le Cancer, the Association pour la Recherche contre le Cancer
(ARC), and the Association Franc¸aise contre les Myophathies (AFM).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
i
To whom correspondence should be addressed. Tel.: 33 1 69 82 34 65;
Fax: 33 1 69 82 31 29; E-mail: carlier@pegase.enzy.cnrs-gif.fr.
1
The abbreviation used is: T
b
4
, thymosin
b
4
.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 21, Issue of May 24, pp. 12302–12309, 1996
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
12302
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purified by poly-L-proline chromatography as described,
2
followed by
anion exchange chromatography (Resource Q, 6 ml, Pharmacia Biotech
Inc.) using a linear gradient of NaCl in 20 m
M Tris-Cl
2
, pH 7.5. The
purified profilins were then concentrated using centricon 10 (Amicon)
and exchanged into the desired buffer.
Fluorescence Measurements—Actin polymerization was monitored by
pyrenyl fluorescence, using a Spex Fluorolog 2 spectrofluorimeter. A
maximum amount of 1% pyrenyl-actin was present in all samples to
minimize the bias in the data due to the fact that profilin does not bind
to pyrenyl-labeled actin.
Polymerization of CaATP-actin was carried out in a Ca-F-polymeri-
zation buffer made up by adding 1 m
M CaCl
2
and 0.1 M KCl to G-buffer
(5 m
M Tris-Cl
2
,pH7.5,1mMdithiothreitol, 0.2 mM ATP, 0.1 mM CaCl
2
,
0.01% NaN
3
). Polymerization of MgATP-actin was carried out in a
Mg-F-buffer (2) made up by supplementing G-buffer with 2 m
M MgCl
2
and 0.1 M KCl (physiological ionic conditions).
All experiments were performed using G-actin in which Cys
374
was
checked to be thoroughly reduced, to be sure that all of the actin was
able to bind profilin with high affinity.
Steady-state fluorescence measurements of F-actin in the presence of
T
b
4
or profilin were carried out as described (2) following overnight
incubation at room temperature in the dark.
Nucleotide Exchange Measurements—The kinetics of nucleotide ex-
change on G-actin was monitored by the change in fluorescence of
e
ATP
bound to G-actin, using a stopped-flow, as described previously (12).
Measurement of Filament Treadmilling at Steady State by an ATP
Chase Experiment—G-actin (20
m
M) was equilibrated overnight in G-
buffer containing 4
m
M
3
H-labeled ATP, polymerized by the addition of
2m
MMgCl
2
and 0.1 M KCl, and diluted 2-fold in 5 mM Tris-Cl
2
, pH 7.8,
1m
Mdithiothreitol, 0.1 mM CaCl
2
,2mMMgCl
2
, 0.1 M KCl (polymeri-
zation buffer without ATP). After 2 h, the F-actin solution was supple-
mented with a chase of 0.2 m
M unlabeled ATP and gently split into
several samples containing different concentrations of profilin. Aliquots
of 150
m
l of each sample were spun at 400,000 3 g for 20 min at 20 °C
at different time intervals. The
3
H radioactivity was measured in the
supernatant as a function of time, and the slope of the initial linear
increase was taken as an indication of the treadmilling rate of actin
filaments.
Dependence of the Rate of Filament Growth on G-actin Concentration
in the Presence of a Constant Amount of Profilin-actin—The initial rate
of filament growth at barbed ends was measured as a function of
G-actin concentration using pyrene fluorescence, as described previ-
ously (16). The 300-
m
l sample contained MgATP-G-actin (1% pyrenyl-
labeled) and profilin at different concentrations, in G-buffer supple-
mented at time 0 with 2 m
M MgCl
2
and 0.1 M KCl. The contents of the
cuvette were mixed, and 30
m
lofa10
m
MF-actin solution (also contain-
ing 1% pyrenyl-labeled actin) were gently added as seeds to the solu-
tion. The amounts of G-actin and profilin were adjusted as follows so
that the concentration of profilin-actin was constant at all concentra-
tions of G-actin. The total concentrations of profilin, [P
o
], and of G-actin,
[A
o
], are linked by the law of mass action,
~@P
o
# 2 @PA#!~@A
o
# 2 @PA#!
@PA#
5 K
P
(Eq. 1)
Hence the total concentration of profilin [P
o
] added to each sample
was derived by rearranging Equation 1 as follows.
@P
o
# 5 @PA#
S
1 1
K
P
@A
o
# 2 @PA#
D
(Eq. 2)
In different series of experiments, [PA] was maintained constant at
each total G-actin concentration [A
o
]. This condition was realized by
adding the amount [P
o
] of profilin, given by Equation 2, using a value of
0.3
m
M for K
p
(2, 12). For instance, in a series of samples in which [PA]
was fixed at 0.5
m
M, the values of [A
o
] and [P
o
] present in the samples
were linked by the following.
@P
o
# 5 0.5
S
1 1
0.3
@A
o
# 2 0.5
D
(Eq. 3)
Profilin is known to bind to Cys
374
-labeled actin with a 2 orders of
magnitude lower affinity than to unlabeled actin (17). Since actin in
these experiments was only 1% pyrenyl-labeled, the binding of profilin
to pyrenyl-actin was practically negligible. As a consequence, the par-
ticipation of profilin-actin complex to filament elongation does not give
any fluorescence signal, and the specific increase in pyrene fluorescence
linked to polymerization of G-actin is proportional to [A
o
]/([A
o
] 2 [PA]).
Hence the initial rates of filament growth measured in fluorescence
units/s were multiplied by ([A
o
] 2 [PA])/[A
o
] to be compared quantita-
tively, in a meaningful fashion, with the rates measured in the control
curve carried out in the absence of profilin. The corrected rates were
then plotted as a function of free ATP-G-actin concentration [A] 5 [A
o
]
2 [PA]. The corrected rate therefore represents the sole contribution of
unliganded G-actin to the growth of filaments from G-actin and profilin-
actin units.
RESULTS
Rate of Filament Growth from MgATP-G-actin Subunits in
the Presence of Profilin-Actin Complex—The following experi-
ment was designed to understand how the kinetic parameters
for barbed end elongation where affected by profilin. The con-
centration of profilin-actin complex, which participates in fila-
ment assembly, was kept constant at 0, 0.5, and 1
m
M in three
series of experiments, while the concentration of free G-actin
was varied (see “Materials and Methods”). Data, displayed in
Fig. 1, show that in the presence of profilin-actin, MgATP-G-
actin undergoes net positive barbed end growth at concentra-
tions below the critical concentration measured in the absence
of profilin in the control J(c) plot. The critical concentration of
MgATP-G-actin was decreased at least 5-fold in the presence of
0.5 or 1
m
M profilin-actin, in agreement with steady state meas-
urements (2). This piece of data testifies that the decrease in
critical concentration observed previously (2) was truly a de-
crease in ATP-G-actin steady state concentration. The slope of
the J(c) plot was not modified in the presence of profilin-actin,
which demonstrated that the rate constant for association of
G-actin to barbed ends is not affected by the participation of
profilin-actin in barbed end assembly. On the other hand, the
extrapolated value of the dissociation rate constant of G-actin
from filament ends (ordinate intercept of the J(c) plot) is de-
creased by profilin.
Profilin Increases the Treadmilling Rate of Actin Fila-
ments—Treadmilling of actin filaments has been shown by
Wegner (18) to result from the difference in critical concentra-
2
H. E. M. Christensen, C. T. Tan, S. Ramachandran, U. Surana, C.
H. Dong, and N. H. Chua, submitted for publication.
FIG.1.Kinetic evidence for the decrease in the partial critical
concentration of ATP-G-actin due to the participation of profi-
lin-actin in filament assembly at the barbed ends. The initial rate
of growth of actin filaments at the barbed end was derived from the
initial increase in pyrenyl-actin fluorescence upon addition of F-actin
seeds (10
m
M F-actin) to a solution of ATP-G-actin in physiological ionic
strength. Measurements were made in the absence of profilin (●) and in
the presence of 0.5
m
M (E)or1
m
M(M) profilin-actin. Rates were
corrected for the fact that profilin binding is restricted to unlabeled
G-actin, which affects the specific fluorescence of free ATP-G-actin (see
“Materials and Methods”). Note that in the presence of profilin-actin,
only the partial contribution of free ATP-G-actin in barbed end growth
is measured in this experiment.
Nucleotide Exchange and Hydrolysis in Profilin Function 12303
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tion at the pointed and barbed ends of actin filaments, which is
itself due to the involvement of ATP hydrolysis in actin assem-
bly. Since profilin, as shown in Fig. 1, increases the energetic
difference between the two ends, an effect of profilin on the
treadmilling rate was expected.
The rate of treadmilling can be appreciated from the rate at
which [
3
H]ADP-F-actin subunits, in filaments assembled from
Mg[
3
H]ADP-G-actin, are released in the medium following a
chase of unlabeled ATP (see “Materials and Methods”). Fig. 2
shows that the rate of filament treadmilling is increased by
25–30% at most by profilin, in a concentration-dependent fash-
ion, consistent with a high affinity of profilin. Although the
turnover of actin filaments is essentially due, for Mg-actin, to
the treadmilling process, diffusional monomer-polymer ex-
change may also contribute to the observed loss of [
3
H]ADP-F-
actin subunits. In order to verify that the observed increase in
filament turnover is an actual increase in treadmilling medi-
ated by the profilin-induced larger difference in critical concen-
tration at the two ends, a control chase experiment was carried
out, in which the barbed ends were capped by gelsolin. Under
these conditions treadmilling does not occur. Because pointed
ends are 1 order of magnitude less dynamic than the barbed
ends, a larger amount of F-actin (25
m
M) was used in that
experiment, and the gelsolin-actin ratio was set at 1:100, so
that the end number concentration was 1 order of magnitude
higher than in the previous experiment carried out in the
absence of gelsolin. A slow release of [
3
H]ADP in the superna-
tant of F-actin was observed subsequent to the ATP chase, due
to diffusional monomer-polymer exchange at the pointed ends.
The rate of diffusional exchange was not affected by profilin.
Therefore, the increase in filament turnover rate recorded
when both ends are free in solution is an actual increase in the
rate of treadmilling. In conclusion, profilin increases the rate of
treadmilling of actin filaments as a consequence of the in-
creased energetic difference between the two ends.
Profilin Does Not Promote Actin Assembly at the Barbed End
When the Polymerization of ATP-actin Is Quasireversible
(CaATP-actin)—We have previously shown (2) that in the pres-
ence of ADP profilin was not able to participate effectively in
assembly at the barbed end and to decrease the critical concen-
tration but behaved strictly as an ADP-G-actin-sequestering
protein. This finding was interpreted as evidence that ATP
hydrolysis was involved in the function of profilin in barbed end
assembly. If polymerization of ATP-actin was reversible, profi-
lin would be expected to behave as a purely ATP-G-actin se-
questering agent. This prediction was tested in the following
way. It is well known that the structural and functional prop-
erties of actin are dependent on the nature of the divalent
metal ion (Mg
21
or Ca
21
) that interacts with the
b
- and
g
-phos-
phates of ATP bound to actin (for reviews, see Refs. 19 and 20).
In particular, nucleation of filament is facilitated, and the
critical concentration for filament assembly is lower, when
Mg
21
(which is the physiologically actin-bound metal ion) is
bound to ATP (21–23). The rate and the mechanism of ATP
hydrolysis on F-actin are also different on Mg-actin and Ca-
actin (24, 25), a conclusion supported by recent molecular mod-
elling studies of the metal-nucleotide binding site on G-actin
(26). Our earlier work showed that in the polymerization of
MgATP-actin, ATP hydrolysis appeared closely coupled to the
growth of filament in a large range of monomer concentration
(24, 25). The nature of the nucleotide bound to the barbed end
terminal subunits varied with the concentration (c) of MgATP-
G-actin. Essentially ATP or ADP-P
i
was bound well above the
critical concentration, while at steady state the proportion of
terminal F-ADP subunits increased. As a result, the depend-
ence of the rate of filament growth on monomer concentration
(J(c) plot) curved downwards in the region of the critical con-
centration, as the proportion of rapidly dissociating terminal
F-ADP subunits increased. In the presence of P
i
, the J(c) plot
was identical at high rates of growth but remained linear and
extrapolated to a somewhat lower critical concentration be-
cause terminal F-ADP-P
i
subunits dissociate more slowly than
F-ADP subunits (27). Similarly, phosphate analogs BeF
3
2
and
AlF
4
2
decreased the critical concentration at the barbed ends
(28). In contrast, in the polymerization of CaATP-actin, ATP
was hydrolyzed very slowly, and filaments grew with a large
cap of ATP subunits even in a range of low G-actin concentra-
tions (25). The J(c) plot displayed no appreciable change in
slope at the critical concentration, as a result of the exclusive
presence of F-ATP ends at the critical concentration (24). In
other words, due to the strong uncoupling between actin as-
sembly and ATP hydrolysis, the polymerization of CaATP-actin
may be considered as quasireversible. Accordingly, the tread-
milling rate of filaments assembled from CaATP-actin in the
presence of only KCl appears to be very slow (29, 30). We
confirmed this observation and further checked that this slow
rate was not affected by profilin, in contrast with the result
obtained for Mg-actin. Treadmilling is triggered by the differ-
ence in critical concentrations between the two ends of the
filament, which is established because ATP hydrolysis is asso-
ciated with actin incorporation into the filament (18). If the
polymerization of CaATP-actin is quasireversible, critical con-
centrations are expected to be identical at the two ends, as is
observed for the reversible polymerization of ADP-actin (31).
Fig. 3 shows that this is indeed the case. At most 10% differ-
ence in critical concentrations at the barbed and pointed ends
was measured for Ca-actin polymerized in the presence of 0.1
M
KCl, while in a parallel experiment carried out with Mg-actin
under the same ionic conditions, the well known 5-fold differ-
ence in critical concentrations between the two ends was re-
corded. In an early work using electron microscopy to measure
of the rates of growth of actin filaments at the barbed and
pointed ends, Pollard and Mooseker (32) also noticed that the
critical concentrations were very similar at the two ends in 20
m
M KCl and very different in 5 mM MgSO
4
1 0.1 M KCl.
FIG.2.Profilin increases the turnover rate of actin filaments.
A solution of 10
m
M [
3
H]ADP-F-actin prepared as described under
“Materials and Methods” was split in two samples supplemented or not
with the indicated amount of profilin. A chase amount of ATP (0.2 m
M)
was applied to each sample at time 0. Aliquots were centrifuged at
different times over a period of 8 h. The linear increase in [
3
H]ADP in
the supernatant was measured and normalized to 1 for the control run
in the absence of profilin. The increase in turnover rate in the presence
of profilin is plotted as percentage increase of the rate measured in the
control. Different symbols refer to independent experiments. The inset
represents typical time courses of [
3
H]ADP increase in the supernatant
in the absence (●) or in the presence (E)of5
m
Mprofilin.
Nucleotide Exchange and Hydrolysis in Profilin Function12304
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Although these conditions were not, at that time, explicitly
referred to as describing Ca-actin and Mg-actin assembly, re-
spectively, the results obtained (32) are similar to the ones
reported here. The ability of profilin to depolymerize F-actin
was further investigated for Ca-actin or Mg-actin compara-
tively, with capped and uncapped barbed ends. The data, dis-
played in Fig. 4a, show that upon addition of increasing
amounts of profilin to Ca-F-actin, only F-actin depolymeriza-
tion due to “sequestration” of Ca-G-actin by profilin was ob-
served, both with capped and uncapped barbed ends. The de-
crease in F-actin at steady state, i.e. the increase in the
concentration of profilin-actin complex, [PA], was linear versus
the total amount of profilin P
o
, as described by the equation,
@PA# 5 @P
o
#
A
c
A
c
1 K
p
(Eq. 4)
where A
c
represents the critical concentration and K
p
the equi-
librium dissociation constant for the profilin-actin complex.
Since the critical concentrations for assembly of Ca-actin are
almost identical at the two ends, almost the same slopes were
observed whether the barbed ends were free or capped. The
derived value of K
p
for binding of profilin to CaATP-actin was
0.8
m
M.
In contrast, when the same experiment was done with Mg-
actin, profilin only sequestered G-actin when barbed ends were
capped by gelsolin. With uncapped barbed ends, no effective
depolymerization of F-actin was observed upon addition of pro-
filin, as previously demonstrated and explained by the profilin-
induced decrease in critical concentration for assembly of Mg-
actin at the barbed ends.
The interpretation of the above fluorescence data in the
presence of profilin (Fig. 4a) in terms of reliable estimates of
assembled and unassembled actin is questionable, since profi-
lin does not bind to pyrenyl-actin (17). For that reason, one
would logically expect that the fluorescence of samples of F-
actin containing different concentrations of profilin be the same
in all samples at equilibrium, because pyrenyl-labeled actin
partitions between F-actin and free G-actin, while the profilin-
actin complex contains only unlabeled actin (33). As discussed
under “Materials and Methods,” the specific fluorescence of
F-actin should increase as the concentration of profilin-actin
complex increases. In fact, we checked that the fluorescence
readings shown in Fig. 4a remained stable over a period of
12–24 h following the addition of profilin to F-actin and in-
creased thereafter very slowly, over a period of 2–3 days, to
reach the expected constant fluorescence level at all profilin
concentrations. We propose that the reason why pyrenyl fluo-
rescence measurements truly reflect the amount of F-actin in
the presence of profilin is the following. The addition of profilin
to preassembled F-actin results in endwise depolymerization of
pyrenyl-labeled filaments until steady state is reached, at
which point the remaining filaments keep their initial intrinsic
fluorescence, while the monomer pool consists of G-actin at the
critical concentration (with a higher pyrenyl-labeling ratio
than the filaments) and unlabeled profilin-actin complex. Since
monomer-polymer exchange reactions are very slow, the label-
ing of filaments remains essentially unchanged for many
hours. Over much longer periods of time (days), pyrenyl-actin
redistributes over F-actin and G-actin. To check this interpre-
tation of the data in Fig. 4a, a sedimentation assay of assem-
bled and unassembled actin in the presence of profilin was
carried out. Mg-actin was assembled at 6
m
M in the presence or
absence of gelsolin, and different amounts of profilin were
added to the two series of samples. The samples were centri-
fuged at 400,000 3 g after 16 h of incubation. The amount of
unassembled actin in the supernatant was evaluated by SDS-
polyacrylamide gel electrophoresis followed by Coomassie Blue
staining. The data shown in Fig. 4b demonstrate that when
barbed ends were capped, profilin caused actin depolymeriza-
tion in proportion to the amount of profilin added. 80% of total
actin was found in the supernatant in the presence of 10
m
M
profilin. This is consistent with a value of 0.66
m
M for K
p
,
according to Equation 4, using C
c
5 0.5
m
M. In contrast, when
barbed ends were free, actin was barely detectable in the su-
pernatant. Note that if profilin displayed a Mg-actin seques-
tering activity when barbed ends are free (C
C
B
5 0.1
m
M) one
would expect to measure an amount of unassembled actin
equal to C
C
B
1 [P
tot
](C
C
B
/(C
C
B
1 K
p
) in the supernatant. In the
presence of 10
m
M profilin, and using K
p
5 0.66
m
M, the amount
of unassembled actin should be 0.1 1 10(0.1/0.76) 5 1.42
m
M,
which represents 24% of total actin in the experiment shown in
Fig. 4b. Clearly the amount of actin in the supernatant is much
lower than 5% of total actin, which demonstrates that C
C
B
is
lowered by profilin. In conclusion, the sedimentation data con-
firm that the fluorescence measurements in the presence of
profilin truly reflect the amount of F-actin.
The difference between the effects of profilin in the assembly
of Ca-actin and Mg-actin at the barbed ends is further illus-
trated in the presence of thymosin
b
4
, in Fig. 4c. Profilin pro-
moted assembly off the pool of T
b
4
-Mg-actin complex, due to
the decrease in critical concentration, as previously demon-
strated (2), but only depolymerization of Ca-F-actin by profilin
was observed in addition to the depolymerization elicited by
T
b
4
.
In summary, the above results demonstrate that when ATP-
actin polymerizes in a reversible fashion, profilin only shows a
G-actin-sequestering activity. The same result had been ob-
tained with ADP-actin (2). The new evidence with CaATP-actin
confirms the view that ATP hydrolysis, and not simply ATP, is
necessary to explain that profilin lowers the critical concentra-
tion at the barbed end, thereby promoting actin assembly off
the pool of sequestered actin.
A consequence of the above results is that when Mg
21
ions
are added to Ca-F-actin assembled at steady state (with free
barbed ends) in the presence of profilin, repolymerization of
actin occurs to a much larger extent than expected from the
FIG.3.Critical concentrations for assembly of CaATP-actin or
MgATP-actin at the barbed and pointed ends under physiolog-
ical ionic conditions. Solutions of 3
m
M F-actin (1% pyrenyl-labeled)
were assembled in buffer F containing 5 m
M Tris-Cl
2
, pH 7.5, 0.1 M KCl,
0.2 m
M ATP, 0.2 mM dithiothreitol, 0.1 mM CaCl
2
supplemented with
either 0.2 m
M CaCl
2
(Ca-F-buffer, circles)or2mMMgCl
2
(Mg-F-buffer,
squares) in the presence (open symbols) or absence (closed symbols)of
10 n
M gelsolin. Serial dilutions of the four samples in homologous Ca-F
or Mg-F-buffer were carried out. The fluorescence of pyrenyl F-actin
was read after 16 h of incubation in the dark.
Nucleotide Exchange and Hydrolysis in Profilin Function 12305
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small difference in critical concentration for assembly of Ca-
actin (0.5
m
M) and Mg-actin (0.1
m
M) (data not shown). This
result is quantitatively explained by the exchange of Mg
21
for
bound Ca
21
on G-actin, which leads to different steady state
conditions and to a change in the function of profilin from
sequestering to assembly-promoting agent. This enhanced po-
lymerization has previously been observed by others (34, 35)
and was attributed to a dissociation of the profilin-actin com-
plex by Mg
21
ions. In fact Mg
21
ions do not decrease the
affinity of profilin for G-actin, as shown here (Fig. 4a) as well as
in previous works (12, 14). The present interpretation hence
provides a satisfactory account of the observed reaction, con-
sistent with the above differences in polymerization of CaATP-
actin and MgATP-actin.
The Ability of Profilin to Increase the Rate of Nucleotide
Dissociation from Actin Is Not Required for Its Function in
Actin Assembly—In showing that the nature of the nucleotide
bound to the barbed end terminal subunits is important in
profilin function, the above results suggest that when the pro-
filin-actin complex associates to a barbed end, profilin might
modify the rates of nucleotide exchange or hydrolysis or of P
i
release on that terminal subunit, which might mediate the
function of profilin in actin assembly. It is therefore important
to establish whether the property of profilin to decrease the
critical concentration for actin assembly at the barbed end is or
is not linked to its property to enhance the rate of nucleotide
dissociation from actin. We have been helped, in this issue, by
a systematic search among different profilin species in nature.
Profilins are ubiquitous proteins in eukaryotes from protozoa
to vertebrates. They seem to share structural and functional
properties regarding actin binding, although the sequences
show a moderate degree of similarity. Three large categories of
known profilins can be distinguished, the lower eukaryote pro-
filins (amoebae, molds, yeasts, and others), the vertebrate pro-
filins, and the plant profilins. The degree of sequence homology
within each class is at least 70%, while it is only 25–40%
between profilins from two different classes. While profilins
from Acanthamoeba castellanii (9) and from vertebrates (10–
12) have been shown to increase the rate of nucleotide ex-
change on G-actin, no corresponding information exists regard-
ing plant profilins. In the plant A. thaliana, four profilin
species have been found, two vegetative forms (profilins 1 and
2) and two floral forms (profilins 3 and 4). The sequences of
profilins 1 and 2, on the one hand, and of profilins 3 and 4, on
the other hand, show 89 and 91% identity, respectively. The
vegetative and floral forms are 71–75% identical. The com-
pared sequences of profilins from bovine spleen, A. castellanii,
birch pollen, and A. thaliana are shown in Table I. Arabidopsis
profilins show 90% homology with birch pollen profilin.The
alignment shows that plant profilins are closer to amoeba than
to vertebrates and are characterized by an insertion of 3–6
amino acids after position 15, i.e. between helix 1 and strand 1,
opposite to the actin-binding region in the three-dimensional
structure.
We have assayed A. thaliana profilins 1 and 3 for their
binding to G-actin, Mg-G-actin sequestration (with capped
barbed ends), promotion of MgATP-actin assembly at the
barbed ends, and effect on the rate of nucleotide exchange on
G-actin. In low ionic strength buffer, formation of the plant
profilin-actin complex was associated with a quenching of tryp-
tophan fluorescence, quantitatively identical (25% quenching
of actin fluorescence with both profilins 1 and 3, data not
shown) to the one observed upon interaction of bovine spleen
profilin with G-actin (14). From the profilin concentration de-
pendence of the fluorescence change (data not shown), equilib-
rium dissociation constants of 1.8 and 2.3
m
M were derived for
FIG.4.The complex of profilin with CaATP-G-actin does not
productively participate in assembly at the barbed ends and
does not affect the critical concentration. A, fluorescence meas-
urements. Solutions of 3
m
M F-actin (1% pyrenyl-labeled) assembled in
Ca-F-buffer (circles) or Mg-F-buffer (squares) in the presence (open
symbols) or absence (closed symbols)of10n
Mgelsolin were incubated
for 16 h in the presence of profilin at the indicated concentrations. B,
sedimentation measurements. Samples of 6
m
M F-actin, assembled in
Mg-F-buffer in the absence (A) or presence (B)of15n
Mgelsolin, were
supplemented with profilin at the indicated concentrations, incubated
for 16 h and centrifuged at 400,000 3 g, 20 °C, for 30 min. The super-
natants were submitted to SDS-polyacrylamide gel electrophoresis.
Note that the concentration of unassembled actin is insignificant when
barbed ends are free, while a large amount of actin is depolymerized
when barbed ends are capped. The rightmost lane shows the total
amount of actin in each sample before centrifugation. C, profilin does
not lower the critical concentration of CaATP-actin at the barbed end.
Squares, a solution of F-actin (1.5
m
M, 1% pyrenyl-labeled) assembled in
Mg-F-buffer in the presence of 15
m
M T
b
4
was supplemented with
profilin at the indicated concentrations. Circles, a solution of F-actin (3
m
M, 1% pyrenyl-labeled) assembled in Ca-F-buffer in the presence of 20
m
M T
b
4
was supplemented with profilin. Pyrenyl fluorescence was mon-
itored after 16 h of incubation.
Nucleotide Exchange and Hydrolysis in Profilin Function12306
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profilin 1-actin and profilin 3-actin complexes, respectively.
Hence, plant profilins have a 5-fold lower affinity than bovine
spleen profilin for G-actin. The affinity of plant profilins for
G-actin was also derived from the G-actin sequestration assay.
Data displayed in Fig. 5a show that under physiological ionic
conditions (2 m
M MgCl
2
, 0.1 M KCl) and when barbed ends are
capped, plant profilins cause depolymerization of F-actin as
described by Equation 4, with a lower efficiency than bovine
spleen profilin. K
p
values of 1.8 and 2.6
m
M were derived for
profilins 1 and 3, respectively (as compared with K
p
5 0.45
m
M
found for bovine spleen profilin in a parallel assay). In contrast,
Fig. 5b shows that when barbed ends were not capped and a
pool of unpolymerized actin was created by T
b
4
(20
m
M)in
solution, plant profilins, like vertebrate profilin, promoted ac-
tin assembly off the pool of T
b
4
-actin complex, an effect medi-
ated by the decrease in critical concentration at the barbed end
(2). Therefore, despite their lower affinity for G-actin, plant
profilins behave in a manner identical to that of bovine spleen
profilin, regarding their different effects in actin assembly
when barbed ends are capped versus uncapped. On the other
hand, when the rate of nucleotide exchange was assayed under
physiological ionic conditions by monitoring the kinetics of
dissociation of bound
e
ATP upon addition of ATP (12), no
change in the first order rate constant was observed upon
addition of saturating amounts of plant profilins, while a 50-
fold increase from 0.04 s
21
to2s
21
was observed when the
assay was carried out in parallel with spleen profilin. Although
the profilin-actin interface is probably similar for bovine spleen
and plant profilins, as judged from the tryptophan fluorescence
data, plant profilins appear unique among profilins from dif-
ferent species in being unable to enhance the rate of nucleotide
dissociation from monomeric actin. We suggest that these pro-
filins are therefore unlikely to enhance P
i
or nucleotide disso-
ciation from the terminal F-actin subunit as well. In conclu-
sion, the ability of profilins to decrease the critical
concentration for actin assembly at the barbed end is not linked
to their effect on nucleotide exchange on actin.
DISCUSSION
The present results bring new support to the view that
profilin lowers the critical concentration for actin assembly at
the barbed end and thereby enhances assembly at this end.
Direct kinetic evidence is provided for active growth of fila-
ments taking place at concentrations of MgATP-G-actin well
below the critical concentration, when profilin-actin complex is
present even in low amounts (0.5
m
M). The presence of profilin-
actin complex however does not modify the rate constant for
association of MgATP-G-actin to the barbed ends. As a conse-
quence of the decrease in critical concentration at the barbed
ends, the treadmilling rate, which reflects the energetic differ-
ence between the two ends, is increased (25–30%) by profilin.
We had proposed (2) that ATP hydrolysis associated with
actin assembly was involved in the function of profilin. This
proposal relied 1) on the idea that an isoenergetic model for
growth of actin filaments from either G-actin alone or G-actin
1 profilin could not theoretically account for the observed de-
crease in critical concentration, and 2) on the experimental
evidence for the sole G-actin sequestering activity of profilin in
the presence of ADP. The present results bring more experi-
mental support to the view that ATP hydrolysis, rather than
ATP binding, at the barbed ends of actin filaments at steady
state plays a role in profilin function. Advantage was taken of
the different properties of CaATP-actin and MgATP-actin (see
Ref. 19, for review). The hydrolysis of ATP following incorpo-
ration of a CaATP-actin in the filament is so slow that its
polymerization can be considered as quasireversible, as dem-
onstrated by the almost identical critical concentrations at the
barbed and pointed ends and the barely detectable treadmilling
at steady state. The present data, showing that profilin be-
haves with respect to Ca-actin as a purely G-actin-sequestering
protein whether barbed ends are capped or uncapped and
therefore does not affect the treadmilling rate, bring more
support to the role of ATP hydrolysis in the function of profilin
in barbed end assembly.
How is ATP hydrolysis used by profilin in barbed end assem-
TABLE I
Alignment of selected profilin sequences
Nucleotide Exchange and Hydrolysis in Profilin Function 12307
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bly? To examine whether profilin accelerated hydrolysis or P
i
release on a terminal actin subunit, following profilin-actin
association to the filament barbed end, we have shown that
plant profilins, which do not affect the rate of nucleotide ex-
change on actin, still lower the critical concentration at the
barbed end. Therefore, the main physiological function of pro-
filin does not necessitate a faster rate of metal/nucleotide dis-
sociation from actin, a conclusion opposite to previous models
(13, 36, 37).
The present work combined with older results concerning the
mechanism of ATP hydrolysis associated with the polymeriza-
tion of MgATP-actin leads to a model for profilin function,
proposed in Scheme I. Nucleotide hydrolysis is highly favored
on an ATP-F-actin subunit immediately adjacent to an ADP-
F-actin subunit (25). The association of MgATP-G-actin-profi-
lin complex to an F-ATP end has little chance to be followed by
ATP hydrolysis; therefore, in this case profilin dissociation is
not enhanced. In contrast, association of MgATP-G-actin-pro-
filin to an F-ADP end will trigger ATP hydrolysis on the newly
added subunit, which causes profilin dissociation, due to the
lower affinity of profilin for ADP-actin than for ATP-actin (2,
14). In this model, the presence of terminal ADP-actin subunits
at steady state and the coupling of ATP hydrolysis to associa-
tion of profilin-actin to barbed ends are necessary for the net
incorporation of actin in filaments from profilin-actin units.
This is satisfied under physiological ionic conditions (MgATP-
actin, 2 m
M MgCl
2
, 0.1 M KCl), in which ATP hydrolysis ap-
pears closely coupled to filament growth in a range of concen-
trations around the critical concentration. As a result,
treadmilling is relatively efficient under these conditions and is
enhanced by profilin. In contrast, ATP hydrolysis is largely
uncoupled from the assembly of Ca-actin, and the fact that
terminal subunits are only CaATP-F-actin at equilibrium does
not allow any net incorporation of actin from profilin-actin
units. Accordingly, treadmilling is low for Ca-actin and re-
mains unchanged in the presence of profilin.
From a structural point of view, it is interesting to note that
profilin-actin complexes made from different profilin species
(amoebae, plant, vertebrates), which display different equilib-
rium dissociation constants, are equally able to participate in
assembly at the barbed ends. Indeed, the different effect of
these profilins on nucleotide dissociation is likely to be linked to
slight differences in the structure of actin in the complex, i.e. a
more or less pronounced opening of the cleft containing metal-
nucleotide. The fact that despite these different structures all
profilin-actin complexes equally well participate in barbed end
assembly indicates that upon association of a profilin-actin unit
to the barbed end, the constraints imposed by the actin-actin
contacts in the polymer, especially at the interface between the
bottom of subdomain 1 of the barbed end terminal subunit and
the tops of subdomains 2 and 4 of the newly added subunit, are
sufficient to counteract the profilin-induced deformation of the
FIG.5.Plant profilins behave like vertebrate profilins in pro-
moting assembly of MgATP-actin at the barbed ends and like
sequestering actin when barbed ends are capped. A, capped
barbed ends. F-actin (1.5
m
M, 1% pyrenyl-labeled) was assembled in
Mg-F-buffer containing 5 n
M gelsolin, and supplemented with bovine
spleen profilin (●), A. thaliana profilin 1 (E), or A. thaliana profilin 3
(å) at the indicated concentrations. Pyrenyl fluorescence was monitored
after 16 h incubation. B, free barbed ends. F-actin (1.5
m
M 1% pyrenyl-
labeled) was assembled in Mg-F-buffer containing 20
m
M T
b
4
, and
supplemented with bovine spleen profilin (●), A. thaliana profilin 1 (E),
A. thaliana profilin 3 (å) at the indicated concentrations.
SCHEME 1. Involvement of ATP hydrolysis in the participation of profilin-actin in filament assembly. a, reversible, nonproductive
association of profilin-actin to barbed ends carrying a terminal ATP subunit. b, net incorporation of actin from profilin-actin linked to ATP
hydrolysis at the F-ATP/F-ADP boundary.
Nucleotide Exchange and Hydrolysis in Profilin Function12308
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actin molecule.
The very high homology between Arabidopsis profilins and
birch pollen profilin indicates that most probably all plant
profilins fail to enhance the rate of nucleotide exchange on
G-actin. Interestingly, it has been recently shown that 1) birch
pollen profilin can functionally replace mammalian profilin in
living cells (35); 2) Arabidopsis profilins can replace yeast pro-
filin
2
; 3) the failure of profilin-deficient Dictyostelium cells to
form a fruiting body (38) can be rescued by maize pollen profilin
(39). These results, combined with the present data, demon-
strate that the function of profilin in vivo is not mediated by its
effect on nucleotide exchange on G-actin.
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