(Received for publication, July 24, 1995; and in revised form, September 15, 1995)
From the
Profilin is an actin- and phosphatidylinositol 4,5-bisphosphate-binding protein that plays a role in the organization of the cytoskeleton and may be involved in growth factor signaling pathways. The subcellular localization of profilin was examined in the yeast Saccharomyces cerevisiae. Immunoblot analysis showed that profilin was localized in both the plasma membrane and cytosolic fractions of the cell. Actin was bound to the profilin localized in the cytosol. The association of profilin with the membrane was peripheral and mediated through interaction with phospholipid. The phospholipid dependence of profilin for membrane binding was examined in vitro using pure profilin and defined unilamellar phospholipid vesicles. The presence of phosphatidylinositol 4,5-bisphosphate in phospholipid vesicles was required for maximum profilin binding. Moreover, the binding of profilin to phospholipid vesicles was dependent on the surface concentration of phosphatidylinositol 4,5-bisphosphate. The subcellular localization of profilin was examined in vivo under growth conditions (i.e. inositol starvation of ino1 cells and glucose starvation of respiratory deficient cells) where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were depleted. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate levels resulted in a translocation of profilin from the plasma membrane to the cytosolic fraction. Profilin translocated back to the membrane fraction from the cytosol under growth conditions where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were replenished. These results suggested that phosphoinositide metabolism played a role in the localization of profilin.
Profilin is a ubiquitous actin- and
PIP(
)-binding protein found in eucaryotic
cells(1) . It is believed that profilin assists in the
regulation of the reorganization of the microfilament
cytoskeleton(2) . Profilin competes with other actin-binding
proteins for globular actin(3) , catalyzing the exchange of
nucleotide phosphates on monomeric actin, thereby activating actin for
polymerization(4) .
Profilin has also been shown to bind
anionic phospholipids(5) . However, at physiological salt
concentrations, profilin binds exclusively to
PIP(6) . Profilin cannot bind to actin and
PIP
simultaneously(7) , as the actin- and
PIP
-binding sites are sufficiently close as to impart
mutually exclusive binding(7) . The interaction of profilin
with PIP
may regulate the availability of profilin for
interaction with actin(5) . Another role proposed for
profilin's association with PIP
is to prevent the
phospholipase C
1-mediated hydrolysis of
PIP
(8) , and thus the generation of the second
messengers inositol trisphosphate and diacylglycerol(9) .
However, tyrosine phosphorylation of phospholipase C
1 allows
hydrolysis of PIP
even in the presence of
profilin(10) .
We are using the yeast Saccharomyces
cerevisiae as a model eucaryote to study profilin. The gene (PFY1) encoding for profilin has been cloned from S.
cerevisiae(11) . S. cerevisiae profilin resembles
profilins from higher eucaryotes in size (15 kDa), ability to bind
actin, PIP
, and polyproline, and inhibit ATP hydrolysis by
monomeric actin (11, 12, 13) . Moreover,
profilin is required for the proper organization of the actin
cytoskeleton into actin cables, which occur at regions of active growth
and for proper maintenance of cell polarity(12) . It is unclear
whether these roles for profilin are related to its ability to bind
actin and/or PIP
(13) . The physiological relevance
of profilin's ability to bind polyproline is also unclear (13) .
The fact that binding to actin and PIP are mutually exclusive in profilin, and that actin is localized
in the cytosol (14) whereas PIP
is localized in the
plasma membrane(15) , raises the suggestion that profilin is
localized in these two compartments of the cell. In this report we
demonstrated that profilin was indeed localized to the plasma membrane
and cytosolic fractions of S. cerevisiae. The majority of
profilin was bound to plasma membranes. However, under growth
conditions where the plasma membrane levels of PIP
were
depleted, profilin was found to translocate from the membrane to the
cytosol. Profilin translocated back to the membrane fraction from the
cytosol under growth conditions where plasma membrane levels of
phosphatidylinositol 4,5-bisphosphate were replenished. The results of
our studies were consistent with the notion that the metabolism of
PIP
played a role in the localization of profilin.
Figure 1: Subcellular localization of profilin. Panel A, equal volumes of the total membrane (lane 1) and cytosolic (lane 2) fractions were subjected to immunoblot analysis using anti-profilin antibodies. The position of profilin is indicated by the arrow in the figure. Panel B, the density of the profilin bands shown in panel A was quantified by scanning densitometry. Panel C, equal volumes of the plasma membrane (PM), mitochondrial (Mito), microsomal (Micro), and cytosolic (Cyto) fractions were subjected to immunoblot analysis using anti-profilin antibodies. The density of the profilin bands was quantified by scanning densitometry. The data shown are representative of two independent experiments.
We next questioned whether the association of profilin to membranes was integral or peripheral. Peripheral membrane proteins are associated with membranes through noncovalent interactions and can be released by treatment with high ionic strength. Integral membrane proteins are more tightly associated and cannot be released by changes in ionic strength. Membranes were treated with NaCl, and the amount of profilin remaining bound to the membrane was analyzed. Treatment with NaCl resulted in a dose-dependent dissociation of profilin from the membranes (Fig. 2). Maximum dissociation was obtained with 0.1 M NaCl. Thus, profilin was a peripheral membrane protein.
Figure 2:
Effect of NaCl on membrane association of
profilin. Total membranes were incubated with the indicated
concentrations of NaCl in buffer A for 30 min. After incubation, the
membranes were collected by centrifugation at 100,000 g for 1 h and resuspended in buffer A. The amount of profilin bound
to the membranes was determined as described under ``Experimental
Procedures.'' The amount of profilin bound to membranes incubated
in buffer without NaCl was set at 100% in the figure. The data shown
are representative of two independent
experiments.
Since profilin is an actin-binding protein(1) , we questioned whether the cytosolic-associated profilin was bound to actin in the cytosol. Profilin was purified from the cytosolic fraction by affinity chromatography with polyproline-Sepharose(12) . The polyproline-Sepharose chromatography procedure was modified such that the column was not washed with buffer containing 3 M urea prior to the elution of profilin with 6 M urea(12) . In this way, if profilin was bound to actin in the cytosol, it should remain bound to actin until dissociated from the polyproline-Sepharose column with 6 M urea. The actin- and polyproline-binding sites on profilin are not the same(13) . The purified profilin preparation was subjected to immunoblot analysis using affinity-purified anti-actin antibodies. Indeed, actin was present in the profilin preparation purified from the cytosolic fraction (Fig. 3).
Figure 3: Immunoblot analysis of actin from the profilin preparation purified from the cytosol. Profilin was purified from the cytosolic fraction by polyproline-Sepharose chromatography under conditions where actin would remain bound to profilin prior to elution with 6 M urea as described under ``Results.'' The purified profilin preparation was subjected to immunoblot analysis using affinity-purified anti-profilin (A) and anti-actin (B) antibodies. The positions of profilin and actin are indicated in the figure.
Figure 4: Effect of plasma membrane concentration on profilin binding. Plasma membrane-associated profilin was removed from membranes by washing with 0.5 M NaCl. Pure profilin (5 µg) was incubated with the indicated concentrations of NaCl-washed plasma membranes. The amount of profilin bound to the membranes was determined as described under ``Experimental Procedures.'' The data shown are representative of two independent experiments.
Phospholipids were extracted from plasma membranes and used for the preparation of phospholipid vesicles. These vesicles were used to examine profilin binding using phospholipid phosphate concentrations similar to that used in the experiment shown in Fig. 4. Profilin bound to these phospholipid vesicles in a concentration-dependent manner similar to that found with the salt-washed plasma membranes (Fig. 5). Profilin binding to these vesicles saturated at 80 µg/ml phospholipid phosphate and half-maximum binding was at 38 µg/ml phospholipid phosphate. The results of this experiment indicated that the association of profilin with plasma membranes was not through interaction with integral membrane proteins. Thus, the association of profilin with the plasma membrane was through interaction with membrane phospholipids.
Figure 5: Binding of profilin to phospholipid vesicles prepared with plasma membrane phospholipids. Phospholipid vesicles were prepared with phospholipids extracted from plasma membranes. Pure profilin (5 µg) was incubated with the indicated concentrations of phospholipid vesicles. The amount of profilin bound to the phospholipid vesicles was determined as described under ``Experimental Procedures.'' The data shown are representative of two independent experiments.
Figure 6: Effect of phospholipid composition on the binding of profilin to phospholipid vesicles. Pure profilin (5 µg) was incubated with the indicated phospholipid vesicles. The amount of profilin bound to the phospholipid vesicles was determined as described under ``Experimental Procedures.'' The molar ratio of the phospholipids in the vesicles is indicated. The final phospholipid concentration (100 µg/ml phospholipid phosphate) of all vesicles was the same. The data shown are representative of two independent experiments.
Figure 7:
Effect of profilin concentration on the
binding of profilin to PC:PIP vesicles. PC:PIP
vesicles were incubated with the indicated concentrations of pure
profilin. The final phospholipid concentration of the vesicles was 100
µg/ml phospholipid phosphate. The amount of profilin bound to the
phospholipid vesicles was determined as described under
``Experimental Procedures.'' The data shown are
representative of two independent experiments. The inset is
Scatchard plot of the data.
Figure 8:
Effect of PIP concentration on
the binding of profilin to PC:PIP
vesicles. Pure profilin
(5 µg) was incubated with PC:PIP
vesicles with the
indicated surface concentrations of PIP
. The final
phospholipid concentration of the vesicles was 100 µg/ml
phospholipid phosphate. The amount of profilin bound to phospholipid
vesicles was determined as described under ``Experimental
Procedures.'' The data shown are representative of two independent
experiments.
Figure 9: Effect of inositol starvation on the localization of profilin in ino1 cells. Inositol-supplemented ino1 cells were grown to the exponential phase of growth. Cells were washed and incubated for 4 h in fresh growth medium without inositol. Control cells were incubated in growth medium with inositol. Following the 4-h incubation, the subcellular localization of profilin from inositol-supplemented and inositol-starved cells was determined as described in the legend to Fig. 1. The data shown are representative of two independent experiments.
Figure 10: Effect of glucose starvation on the localization of profilin in respiratory deficient cells. Glucose-supplemented cells were grown to the exponential phase of growth. Cells were washed and incubated for 30 min in fresh growth medium without glucose. Following the 30-min incubation, the culture was divided and glucose was added back (indicated by the arrow) to one of the cultures. The incubation of the cultures was continued for an additional 30 min. At the indicated time intervals, samples of the cultures were taken and analyzed for the association of profilin with the membrane and cytosolic fractions of the cell as described in the legend to Fig. 1. The data shown are representative of two independent experiments.
In this work we examined the regulation of profilin
localization by PIP metabolism in S. cerevisiae.
The initial aim of this study was to examine the subcellular
localization of profilin. About 80% of the cellular profilin was
localized to the plasma membrane, whereas the remainder was localized
to the cytosol. That profilin could be dissociated from membranes with
high ionic strength buffer indicated that profilin was a peripheral
membrane protein. Pure profilin bound to salt-washed plasma membranes
and to unilamellar phospholipid vesicles prepared from phospholipids
extracted from plasma membranes. These results raised the suggestion
that profilin's association with plasma membranes was not
mediated through interaction with other peripheral membrane proteins or
integral membrane proteins. Instead, profilin's peripheral
associated with plasma membranes was through interaction with membrane
phospholipids. This conclusion would be expected based on
profilin's ability to bind PIP
vesicles(13) .
PIP
is a minor phospholipid found in S. cerevisiae that is localized to plasma membranes(15) . Thus, the
localization of profilin to plasma membranes would also be expected.
Actin was associated with profilin purified from the cytosol. This was
consistent with profilin's role in the organization of the actin
cytoskeleton (12) .
The phospholipid requirement for
profilin binding to membranes was examined with pure profilin and well
defined unilamellar phospholipid vesicles. The vesicles were prepared
with phospholipids found in S. cerevisiae plasma membranes.
Under the conditions of our experiments, binding of profilin to these
vesicles was dependent on the presence of PIP. The
dissociation constant for profilin binding to PC:PIP
vesicles (0.13 µM) was in the range reported for
profilin from higher eucaryotes(5, 8) . In addition,
profilin binding to PC:PIP
vesicles was dependent on the
surface concentration of PIP
. Thus, profilin followed
surface dilution kinetics(39) , a characteristic common to
lipid-dependent enzymes that bind lipids at a membrane
surface(38) .
The physiological relevance of the dependence
of profilin binding on the surface concentration of PIP was
addressed by two independent approaches using S. cerevisiae strains where the plasma membrane levels of PIP
are
known to fluctuate under certain growth conditions. Inositol starvation
of ino1 cells (36, 43) and glucose starvation
of respiratory deficient cells (45) were used to deplete plasma
membrane levels of PIP
. After a 4-h starvation period of ino1 cells for inositol, a small but reproducible amount of
profilin was found to translocate from membranes to the cytosol.
Inositol starvation also causes fluctuations in the plasma membrane
levels of phosphatidylcholine, phosphatidylethanolamine,
phosphatidylinositol, phosphatidylserine, and phosphatidate, and PIP (36) . However, it was unlikely that the translocation of
profilin was due to changes in these phospholipids. None of these
phospholipids had a significant effect on binding of pure profilin to
vesicles. In a more dramatic fashion, glucose starvation of respiratory
deficient cells over a 20-min period caused a significant amount of
profilin to translocate from membranes to the cytosol. Moreover, the
addition of glucose back to glucose-starved cells caused profilin to
translocate back to membranes from the cytosol. These translocations
occurred too rapidly to be attributed to a genetic mechanism. Taken
together, these results raised the suggestion that the plasma membrane
concentration of PIP
played a role in the regulation of
profilin localization. Our studies did not rule out the possible
regulation of profilin localization by other plasma membrane-associated
components or other cellular factors (e.g. cytoskeleton),
which could have changed in response to inositol starvation of ino1 cells or glucose starvation of respiratory deficient cells.
In
summary, this work addressed the fundamental question of where profilin
was localized in the cell. We demonstrated that profilin was localized
to the plasma membrane and cytosolic fractions and that profilin
binding to the membrane was dependent on PIP. It is known
that PIP
levels change under various growth conditions. Our
studies showed that conditions which affect PIP
metabolism
played a role in profilin's localization. Future studies will be
directed toward gaining insight into the role PIP
metabolism plays in profilin function.