* Banting and Best Department of Medical Research, Department of Biochemistry, University of Toronto, Toronto, Ontario
M5G 1L6, Canada
DdCAD-1 is a 24-kD Ca2+-dependent cell- cell adhesion molecule that is expressed soon after the initiation of development in Dictyostelium cells. DdCAD-1 is present on the cell surface as well as in the cytosol. However, the deduced amino acid sequence of DdCAD-1 lacks a hydrophobic signal peptide or any predicted transmembrane domain, suggesting that it may be presented on the cell surface via a nonclassical transport mechanism. Here we report that DdCAD-1 is transported to the cell surface via contractile vacuoles, which are normally involved in osmoregulation. Immunofluorescence microscopy and subcellular fractionation revealed a preferential association of DdCAD-1 with contractile vacuoles. Proteolytic treatment of isolated contractile vacuoles degraded vacuole-associated calmodulin but not DdCAD-1, demonstrating that DdCAD-1 was present in the lumen. The use of hyperosmotic conditions that suppress contractile vacuole activity led to a dramatic decrease in DdCAD-1 accumulation on the cell surface and the absence of cell cohesiveness. Shifting cells back to a hypotonic condition after hypertonic treatments induced a rapid increase in DdCAD-1-positive contractile vacuoles, followed by the accumulation of DdCAD-1 on the cell membrane. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a specific inhibitor of vacuolar-type H+-ATPase and thus of the activity of contractile vacuoles, also inhibited the accumulation of DdCAD-1 on the cell surface. Furthermore, an in vitro reconstitution system was established, and isolated contractile vacuoles were shown to import soluble DdCAD-1 into their lumen in an ATP-stimulated manner. Taken together, these data provide the first evidence for a nonclassical protein transport mechanism that uses contractile vacuoles to target a soluble cytosolic protein to the cell surface.
THE cellular slime mold Dictyostelium discoideum
transits from the solitary amoeboid state to an organized multicellular structure during development.
This process is initiated in cells upon the depletion of nutrients, leading to the expression of many developmentally regulated genes and the chemotactic migration of cells in
response to extracellular cAMP. Cells stream in concentric
rings and/or spirals toward aggregation centers, giving rise
to multicellular entities called pseudoplasmodia or slugs.
The migrating slugs eventually culminate in the formation
of fruiting bodies consisting of primarily spores and stalk
cells (for review see Loomis, 1975 Multicellularity during development is maintained by
the expression of cell-cell adhesion molecules, which fall
into two broad categories based on their sensitivity to
EDTA (for reviews see Gerisch, 1980 DdCAD-1 is expressed by cells soon after the initiation
of development (Knecht et al., 1987 Recent cloning of the DdCAD-1 cDNA predicts a protein of 23,924 daltons (Wong et al., 1996 In this report we present morphological and biochemical evidence that DdCAD-1 is transported to the cell surface from the cytosol via contractile vacuoles, which is
known so far to function exclusively in osmoregulation in
cells. Furthermore, we show that isolated contractile vacuoles selectively take up soluble DdCAD-1 into their lumen
in a cell-free system. Our results demonstrate, for the first
time, a protein targeting function for contractile vacuoles
and a novel nonclassical protein transport mechanism.
Cell Strains and Culture Conditions
NC4 cells were cultured on agar dishes in association with Klebsiella aerogenes as the food source (Sussman, 1987 Laser Scanning Confocal Microscopy
Cells were fixed according to Fukui et al. (1987) Subcellular Fractionation
Subcellular fractionation was performed following the method of Nolta
and Steck (1994) For proteolytic digestion, proteinase K was added to give a final concentration of 100 µg/ml in 100 µl of the contractile vacuole fraction in the
presence or absence of 1% Triton X-100 and then was incubated at 37°C
for 2 h. After the addition of PMSF at 2 mM, samples were boiled for 10 min and subjected to SDS-PAGE, followed by Western blot analysis using
anti-DdCAD-1 and anti-calmodulin antibodies.
Quantification of DdCAD-1 on the Cell Surface
All steps were carried out on ice unless noted otherwise. After development for a specific time period, 7.5 × 106 cells were resuspended in 1 ml of
0.1% BSA, 2 mM EGTA, and 17 mM phosphate buffer, pH 6.4, and rotated at 180 rpm for 10 min to block nonspecific binding sites. Next, anti-
DdCAD-1 IgG (8.2 µg) was added to the cell suspension and incubated
for 15 min. Cells were washed three times using the same buffer solution
and then incubated with HRP-conjugated goat anti-rabbit IgG (1:50,000 dilution) for 15 min on a platform shaker. After three washes, cells were
resuspended in 0.5% Triton X-100, 0.5 mg/ml 2,2 Inhibition of Contractile
Vacuole Activity by Hypertonic Treatments and
7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole
For hypertonic treatments, NC4 cells were developed for 3 h in the presence or absence of 0.1 M sorbitol or 0.1 M KCl. Then cells were subjected
to quantification of the amount of DdCAD-1 on the cell surface, cell cohesion assay, and Western blot analysis of amounts of DdCAD-1 in the
whole cell lysate and in the membrane fraction. To prepare total cell
membranes, NC4 cells were homogenized as described above. The postnuclear supernatant (2 ml) was placed on top of 1 ml of 15% sucrose in
GMC buffer, and membranes were pelleted at 33,000 rpm for 1 h at 4°C.
Activity of acid phosphatase secreted into the medium was measured as
described by Crean and Rossomando (1979) Alternatively, NC4 cells were developed for 2 h in the presence of 5 and 10 µM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) (ICN Biomedical Inc., Aurora, OH), a potent inhibitor of vacuolar H+-ATPase
(Mellman et al., 1986 Cell Cohesion Assay
Intercellular cohesion was assayed using a modified method (Lam et al.,
1981 DdCAD-1 Null Mutant
The cadA gene encoding DdCAD-1 was disrupted using the restriction
enzyme-mediated integration method (Kuspa and Loomis, 1992 In Vitro Reconstitution of DdCAD-1 Import into
Contractile Vacuoles
A membrane-free cytosolic fraction prepared from KAX3 cells was used
as the source of soluble DdCAD-1 molecules. Cells (~2 × 109 cells) were
developed for 6 h in suspension and then lysed at room temperature in 12 ml of 2 mM MgCl2, 10 mM Tris-HCl, pH 7.5 (TM buffer) by two passages
through a membrane filter with 5-µm-diam pores. DTT was added to the
postnuclear supernatant at a final concentration of 2 mM and then centrifuged at 40,000 rpm using a Beckman SW40 rotor for 1 h at 4°C. Aliquots
of the supernatant were frozen rapidly in liquid nitrogen and stored at
After 6 h of development in a suspension culture, cadA To carry out the in vitro import assay, the cytosolic fraction was clarified by centrifugation at 40,000 rpm for 1 h at 4°C. Then, one-third volume
of 60% sucrose in TM buffer was added to the soluble protein fraction.
The contractile vacuole fraction was thawed on ice and diluted 1:1 with
TM buffer containing 4 mM DTT. 100 µl of the cytosol fraction was mixed
with 200 µl of the contractile vacuole fraction and incubated for 1 h at
room temperature. Contractile vacuoles were separated from free proteins by centrifugation through 3 ml of 25% sucrose in TM buffer, on top
of a cushion of 0.2 ml of 60% sucrose. Samples were centrifuged at 40,000 rpm using a Beckman SW50.1 rotor for 1 h at 4°C. Contractile vacuoles
(500 µl) were collected from the 25-60% interface of the sucrose gradient.
As a control, purified GST was added to the cytosolic fraction at 0.2 mg/
ml. In some experiments, 1 mM ATP and/or an ATP regenerating system
containing 40 mM creatine phosphate and 0.2 µg/ml creatine kinase (Scott
and Klionsky, 1995 Immunolocalization of DdCAD-1 in
Contractile Vacuoles
DdCAD-1 is a soluble protein that is localized primarily in
the cytosol of Dictyostelium cells (Brar and Siu, 1993
Contractile vacuoles are intracellular membrane organelles involved in osmoregulation by excreting water
into the extracellular medium during transient fusion with
the plasma membrane (Zhu and Clarke, 1992
Intravacuolar signals of DdCAD-1 were frequently observed in contractile vacuoles (Fig. 3 a). Occasionally, they
displayed a punctate staining pattern with regular intervals
along the luminal surface of the vacuole (Fig. 3 b). In contrast, calmodulin staining was never detected inside contractile vacuoles, suggesting that DdCAD-1 was selectively
taken up by contractile vacuoles. Interestingly, strong
DdCAD-1 signals were also seen spreading between the
lumen of the contractile vacuole and the exterior of the
plasma membrane (Fig. 3, c and d), implying translocation
of DdCAD-1 between these two membranes.
Copurification of DdCAD-1 with Contractile Vacuoles
The association of DdCAD-1 with contractile vacuoles
was confirmed by subcellular fractionation (Fig. 4). Cells
were collected at 12 h of development and homogenized.
The postnuclear supernatant was fractionated on a continuous sucrose density gradient. Distribution of intracellular
organelles, including contractile vacuoles, mitochondria, lysosomes, and plasma membranes, over the gradient were
determined by measuring the activity of specific enzyme
markers (Fig. 4 A) (Padh et al., 1989
Consistent with our morphological data, DdCAD-1 cofractionated with the peak activity of the contractile vacuole marker enzyme alkaline phosphatase (Nolta and Steck,
1994 To demonstrate that DdCAD-1 was present in the lumen of contractile vacuoles, contractile vacuole preparations were subjected to proteolytic digestion by proteinase
K in the presence or absence of 1% Triton X-100 at 37°C
for 2 h, and then analyzed by Western blotting using anti-
DdCAD-1 and anti-calmodulin antibodies (Fig. 4 C). The
level of DdCAD-1 was reduced by <30% when proteolysis was carried out in the absence of detergent. However,
DdCAD-1 was digested completely in the presence of Triton X-100. In contrast, calmodulin, which is known to associate with the cytoplasmic surface of contractile vacuoles (Zhu and Clarke, 1992 Inhibition of the Surface Expression of DdCAD-1 by
Hypertonic Treatment
It has been reported that incubation of cells in hypertonic
media leads to the collapse of contractile vacuoles (Zhu
and Clarke, 1992
Whether a lower level of DdCAD-1 expression on the
cell surface would result in the loss of cell-cell adhesion
was also investigated. Treated and untreated cells were assayed for cell cohesion in phosphate buffer. Whereas control cells achieved 75% cell aggregation within 20 min,
<30% of cells developed in the hypertonic solutions were
able to form aggregates (Fig. 5 A). Also, the average size of aggregates was much smaller than those of control cells
(data not shown).
To distinguish between the effects of hypertonic treatment on overall DdCAD-1 expression and its effects on
DdCAD-1 transport to the plasma membrane, Western
blot analysis was carried out on total cell homogenates and
the membrane fraction (Fig. 5 B). No significant difference in the total amount of DdCAD-1 was observed in
cells with or without hypertonic treatments. However, the
level of DdCAD-1 in the membrane fraction was much reduced in cells treated with either sorbitol or KCl. Therefore, the data indicate that hypertonic conditions affect the
transport of DdCAD-1 to the cell surface and not its accumulation in the cytosol. It is therefore evident that the surface expression of DdCAD-1 is dependent on contractile
vacuoles.
Lysosomes have been shown to secrete lysosomal enzymes during development (Cardelli, 1993 Induction of Surface Expression of
DdCAD-1 by Hypotonic Shift
The reverse shift of environmental osmolarity from a
higher strength to a lower one induces reappearance of
contractile vacuoles (Zhu and Clarke, 1992 Inhibition of the Surface Expression of DdCAD-1
by NBD-Cl
Since it has been reported that NBD-Cl is a specific inhibitor to vacuolar-type H+-ATPase (Mellman et al., 1986
The levels of surface-associated DdCAD-1, in cells developed in the presence of 5 and 10 µM NBD-Cl, were
quantified. NBD-Cl-treated cells showed significant reductions in their levels of DdCAD-1 on the cell surface
(Fig. 7 A). In the presence of 10 µM NBD-Cl, the cell surface level of DdCAD-1 dropped by 65%. However, no significant difference in the total cellular level of DdCAD-1 was observed in cells with or without NBD-Cl treatments
(Fig. 7 B). When the effects of NBD-Cl on the secretion of
the lysosomal enzyme acid phosphatase were examined,
the amount of secreted acid phosphatase increased 1.5-fold in 10 µM NBD-Cl (Fig. 7 A). These results indicate
that NBC-Cl inhibited contractile vacuole activity and the
transport of DdCAD-1, but it had no effect on DdCAD-1
accumulation and the lysosomal secretion pathway.
In Vitro Reconstitution of DdCAD-1 Import into
Contractile Vacuoles
To test whether isolated contractile vacuoles could selectively import DdCAD-1, a cell-free reconstitution assay
was developed using a mutant strain with the cadA gene,
which encodes DdCAD-1, disrupted by the blasticidin-
resistant cassette. The cadA
The reconstitution assay was carried out as illustrated
schematically in Fig. 9 A. Contractile vacuoles were isolated from the cadA
Several controls were carried out to demonstrate that
the import of DdCAD-1 into contractile vacuoles was a selective process. First, when the wild-type cytosolic fraction
was incubated with buffer, no detectable amount of DdCAD-1
was recovered from the interface region of the sucrose
gradient, demonstrating that the cytosolic fraction was devoid of wild-type contractile vacuoles (Fig. 9 B, lane e).
Second, when GST, a soluble protein with a molecular size
similar to DdCAD-1, was added to the cytosol, no GST
was found associated with or imported into contractile
vacuoles (Fig. 9 B). Also, calmodulin, which is present
both in the cytosol and on the vacuole surface, did not enter the vacuole lumen. Finally, when the contractile vacuole membrane was disrupted by low osmotic shock before
the import reaction, DdCAD-1 was degraded by proteinase K in the absence of SDS (data not shown). Taken together, these results ruled out the presence of contaminating wild-type contractile vacuoles or nonspecific uptake of
soluble DdCAD-1 by damaged contractile vacuoles.
Since the contractile vacuole fraction used in the DdCAD-1
import assay was contaminated by plasma membrane and
lysosomes, experiments were carried out to determine
whether plasma membrane vesicles and lysosomes also
contributed to DdCAD-1 uptake. A series of discontinuous sucrose density gradients were performed to obtain membrane fractions that contained various amounts of
contractile vacuoles, plasma membrane, and lysosomes,
and the import of DdCAD-1 into protease-resistant compartments in these fractions was assessed. As shown in Table I, the DdCAD-1 import activity was not affected by
the increase or decrease in the relative amounts of plasma membrane and lysosomes. In contrast, the DdCAD-1 import activity was dependent on the amount of contractile
vacuoles in these fractions. When the relationship between
contractile vacuoles and DdCAD-1 import was subjected
to linear regression analysis, a strong positive correlation
was observed between DdCAD-1 import activity and the activity of the contractile vacuole marker alkaline phosphatase, with a correlative coefficient of +0.997. Neither the
vanadate-sensitive H+-ATPase activity nor the acid phosphatase activity showed positive correlation with DdCAD-1
import.
Table I.
Distributions of Organelle Markers and DdCAD-1 Import Activity in Different Membrane Fractions
).
; Siu et al., 1988
; Siu,
1990
; Fontana, 1995
; Bozzaro and Ponte, 1995
). There are
two types of EDTA-sensitive cell adhesion sites. The
EDTA/EGTA-sensitive cell adhesion sites, also known as
contact sites B, are mediated by the Ca2+-dependent cell
adhesion molecule gp24/DdCAD-1 (Knecht et al., 1987
;
Brar and Siu, 1993
), while the EDTA-sensitive/EGTA-
resistant sites are probably mediated by a Mg2+-dependent cell adhesion molecule (Fontana, 1993
). The molecular nature of the latter sites is not yet known. Both types of
adhesion sites are responsible for cell-cell interactions in
the early stages of development. Coinciding with the aggregation stage is the rapid accumulation of the cell adhesion molecule gp80, which mediates the EDTA-resistant
cell adhesion sites or contact sites A (Muller and Gerisch,
1978
; Siu et al., 1985
; Kamboj et al., 1988
, 1989
). In postaggregation stages, the EDTA-resistant adhesion sites are
mediated by the membrane glycoprotein gp150 (Geltosky et al., 1979
; Siu et al., 1983
; Gao et al., 1992
).
). Antibodies raised
against gel-purified DdCAD-1 specifically inhibit the
EDTA/EGTA-sensitive cell-cell adhesion sites and block
development (Loomis, 1988
). We have purified DdCAD-1
to homogeneity and demonstrated that labeled soluble
DdCAD-1 binds to cells in an EDTA/EGTA-sensitive
manner (Brar and Siu, 1993
). Binding of DdCAD-1 to
cells is prevented when cells are precoated with anti-
DdCAD-1 antibodies, consistent with a homophilic mode
of interaction. In addition, binding of DdCAD-1 to cells inhibits cell reassociation, indicating that it contains only a
single cell binding site.
). The deduced
amino acid sequence of DdCAD-1 shows significant sequence
similarities with members of the cadherin family, and it
contains a Ca2+-binding motif residing in the carboxy-terminal region. Indeed, Ca2+ overlay experiments have
shown that DdCAD-1 is a Ca2+-binding protein with multiple binding sites (Brar and Siu, 1993
; Wong et al., 1996
).
It is therefore conceivable that DdCAD-1 is a primitive
member of the cadherin superfamily and it may mediate cell-cell adhesion in a manner similar to that of cadherins
(Shapiro et al., 1995
; Nagar et al., 1996
). Another novel
feature of the predicted sequence is that it lacks an amino-terminal hydrophobic signal peptide or a transmembrane
domain, suggesting that DdCAD-1 is a soluble protein.
Consistent with this observation, both subcellular fractionation and immunofluorescence microscopy have revealed a predominant cytoplasmic localization of DdCAD-1, indicating that 60-80% of DdCAD-1 is soluble (Brar and Siu,
1993
; Sesaki and Siu, 1996
). However, IgG binding and
capping experiments clearly demonstrate that a substantial amount of DdCAD-1 is present on the cell surface
(Brar and Siu, 1993
; Wong et al., 1996
). Interestingly, DdCAD-1 undergoes rapid translocation from the cytoplasm to the plasma membrane in the preaggregation
stage of development (Sesaki and Siu, 1996
), and then it
becomes concentrated on filopodial structures and in cell-
cell contact regions. These observations thus raise the
question of how DdCAD-1 is transported and anchored to
the cell surface.
Materials and Methods
). Cells were grown to a density of
108 cells per 100-mm-diam plate and then collected for experiments. Bacteria were removed by differential centrifugation. The axenic strain
KAX3 was cultured in HL-5 medium (Sussman, 1987
). The KAX3 cells
overproduced DdCAD-1 and provided excellent signals for immunofluorescence microscopy. These cells were used in most experiments. For development under submerged conditions, cells were washed three times
with 17 mM phosphate buffer, pH 6.4, and resuspended at 2 × 106 cells
per ml in the same buffer. Approximately 106 cells were deposited on a
coverslip coated with 0.1% poly-L-lysine, and 0.4 ml of buffer was removed after 10 min. Coverslips were placed in a moist chamber, and development of these cells was carried out at room temperature. For development in suspension, cells were suspended at 1-1.5 × 107 cells per ml in
17 mM phosphate buffer, pH 6.4, and rotated at 180 rpm on a platform
shaker at room temperature.
and then processed for laser scanning confocal microscopy (LSCM)1 as described previously
(Sesaki and Siu, 1996
). Cells were developed on coverslips for different
time periods and then fixed with 3.7% formaldehyde in 17 mM phosphate
buffer for 15 min at room temperature, followed by permeabilization with
cold methanol (
20°C) containing 1% formaldehyde for 5 min. Nonspecific binding was blocked by incubation with 1% (wt/vol) BSA in PBS for
10 min. Samples were incubated with the anti-DdCAD-1 antiserum (1:200
dilution in PBS containing 0.1% BSA) for 1 h, washed three times with
PBS containing 0.05% Tween-20, and then stained with FITC-conjugated
goat anti-rabbit IgG (1:300 dilution) for 1 h. For double immunofluorescence labeling, anti-gp80 mAb 80L5C4 (1:100) (Siu et al., 1985
), anti-calmodulin mAb mixture, 6D4, 1F11, and 2D1 (Sigma Chemical Co., St.
Louis, MO) (1:100 dilution) (Zhu and Clarke, 1992
), and anti-H+-ATPase
mAb N2 (1:50) (Fok et al., 1993
) were used, followed by Texas red-conjugated goat anti-mouse IgG (1:300 dilution). For double immunostaining
for DdCAD-1 and the Dictyostelium lysosomal enzyme
-mannosidase,
cells were developed for 6 h in suspension and then placed on coverslips.
After a 10-min incubation at room temperature, cells were fixed with
3.7% formaldehyde for 2 h, permeabilized with 1% saponin in PBS, and
then stained with anti-DdCAD-1 antibody and anti-
-mannosidase mAb
(2H9) (1:100) (Bush and Cardelli, 1989
). Coverslips were mounted in PBS
containing 80% glycerol, 0.2% p-phenylenediamine, and 2.5% 1,4-diazabicyclo-[2,2,2]-octane. Images were acquired using an MRC 600 confocal
imaging system (Bio Rad Laboratories, Hercules, CA) on an Optiphot microscope (Nikon, Tokyo, Japan) equipped with a 40× objective. Alternatively, an Axiovert 135 inverted microscope equipped with a 63× Neofluar objective and LSM 410 confocal attachment was used (Carl Zeiss, Inc.,
Thornwood, NY).
with minor modifications. After development for 12 h in
liquid culture, KAX3 cells (1.5 × 108 cells in 10 ml) were disrupted at
room temperature in GMC buffer (5 mM glycine-NaOH, 1 mM MgCl2, 0.1 mM CaCl2, pH 8.5) using membrane filters with 5-µm-diam pores (Millipore Corp., Bedford, MA). A postnuclear supernatant (1 ml), which was
prepared by centrifugation of homogenates at 1,500 rpm for 5 min at 4°C,
was layered on top of a 12-ml 30-60% (wt/vol) sucrose gradient prepared
in GMC buffer. Gradients were centrifuged at 33,000 rpm using an SW40
rotor (Beckman Instruments, Inc., Palo Alto, CA) at 4°C for 3 h. Fractions
(0.72 ml each) were collected from the top. Distribution of organelles in
gradients was determined using specific enzyme markers (Padh et al.,
1989
; Nolta et al., 1991
): alkaline phosphatase for contractile vacuoles,
vanadate-sensitive H+-ATPase for the plasma membrane, F1F0-ATPase
for mitochondria, and acid phosphatase for lysosomes. To determine the
distribution of DdCAD-1, 0.5 ml of each fraction was diluted with 2.5 ml
of GMC buffer containing 30 mM NaOH, incubated on ice for 30 min, and
centrifuged at 33,000 rpm for 1 h at 4°C (Luna et al., 1981
). Pelleted membrane-associated proteins were subjected to SDS-PAGE (Laemmli, 1970
),
transferred onto nitrocellulose membrane, and stained with anti-
DdCAD-1 antibody.
-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 0.05% H2O2, and 100 mM citrate-NaOH, pH
4.0, and then incubated for 15 min at room temperature. Samples were
clarified by centrifugation and absorbance of the supernatant was measured at OD405.
.
; Padh et al., 1989
). Since NBD-Cl was prepared as a
5 mM stock solution in ethanol, control cells were developed in the presence of 0.2% ethanol. Cells were subjected to quantification of the cell
surface-associated DdCAD-1, Western blot analysis, and measurement of
acid phosphatase activity secreted into the medium. Morphological observations were carried out using a Zeiss Axiovert 135 inverted microscope.
) of the original roller tube assay of Gerisch (1961)
. After development for a specific period in either hypertonic medium or NBD-Cl, NC4
cells were resuspended in 17 mM phosphate buffer, pH 6.4, at 3 × 106 cells
per ml. Cell aggregates were dispersed by gentle pipetting. Cells were allowed to re-form aggregates by rotating at 180 rpm on a platform shaker
at room temperature. After 20 min, the number of nonaggregated cells,
including both singlets and doublets, was scored using a hemocytometer.
). A plasmid containing the cadA gene (Wong et al., 1996
) and the blasticidin S-resistance cassette (Sutoh, 1993
) was constructed for DNA integration. DNA
transfection was carried out by electroporation, and blasticidin S-resistant
transformants were isolated and cloned. The disruption of the cadA gene
was confirmed by Southern and Western blot analyses.
70°C. Protein concentration was determined using the bicinchoninic acid
assay kit (Pierce Chemical Co., Rockford, IL) and the protein concentration was adjusted to ~6 mg/ml. DdCAD-1 concentration in the cytosol was 0.2 mg/ml, determined by Western blot analysis using purified DdCAD-1 as a standard.
cells (5 × 108)
were collected and homogenized. The postnuclear supernatant (5 ml) was
loaded on a discontinuous sucrose density gradient (28% and 42%, 4 ml
each) and centrifuged at 40,000 rpm for 1 h at 4°C. Contractile vacuoles
enriched at the interface were collected (~300 µl), frozen in aliquots in
liquid nitrogen, and stored at
70°C. Protein concentration of this fraction was 2-3 mg/ml. Purified glutathione-S-transferase (GST) protein and
anti-GST antibody were prepared as described previously (Zhao and Siu,
1996
).
) were added to the reaction mixture.
Results
).
However, a substantial amount of DdCAD-1 is also present
on the cell surface (Sesaki and Siu, 1996
). To investigate
the mechanism by which DdCAD-1 is transported to the
cell surface, immunofluorescence labeling was carried out
to determine whether DdCAD-1 was associated with intracellular membrane structures. LSCM revealed that
DdCAD-1 was preferentially associated with large cytoplasmic vacuoles, with diameters varying between 1 and 4 µm
(Fig. 1, a and b). Usually, more than one DdCAD-1-positive vacuole was present in each cell. DdCAD-1-positive
vacuoles appeared at as early as after 3 h of development.
The morphology and size of the stained vacuoles suggested that they might be contractile vacuoles. To determine whether DdCAD-1 was synthesized on the RER and
then targeted to the plasma membrane via the Golgi apparatus, double immunofluorescence labeling was carried
out with antibodies directed against DdCAD-1 and gp80. The latter cell adhesion molecule is synthesized and glycosylated using the ER-Golgi pathway (Hohmann et al.,
1985
, 1987
) and was therefore used as a marker for these
organelles. The results show that DdCAD-1 did not colocalize with gp80 (Fig. 1, c and d), suggesting that DdCAD-1
does not associate with either the ER or the Golgi apparatus. Since the Dictyostelium lysosome has been shown to be a secretory organelle (Cardelli, 1993
; Ruscetti et al.,
1994
), double immunostaining was carried out to determine whether DdCAD-1 was enriched in lysosomes. Using
-mannosidase as a marker for lysosomes (Bush and
Cardelli, 1989
), we found that DdCAD-1 did not colocalize
with
-mannosidase in lysosomes.
Fig. 1.
Subcellular localization of DdCAD-1 by immunofluorescence microscopy. KAX3 cells were cultured axenically and then developed for 12 h on coverslips under submerged conditions. Cells were fixed and stained with rabbit antibodies against DdCAD-1 for
LSCM (a and b). Preferential association of DdCAD-1 with large cytoplasmic vacuoles as well as the plasma membrane was observed.
Similar results were obtained with cells developed for 6 and 9 h (data not shown). Cells were subjected to double immunostaining using rabbit antibodies against DdCAD-1 (c and e) and mouse anti-gp80 mAb (d) or mouse anti--mannosidase mAb (f). Bars, 5 µm.
[View Larger Version of this Image (90K GIF file)]
; Heuser et
al., 1993
; Nolta and Steck, 1994
). Vacuolar H+-ATPase
(Fok et al., 1993
) and calmodulin (Zhu and Clarke, 1992
; Zhu
et al., 1993
) are known to associate with contractile vacuoles. To demonstrate that DdCAD-1 was indeed associated with contractile vacuoles, double immunofluorescence
staining was carried out. Results showed that DdCAD-1
was colocalized to contractile vacuoles with both H+-ATPase
(Fig. 2, a and b) and calmodulin (Fig. 2, c-f). Serial confocal images through single cells showed that contractile vacuoles were located close to the plasma membrane and vacuoles fused with the plasma membrane were frequently
observed (data not shown). Contractile vacuoles fused with
the plasma membrane often displayed strong DdCAD-1
and calmodulin staining (Fig. 2, e and f).
Fig. 2.
Colocalization of DdCAD-1 with contractile vacuole
markers. After development for 12 h under submerged conditions, cells were fixed and then double labeled with rabbit anti-
DdCAD-1 antibody (a, c, and e) and mouse anti-calmodulin
mAb (d and f) or mouse anti-H+-ATPase mAb (b) for LSCM.
e and f show the fusion of contractile vacuoles with the plasma
membrane. Bars, 5 µm.
[View Larger Version of this Image (93K GIF file)]
Fig. 3.
Different patterns of DdCAD-1 signal associated with
contractile vacuoles. Images taken at a higher magnification frequently revealed intravacuolar signals of DdCAD-1 (a). Occasionally, a punctate staining pattern of DdCAD-1 localization on
the luminal surface of contractile vacuoles was observed (b).
Contractile vacuoles were frequently observed in contact with the
plasma membrane (a and b). Upon fusion of contractile vacuoles
with the cell membrane, a contiguous staining pattern of
DdCAD-1 spanning between the contractile vacuole membrane
and the plasma membrane was observed (c and d). Bars, 3 µm.
[View Larger Version of this Image (96K GIF file)]
; Nolta et al., 1991
).
To determine DdCAD-1 distribution, membranes were
pelleted from gradient fractions by ultracentrifugation in
the presence of 25 mM NaOH, which was included to remove proteins loosely associated with membranes (Luna
et al., 1981
), and then analyzed by Western blotting (Fig. 4 B).
Fig. 4.
Copurification of DdCAD-1 with contractile vacuoles.
KAX3 cells were developed for 12 h in liquid culture and then
homogenized. The postnuclear supernatant was fractionated on a
30-60% continuous sucrose density gradient by centrifugation at
35,000 rpm for 3 h at 4°C. (A) The distribution of different organelles was determined by assaying specific enzyme markers: alkaline phosphatase for contractile vacuoles (), vanadate-sensitive H+-ATPase for the plasma membrane (
), F1F0-ATPase for
mitochondria (
), and acid phosphatase for lysosomes (
). (B)
Cofractionation of DdCAD-1 with the contractile vacuole
marker enzyme was determined by Western blot analysis. A sample of 500 µl was taken from each fraction and diluted with 5 vol
of the homogenization buffer. After incubation for 30 min on ice
in the presence of 25 mM NaOH, membranes were pelleted, subjected to SDS-PAGE, and then blotted against the anti-DdCAD-1
antibody. The relative amounts of DdCAD-1 in these fractions
were estimated by densitometry (
) and shown in A. (C) Presence of DdCAD-1 in the lumen of contractile vacuoles. Proteolytic digestion of DdCAD-1 associated with the contractile vacuole peak fractions in a sucrose density gradient was carried out. A 100-µl sample of the contractile vacuole fraction was incubated with 0.1 mg/ml proteinase K in the presence or absence of
1% Triton X-100 for 2 h at 37°C. After boiling, proteins were separated by SDS-PAGE, followed by Western blot analysis using
anti-DdCAD-1 and anti-calmodulin antibodies.
[View Larger Version of this Image (30K GIF file)]
). In addition to contractile vacuoles, the fractions
(15-17) corresponding to the peak of vanadate-sensitive H+-ATPase activity, a marker for the plasma membrane,
contained a substantial amount of DdCAD-1, indicating its association with the cell membrane. Acid phosphatase released from lysosomes can be found at the top of the sucrose gradient (Nolta et al., 1991
). Interestingly, both acid
phosphatase and DdCAD-1 were found in fractions 1-3.
Since earlier immunostaining results showed that DdCAD-1 was not associated with lysosomes (Fig. 1, e and f),
DdCAD-1 found in the top region of the gradient probably came from the cytosolic pool and not from lysosomes.
), was completely degraded in
the absence of detergent. The protection of DdCAD-1 from proteolysis by the vacuole membrane indicated the
localization of DdCAD-1 in the vacuole lumen.
). If contractile vacuoles represent the
major vehicle by which DdCAD-1 is transported to the
plasma membrane, hypertonic treatment should inhibit
the cell surface expression of DdCAD-1. To test this theory, cells were developed for 3 h in the presence or absence of 0.1 M sorbitol or 0.1 M KCl and the relative level of DdCAD-1 expressed on the cell surface was quantified
by ELISA. The levels of surface-associated DdCAD-1 in
cells developed in the presence of sorbitol and KCl were
~30 and ~10%, respectively, relative to control cells (Fig.
5 A). Under hypertonic conditions, contractile vacuoles
collapsed and DdCAD-1-stained vacuoles were rarely observed (Fig. 6), suggesting that most of the DdCAD-1 failed to enter contractile vacuoles and remained in the cytosol.
Fig. 5.
Inhibition of DdCAD-1 transport and cell-cell adhesion by hypertonic conditions. NC4 cells were collected and developed for 3 h in the presence of 0.1 M sorbitol or 0.1 M KCl.
(A) The relative amounts of DdCAD-1 expressed on the cell surface of the treated and untreated cells were determined by
ELISA (solid bars). Background was subtracted using vegetative
cells that do not contain DdCAD-1 (Knecht et al., 1987). Effects
of hypertonic treatment on cell-cell adhesion were determined
using the cell reassociation assay (see Materials and Methods)
(open bars). Values represent the mean ± SD (n = 3). (B) Total
cellular DdCAD-1 (a) and DdCAD-1 associated with the membrane fraction (b) were examined by Western blot analysis. (C)
The activity of acid phosphatase secreted into the medium was
determined as described (Crean and Rossomando, 1979
).
[View Larger Version of this Image (17K GIF file)]
Fig. 6.
Time course of DdCAD-1 transport after hypotonic
shift. KAX3 cells were developed for 4, 4.5, 5, 5.5, and 6 h in either 0.1 M sorbitol (A) or 0.1 M KCl (B) and then transferred to
17 mM phosphate buffer, pH 6.4. All cell samples were collected
at 6 h, fixed, and stained with anti-DdCAD-1 antibody. The
number of cells with DdCAD-1-positive contractile vacuoles was
scored (). The percentage of positive control cells (
) is shown
along the y-axis of a. Values represent the mean of three experiments; 100 cells were scored in each experiment. The amounts of
DdCAD-1 present on the cell surface were determined by
ELISA, and values were normalized to that of control cells (
).
[View Larger Version of this Image (21K GIF file)]
). Our earlier
data showed that the lysosomal enzyme acid phosphatase
and DdCAD-1 cofractionated in the top fractions of the
sucrose gradient. To assess the possibility that DdCAD-1
might be transported via lysosomes, we examined the secretion of acid phosphatase into the outer media under hypertonic conditions. If DdCAD-1 transport makes use of
the lysosomal secretory pathway, one would predict the inhibition of lysosomal enzyme secretion by hypertonic
treatments. In contrast with DdCAD-1, the secretion of
acid phosphatase was stimulated by 1.3- and 5-fold in the
presence of 0.1 M sorbitol and 0.1 M KCl, respectively. It
is therefore unlikely that lysosomes are involved in the
transport of DdCAD-1.
). It was therefore of interest to examine whether the surface expression
of DdCAD-1 was resumed after a hypoosmotic shift. After
development for various time intervals in the presence of
sorbitol or KCl, cells were transferred to 17 mM phosphate buffer and all cell samples were collected at 6 h for
assays. The appearance of DdCAD-1-positive contractile vacuoles was monitored by fluorescence microscopy, and
the accumulation of DdCAD-1 on the cell surface was determined using ELISA (Fig. 6). Initially, only 5-10% of
the cells contained DdCAD-1-positive contractile vacuoles. Most cells contained one or more DdCAD-1-positive contractile vacuoles by 60 min after the shift. The inhibition of DdCAD-1 expression on the cell surface by hypertonic medium was reversible, and the accumulation of
DdCAD-1 on the cell surface followed the appearance of
DdCAD-1-positive contractile vacuoles. DdCAD-1 accumulated rapidly on the cell surface after an initial delay of
30-60 min. The surface level of DdCAD-1 became indistinguishable from that of control cells after 2 h. The temporal relationships between the appearance of contractile
vacuoles and the surface accumulation of DdCAD-1 are
consistent with the notion that DdCAD-1 is transported to
the plasma membrane via contractile vacuoles.
;
Padh et al., 1989
), we used this reagent to inhibit contractile vacuole activity and its effects on the expression of
DdCAD-1 on the cell surface. Cells were developed for 2 h
in the presence or absence of 5 and 10 µM NBD-Cl.
Treated cells lost their contractile vacuole function and
displayed a round and swollen morphology, whereas control cells spread well and adopted a more elongated shape
(Fig. 7 C). Although treated cells appeared to be swollen,
most cells remained intact during the 2-h incubation period in 17 mM phosphate buffer. Cell lysis began to occur
at 20 µM NBD-Cl.
Fig. 7.
Inhibition of DdCAD-1 transport by NBD-Cl. NC4
cells were collected and developed for 2 h in the presence of
NBD-Cl. (A) The relative amounts of DdCAD-1 expressed on
the cell surface of the treated and untreated cells were determined by ELISA (black bars). Background was subtracted using
vegetative cells that do not contain DdCAD-1. The activity of secreted acid phosphatase in the outer media (gray bars) was determined as described previously (Crean and Rossomando, 1979).
(B) Total cellular DdCAD-1 was examined by Western blot analysis. (C) Morphologies of cells were observed using conventional
phase contrast microscopy. Bar, 50 µm.
[View Larger Version of this Image (37K GIF file)]
mutant cells did not express
DdCAD-1 but contained normal contractile vacuoles. Immunofluorescence microscopy showed the presence of calmodulin-positive contractile vacuoles inside these mutant cells (Fig. 8 A), and this was further demonstrated by
Western blot analysis of contractile vacuoles purified on a
sucrose density gradient (Fig. 8 B).
Fig. 8.
cadA cells contained contractile vacuoles devoid of DdCAD-1. (A)
The DdCAD-1 import assay made use of
cadA
cells that contained contractile
vacuoles devoid of DdCAD-1. The
cadA
cells were double immunostained
with anti-DdCAD-1 (a) and anti-calmodulin (b) antibodies. (B) A Western
blot of the contractile vacuole-enriched fractions isolated from mutant and wild-type (WT) cells was stained with both
anti-DdCAD-1 antibody (
) and anti-calmodulin antibody (*).
[View Larger Version of this Image (39K GIF file)]
cells and then mixed with the soluble
protein fraction obtained from wild-type cells. After 1 h of
incubation at room temperature, the contractile vacuoles
were repurified using a discontinuous sucrose density gradient. The uptake of DdCAD-1 by contractile vacuoles
was examined by proteinase K (10 µg/ml) digestion of the
repurified vacuoles followed by Western blot analysis (Fig. 9 B). If DdCAD-1 was imported from the wild-type cytosol into the contractile vacuoles of cadA
cells during incubation, DdCAD-1 should have become resistant to proteolytic digestion in the absence of detergent. Indeed, the
DdCAD-1 associated with contractile vacuoles was resistant to proteinase K digestion, indicating the translocation
of DdCAD-1 into the lumen of these vacuoles. In contrast,
contractile vacuole-associated calmodulin was degraded
under the same condition, yielding a partially digested
fragment (Fig. 9 B). Addition of either SDS or Triton X-100 resulted in the complete digestion of DdCAD-1.
Fig. 9.
In vitro import of DdCAD-1 into contractile vacuoles.
(A) Schematic drawing of the experimental protocol. The membrane-depleted cytosol fraction and the contractile vacuole-
enriched fraction were prepared from KAX3 cells and cadA
cells, respectively. These fractions were mixed at a ratio of 2:1
and incubated for 1 h at room temperature. As a negative control, GST was added to the assay mixture at 0.2 mg/ml. Then contractile vacuoles were reisolated by centrifugation through a discontinuous sucrose density gradient and subjected to Western
blot analysis. (B) Western blots showing the incorporation of
DdCAD-1 into contractile vacuoles (lanes a-c). After incubation
with the cytosol fraction derived from wild-type cells, repurified
contractile vacuoles were subjected to proteinase K (10 µg/ml)
digestion in the presence or absence of SDS. Additional controls
included the incubation of the cadA
contractile vacuoles with
buffer alone (lane d) and the incubation of wild-type cytosol with
buffer alone (lane e).
[View Larger Version of this Image (30K GIF file)]
The import of DdCAD-1 was enhanced by ATP and the
ATP regeneration system. When 1 mM ATP and the ATP
regeneration system were added to the import mixture, a
twofold increase in DdCAD-1 uptake by contractile vacuoles was observed (Fig. 10 A). However, when only ATP
was added, DdCAD-1 import was indistinguishable from the control. Similarly, the regeneration system itself did
not have any stimulatory effect (data not shown). To exclude the possibility that ATP and the regeneration system
increased the yield of the contractile vacuole membrane,
we measured alkaline phosphatase activity in the repurified contractile vacuole membrane fractions and no significant difference was observed (Fig. 10 B). Also, the association of calmodulin with contractile vacuoles was not
affected by the inclusion of ATP and the ATP regeneration system (Fig. 10 A).
In this paper we have provided evidence for a novel protein transport pathway involved in the presentation of the
cell adhesion molecule DdCAD-1 on the surface of Dictyostelium cells. DdCAD-1 is present primarily in the cytoplasm of the cell (Brar and Siu, 1993), and it begins to accumulate on the cell surface in the preaggregation stage of
development (Sesaki and Siu, 1996
). DdCAD-1 lacks a hydrophobic signal peptide sequence and is not associated with the Golgi apparatus or the ER, suggesting that it is
not transported to the plasma membrane via the classical
pathway. Our morphological observation indicates that
DdCAD-1 is concentrated in contractile vacuoles. Biochemical analysis of purified contractile vacuoles confirms
the presence of a substantial amount of DdCAD-1 inside
contractile vacuoles. Hypertonic treatment of cells leads to
the collapse of contractile vacuoles and inhibits the accumulation of DdCAD-1 on the cell surface. However, hypotonic shift results in the rapid reappearance of DdCAD-1-positive contractile vacuoles and the accumulation of
DdCAD-1 on the cell surface. Furthermore, NBD-Cl, a
specific inhibitor of vacuolar H+-ATPase, represses the
activity of contractile vacuoles and inhibits the accumulation of DdCAD-1 on the cell surface. Taken together, these results indicate that contractile vacuoles represent
the major vehicle by which DdCAD-1 is transported from
the cytoplasm to the plasma membrane.
Contractile vacuoles are intracellular organelles found
in most freshwater protozoa that are responsible for osmoregulation in cells. Both appearance and behavior of contractile vacuoles are tightly correlated with environmental
osmolarity (Patterson, 1980; Zeuthen, 1992
). Light microscopy shows that contractile vacuoles repeat to fill and
empty, and such repetitive behavior is accelerated when
cells are placed in hypotonic media, suggesting that contractile vacuoles function by excreting excess water from the cytosol. On the other hand, placing cells in hypertonic
media leads to the collapse of contractile vacuoles. Furthermore, when a dominant-negative form of rabD, a
small GTPase localized on contractile vacuoles, is overexpressed in Dictyostelium cells, the contractile vacuoles exhibit abnormal morphology and fail to respond to environmental osmolarity, resulting in a swollen cell shape in
hypotonic media (Bush et al., 1996
). In addition to this
classical view of contractile vacuole as a water-excretory organelle, our present data define a new role for contractile vacuoles in protein transport.
Although the lysosome has been shown to be a secretory organelle, capable of releasing lysosomal enzymes
into the medium during growth and development in Dictyostelium (Cardelli, 1993), several lines of evidence rule out
the involvement of lysosomes in DdCAD-1 transport. First,
immunofluorescence microscopy showed that DdCAD-1 was not enriched in lysosomes. Second, hypertonic conditions stimulated secretion of the lysosomal enzyme acid
phosphatase, whereas DdCAD-1 transport to the cell surface was inhibited. Third, NBD-Cl also exerted opposite
effects on DdCAD-1 transport and lysosomal secretion. Fourth, fractions enriched in lysosomes did not show a
corresponding increase in the DdCAD-1 import activity.
Finally, it has been shown that all of the lysosomal enzymes examined so far contain classical signal peptides at
their NH2 termini (Cardelli, 1993
). However, DdCAD-1
does not contain a signal peptide (Wong et al., 1996
).
A growing number of soluble proteins that lack an NH2-terminal signal peptide are known to be transported to the
cell surface or secreted into the medium via nonclassical
pathways. Examples of these proteins include galectin-1
(Cooper and Barondes, 1990; Cleves et al., 1996
),
-factor
(Kuchel et al., 1989), interleukin-1
(Rubartelli et al.,
1990
; Siders and Mizel, 1995
), thioredoxin (Rubartelli et
al., 1992
), and basic FGF (Florkiewicz et al., 1995
). Previous studies have identified several protein transporters that are involved in the secretion of these proteins, including several members of the ATP-binding cassette superfamily localized in the plasma membrane (for reviews see
Higgins, 1992
; Fath and Kolter, 1993
). The use of these
transporters in DdCAD-1 transport cannot be ruled out.
However, our results indicate that, even though a plasma
membrane-associated DdCAD-1 transport mechanism does exist, its contribution to DdCAD-1 transport is probably minimal (see Table I). The major DdCAD-1 transport pathway appears to depend on contractile vacuoles.
Many steps along the classical transport pathway, including targeting of precursor protein to the ER membrane (Andrews et al., 1989), protein translocation across
the membrane (Hansen et al., 1986
), vesicle-mediated intercompartmental protein transport (Balch et al., 1984
;
Baker et al., 1988
), and fusion of secretory vesicles with
the plasma membrane (Crabb and Jackson, 1985
), have been reconstituted successfully in vitro and studied extensively (Pryer et al., 1992
; Rothman, 1994
; Rapoport et al.,
1996
). However, nonclassical protein transport pathways
are much less well characterized. We propose that the contractile vacuole-mediated protein transport pathway may
involve a minimum of four steps, as illustrated schematically in Fig. 11. Initially, DdCAD-1 is recognized by contractile vacuoles and binds to specific components on the
cytoplasmic surface of contractile vacuoles. The binding of
DdCAD-1 on the contractile vacuole membrane will lead
to the translocation of DdCAD-1 across the contractile
vacuole membrane. This is followed by the association of
DdCAD-1 with an "anchoring" protein on the luminal
surface of contractile vacuoles. We envision that one end
of the DdCAD-1 molecule is associated with an integral
membrane protein, whereas the cell adhesion activity is associated with another segment of the molecule. As the contractile vacuole fuses with the plasma membrane, the anchored DdCAD-1 molecules move laterally from the vacuole
membrane to the plasma membrane.
As a first step to characterize this pathway biochemically, we have established an in vitro reconstitution system
and have successfully demonstrated the import of DdCAD-1
using isolated contractile vacuoles and cytosolic proteins.
Our results clearly showed that DdCAD-1 in the wild-type
cytosol was taken up by contractile vacuoles isolated from
cadA cells, whereas GST and calmodulin failed to enter
the contractile vacuole. The import of DdCAD-1 is therefore a highly selective process, which probably involves a
specific recognition mechanism similar to those found in
other organelle import systems (Schatz and Dobberstein,
1996
). The initial binding of DdCAD-1 on contractile vacuoles can be accomplished by a specific signal sequence on DdCAD-1 or by the formation of a complex of DdCAD-1
and other cytosolic proteins, which in turn is recognized by
specific receptors on the contractile vacuole membrane.
The import mechanism is enhanced by exogenously added
ATP and an ATP regeneration system, suggesting the involvement of specific ATP-dependent transporter(s) in
the contractile vacuole membrane. This cell-free system
should facilitate a biochemical approach in our future
characterization of the early steps involved in this protein
transport pathway and allow the identification of membrane components involved in DdCAD-1 translocation.
The fusion of contractile vacuoles with the plasma membrane and their subsequent contraction have been studied
fairly extensively. When contractile vacuoles become filled
with water, the contents are discharged into the medium
through the pore formed by fusion between the contractile
vacuole and the plasma membrane (Heuser et al., 1993). It
has been proposed that forces involved in pushing away
the contents are generated by contraction of the contractile vacuole membrane driven by the actin microfilament
cytoskeleton covering the cytoplasmic surface of contractile vacuoles (Baines et al., 1995
). Myosin I apparently
plays a crucial role in the contraction process, since mechanical loading of anti-myosin I antibodies into Acanthamoeba cells leads to cell burst in a hypotonic solution
(Doberstein et al., 1993
).
-Actinin is known to associate with contractile vacuoles (Furukawa and Fechheimer,
1994
), suggesting that it may also have a role in vacuole
contraction.
The accumulation of DdCAD-1 on the cell surface may
result from the lateral movement of DdCAD-1 from the
luminal surface of contractile vacuoles to the plasma membrane after fusion of these two membranes. Since cells are
also known to release soluble DdCAD-1 into the medium
during development (Siu et al., 1997), it is possible that
some of these molecules will bind back to the cell surface by their association with the DdCAD-1 anchoring protein.
We have obtained preliminary evidence for the involvement of an integral membrane protein that can anchor soluble DdCAD-1 to the plasma membrane (Brar, 1994
).
Whether the same protein is involved in the anchoring of
soluble DdCAD-1 on the luminal side of the contractile vacuole membrane remains to be determined.
DdCAD-1 is expressed soon after the initiation of development. However, it is present on the cell surface only
for a discrete period of time, mediating cell-cell adhesion
in the preaggregation stage (Sesaki and Siu, 1996). Prevention of cell-cell adhesion using anti-DdCAD-1 Fab in the
early stages of development blocks the cAMP-mediated
stimulation of gp80 expression at the aggregation stage,
suggesting that cell-cell adhesion mediated by DdCAD-1 may be involved in signaling pathways that regulate gene
expression (Desbarats et al., 1994
). Interestingly, upon the
formation of stable cell-cell contacts, surface-associated
DdCAD-1 leaves the plasma membrane and becomes internalized (Sesaki and Siu, 1996
). The observation that
DdCAD-1 is enriched in endosomes suggests that internalization of DdCAD-1 may be mediated by an endocytic pathway (Adessi et al., 1995
). The presentation and subsequent removal of DdCAD-1 thus represent a dynamic
process, which may be used to initiate and fine tune the
adhesive interactions among cells during development.
Received for publication 31 January 1997 and in revised form 14 May 1997.
Please address all correspondence to Chi-Hung Siu, Charles H. Best Institute, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6, Canada. Tel: (416) 978-8766. Fax: (416) 978-8528. E-mail: chi.hung.siu @utoronto.caWe thank Dr. David H. MacLennan for advice and comments on the
manuscript, Dr. Margaret Clarke for discussion, Dr. Agnes Fok for the
gift of mAb N2 against the vacuolar H+-ATPase, Dr. James A. Cardelli
for the gift of mAb 2H9 against -mannosidase, and Ms. Tak Yee Lam for
expert assistance. We also thank the Ontario Laser and Lightwave Research Center for access to their confocal microscope facility and Dr. Xijia
Gu for advice on LSCM.
This work was supported by operating grant MT-6140 from the Medical Research Council of Canada. E.F.S. Wong is supported by a Medical Research Council studentship, and H. Sesaki is a research fellow of the Japan Society for the Promotion of Science.
GST, glutathione-S-transferase; LSCM, laser scanning confocal microscopy; NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.
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