(Received for publication, July 20, 1995; and in revised form, September 19, 1995)
From the
Results from a number of laboratories suggest that intracellular
Ca is involved in the regulation of Dictyostelium
discoideum growth and development. To learn more about the
regulation and function of intracellular Ca
in this
organism, we have cloned and sequenced cDNAs that encode a putative
P-type Ca
ATPase designated patA. The
deduced protein product of this gene (PAT1) has a calculated molecular
mass of 120,718 daltons. It exhibits about 46% amino acid identity with
Ca
ATPases of the plasma membrane Ca
ATPase family and lower identity with sarco(endo)plasmic
reticulum Ca
ATPase family members and monovalent
cation pumps. However, PAT1 lacks the highly conserved
calmodulin-binding domain present in the C-terminal region of most
plasma membrane Ca
ATPase-type enzymes. When Dictyostelium amoebae are adapted to grow in the presence of
80 mM CaCl
, both the patA message and
protein product are up-regulated substantially. These cells also
exhibit an increase in the rate and magnitude of intracellular P-type
Ca
uptake activity. Immunofluorescence analysis
indicates that PAT1 colocalizes with bound calmodulin to intracellular
membranes, probably components of the contractile vacuole complex. The
presence of PAT1 on the contractile vacuole suggests that in Dictyostelium this organelle might function in Ca
homeostasis as well as in water regulation.
Intracellular calcium functions in the regulation of a wide
variety of cellular processes in both higher and lower eukaryotic cells (1, 2) . Recently, considerable progress has been made
in understanding Ca-signaling mechanisms in mammalian
cells(3) ; however, much less is known about the role of this
ion in lower organisms.
Calcium seems to be involved in development
of the lower eukaryote Dictyostelium discoideum, as suggested
by a number of observations. When starved of nutrients, amoebae of this
organism synthesize and secrete cAMP in a periodic fashion. The cells
respond to the waves of cAMP by differentiating, aggregating into
multicellular structures, and eventually forming fruiting bodies
comprised of two major cell types, stalk cells and
spores(4, 5) . During early development, treatment of
the amoebae with EGTA, a Ca chelator(6) ,
La
, a Ca
-channel
blocker(7) , or putative intracellular Ca
or
calmodulin antagonists (8, 9, 10) inhibits
cell differentiation and/or aggregation. In addition, depletion of
intracellular Ca
stores with EGTA and A23187 reduces
the stability and secretion of the enzyme cyclic nucleotide
phosphodiesterase and its specific inhibitor(11, 12) .
During late development, Ca
accumulates
preferentially in prestalk regions of migrating aggregates and
influences stalk/spore cell differentiation(13, 14) .
These observations indicate that changes in the distribution of
intracellular Ca might be important in regulating Dictyostelium development. When the amoebae are shaken in
buffer, they accumulate and extrude Ca
ions. The rate
of Ca
uptake is increased dramatically by the
activation of cell surface folate and cAMP receptors (7, 15) through a process that might be G-protein
independent(16) . Receptor activation also results in the
inositol 1,4,5-trisphosphate-induced release of Ca
from a small intracellular Ca
pool(17, 18) . Using a
Ca
uptake assay and isolated cell
fractions, a major inositol 1,4,5-trisphosphate-insensitive
intracellular Ca
pool has been
identified(19, 20) . This pool is associated with
``acidosomal'' membranes(21, 22) , a
component of the contractile vacuole complex(23, 24) .
Transport of Ca
into this pool occurs via a
vanadate-sensitive(19) , thapsigargin-insensitive (22) Ca
ATPase, and ion movement is reported
to be facilitated by a high intravesicular proton
concentration(21, 22) . Using a
Ca
-sensitive electrode and filipin-permeabilized
cells, a small intracellular inositol 1,4,5-trisphosphate-sensitive
Ca
pool has also been detected. Ca
uptake into this pool is vanadate-resistant and might involve a
H
/Ca
antiport(25) . In
addition, a Ca
-stimulated ATPase associated with
plasma membrane fractions has been reported(26) ; this enzyme
might function in Ca
efflux. At present, however,
little is known about the movement of Ca
ions between
intracellular pools or the extrusion of Ca
from the
cells.
One way to dissect complex systems such as this is to clone
genes encoding the various pumps/transporters and to use these clones
to disrupt the endogenous sequences by homologous recombination or to
reduce their expression by antisense RNA strategies. This approach has
been very effective at identifying certain
Ca-signaling pathways in Saccharomyces
cerevisiae(27, 28, 29) .
In all
eukaryotic cells, Ca-translocating ATPases play a
major role in Ca
homeostasis (30) . These
pumps are members of a large family of P-type cation transport ATPases
(so named because they form a phosphoenzyme intermediate during the
catalytic reaction). All ATPases of this family possess regions of high
amino acid sequence homology; however, the different members can be
subclassified according to their cellular localization and function, e.g. plasma membrane Ca
ATPase (PMCA) (
)and sarco(endo)plasmic reticulum Ca
ATPase (SERCA)(31, 32) . In the present work, we
have used the PCR to clone cDNAs of a D. discoideum gene patA (P-type ATPase A) that encode a putative Ca
ATPase. Indirect immunofluorescence analysis suggests that the
product of this gene (PAT1) is associated with an intracellular
organelle, probably the contractile vacuole.
All
procedures involving plasmid and phage amplification (36) were
performed using Escherichia coli strains DH5 and LE392,
respectively.
The cDNA insert in the first clone
isolated (cDNA1) was completely sequenced using a combination of
exonuclease III/mung bean nuclease-generated deletions (36) and
synthetic oligodeoxyribonucleotides. Double-stranded DNA sequencing was
performed using a Sequenase Version 2.0 kit (U. S. Biochemical
Corp./Amersham). The deduced open reading frame of cDNA1 encoded a
putative protein with significant identity to Ca
ATPases, but it lacked both the 5`-initiation and 3`-termination
sequences. A
0.45-kb fragment spanning the 5`-end of cDNA1 to a
unique internal EcoRV restriction site was used to rescreen
the cDNA library, and several additional
clones were obtained.
Two of the new cDNAs (cDNA2 and cDNA3) were found to encode the missing
3`- and 5`-ends, respectively. The combined overlapping sequences of
cDNAs 1-3 encode the full-length patA cDNA sequence
submitted to the GenBank
/EMBL data bank.
A 50-µg sample of GST-PAT1 was transferred to nitrocellulose after SDS-PAGE and used as an affinity matrix to purify antibodies specific for the fusion protein (anti-PAT1) as described(36) .
Immunostained cells
were examined using a Bio-Rad MRC-600 confocal microscope with
xenon-argon laser attached to a Nikon Optiphot microscope with a
60 objective lens (NA = 1.4) and simultaneous dual
channel (split-screen) imaging. For each cell, the background gray
levels and gain were adjusted so that the gray scale levels did not
saturate the detector (i.e. fell within the range
1-255), and a series of optical sections about 0.7 µm apart
were recorded. Confocal images were printed using a Mitsubishi thermal
video printer; phase-contrast images were photographed directly through
the microscope using a 100
NA 1.3 phase contrast objective and a
Nikon F3 camera.
Partial sequencing of the D. discoideum PCR products
identified four unique sequences with relatively high amino acid
sequence identity to known P-type ion pumps (Table 1). One of
these PCR products (0.95 kb) showed appreciable identity to
Ca ATPases; therefore, this sequence was used to
isolate corresponding cDNAs. Analysis of these cDNAs revealed a
complete open reading frame (patA). The proposed ATG start
codon in the patA cDNA is in a sequence (AAAATGA),
which agrees well with the consensus translation initiation sequence
(AXAATGG) of D. discoideum(48) .
Moreover, this ATG is preceded by an in-frame TAA stop codon 12 bp
upstream. The open reading frame terminates after coding for 1115 amino
acids, corresponding to a protein with a calculated molecular mass of
120,718 daltons.
The putative protein product of patA (PAT1, Fig. 1) contains the conserved phosphorylation and
ATP-binding domains present in all P-type ATPases. It shows highest
amino acid sequence identity to PMCA family members and lower identity
to the SERCA family of Ca ATPases and monovalent
cation pumps (Table 2). Hydropathy analysis of the deduced amino
acid sequence (not shown) reveals a profile very similar to those of
PMCA pumps, except for a deletion in the PAT1 sequence between the
second and third transmembrane domains. This is a phospholipid-binding
regulatory region that is subject to alternative splicing and is not
well conserved among the known PMCA isoforms(32) . The vacuolar
Ca
ATPase from S. cerevisiae (PMC1 and its protein product Pmc1p, (28) ), a PMCA family
member, also has a large deletion as well as an insertion in this
region. Like Pmc1p, PAT1 lacks the identically conserved amino acid
sequence associated with calmodulin-binding subdomain A. This sequence,
essential for calmodulin binding, is present near the C terminus of all
mammalian PMCA isoforms (49) but is absent in the yeast
isoform, which is truncated in this region (28) and in PAT1
where sequence conservation is lost (Fig. 2). Calmodulin-binding
subdomain B, which is thought to influence the affinity of calmodulin
binding to subdomain A(49) , also appears to be absent in PAT1.
The amino acid sequence of this subdomain is variable in the different
PMCA isoforms, although some conservation is maintained. PROSITE
analysis of PAT1 reveals a potential cAMP-dependent protein kinase
phosphorylation site (Fig. 1, amino acids 1031-1034),
which is also present in certain PMCA isoforms (e.g. human PMCA1a, (50) ).
Figure 1: Deduced amino acid sequence of patA cDNA. Amino acids are numbered on the left. Amino acid sequences corresponding to primers A1 (amino acids 381-389, phosphorylation domain), B2 (amino acids 677-683, ATP-binding domain), and the amino acid sequence of the putative cAMP-dependent protein kinase phosphorylation site (amino acids 1031-1034) are underlined.
Figure 2: Amino acid sequence alignment of PMCA family members in the calmodulin-binding region. The calmodulin-binding domains A and B (boxed) of human (Hu) and rat (Rt) PMCA isoforms (49) were aligned with the corresponding regions of PAT1 and S. cerevisiae Pmc1p using the CLUSTAL program. Identical amino acids are denoted by asterisks under the sequences. The position in each sequence of the residue at the C-terminal end of the region is indicated in parentheses on the right.
Figure 3:
Expression of patA mRNA. A, poly(A) RNA isolated from AX2 cells grown
in unsupplemented MES/HL-5 medium (lane 1) or in the same
medium supplemented with 80 mM CaCl
(lane
2) or 80 mM MgCl
(lane 3) was
size-fractionated on an agarose gel, transferred to nylon membrane, and
probed at high stringency with a 1367-base pair patA cDNA
fragment containing the sequence from base pair 1 to a unique internal EcoRI site. B, the membrane was stripped and reprobed
with a full-length vatP cDNA, which encodes a Dictyostelium proteolipid.
To detect the PAT1, antibodies were raised against the
C-terminal 176 amino acids of the protein fused to GST and affinity
purified as described under ``Experimental Procedures.''
Total membrane samples prepared from the cells used in Fig. 3were subjected to Western analysis using the
affinity-purified anti-PAT1. The antibodies detected a single band of
approximately 120 kDa, the predicted molecular mass of PAT1 (Fig. 4, lane 1). Membranes from cells grown in the
presence of CaCl possess a significantly higher level of
PAT1 (Fig. 4, lane 2). This elevated level of PAT1
correlates well with the increased abundance of patA message
observed in the Ca
-grown cells. In contrast,
membranes from Mg
-grown cells contain levels of PAT1
comparable to cells grown in unsupplemented MES/HL-5 medium (Fig. 4, lane 3).
Figure 4:
Western blot analysis of PAT1. Membranes
from 2 10
cells (2 µg of protein) grown in
unsupplemented MES/HL-5 medium (lanes 1 and 4) or in
the same medium supplemented with 80 mM CaCl
(lanes 2 and 5) or 80 mM MgCl
(lanes 3 and 6) were subjected to SDS-PAGE on
7.5% gels and transferred to nitrocellulose membranes. Lanes
1-3 were probed with affinity-purified anti-PAT1 antibodies
while lanes 4-6 were probed with preimmune
serum.
Figure 5:
Ca uptake by
filipin-permeabilized cells. Amoebae were grown to late-log phase in
unsupplemented MES/HL-5 medium (
) or in the same medium
supplemented with 80 mM MgCl
(
) or 80
mM CaCl
(
). The cells were permeabilized
with filipin, incubated with mitochondrial inhibitors, and assayed for
ATP-dependent Ca
-transporting activity in uptake
medium containing 100 nM free Ca
.
Ca
uptake values for control (unsupplemented) and
MgCl
-grown cells are an average of results from two
independent experiments; values for CaCl
-grown cells are an
average ± S.D. of results from three
experiments.
Figure 6:
Indirect immunofluorescence microscopy of
PAT1 and insoluble calmodulin in D. discoideum cells. This
figure shows two different cells: A-E is one cell, and F-J is the other. E and J are phase
contrast images of the two cells, and the other illustrations are in
pairs; the left images (A, C, F,
and H) show staining for PAT1 with affinity-purified anti-PAT1
antibodies, while the right images (B, D, G, and I) illustrate staining for calmodulin with 2D1
antibodies. The optical section for A-B is seven sections from
that of C-D; the optical section for F-G is four
optical sections from that for H-I. All images are printed at
2100. In each section, the staining is very similar, if not
identical, using the two different
antibodies.
Like other eukaryotic cells, amoebae of D. discoideum should possess a variety of P-type pumps to regulate the
distribution and concentration of intracellular cations during growth
and development. Using the PCR, we have identified four genes from Dictyostelium that exhibit appreciable identity to P-type
pumps from other organisms (Table 1). In addition, a number of
other cDNAs from this organism have been cloned, which, by virtue of
sequence similarity, seem to be related to these ATPases. ()Therefore, D. discoideum appears to possess at
least as many P-type ion pumps as S. cerevisiae, where five
have been identified to
date(27, 28, 46, 47) . In the
present study, we have focused our work on one Dictyostelium gene, patA.
Several lines of evidence suggest that patA encodes an intracellular P-type Ca ATPase. First, the amino acid sequence deduced from patA cDNAs contains phosphorylation and ATP-binding motifs conserved in
all P-type ATPases as well as a putative cAMP-dependent protein kinase
phosphorylation site present in certain PMCA isoforms (Fig. 1).
Moreover, amino acid sequence alignment (Table 2) and hydropathy
analysis of PAT1 suggest that this protein is a member of the PMCA
family of Ca
ATPases. Second, both patA mRNA (Fig. 3) and PAT1 (Fig. 4) are overexpressed in
Ca
-adapted cells. To grow amoebae in the presence of
relatively high concentrations of Ca
, the
Ca
content of the growth medium must be increased
gradually over a period of several weeks. Thus, growing the cells under
conditions of Ca
stress appears to select variants
that overexpress patA. A similar up-regulation of a SERCA
Ca
ATPase has been observed in Chinese hamster lung
fibroblast DC-3F cells during growth in the presence of thapsigargin,
an inhibitor of the SERCA family of ATPases(54) .
Ca
-grown Dictyostelium cells, permeabilized
with filipin, also exhibit an increase in the rate and magnitude of
non-mitochondrial, vanadate-sensitive Ca
uptake (Fig. 5). The enhanced ability of these cells to sequester
Ca
is probably a consequence of the elevated levels
of PAT1. Interestingly, Ca
accumulation continued
after 6 min in Ca
-grown cells but leveled off in
cells not adapted to 80 mM Ca
. This may be
due to an enhanced Ca
storage capacity in the
Ca
-grown cells. Alternatively, the possibility exists
that other intracellular P-type Ca
ATPases are also
up-regulated in Ca
-stressed cells. Third, cell
localization studies on PAT1 using indirect immunofluorescence indicate
that this ATPase resides on membranes of the contractile vacuole
complex (Fig. 6).
In mammalian cells, PMCA-type
Ca ATPases are situated on the plasma membrane,
whereas in Dictyostelium, PAT1, a PMCA homolog, appears to be
a component of the contractile vacuole. This finding is consistent with
the biochemical evidence for intracellular PMCA activity in this
organism(19, 20, 21, 22) . Recently,
intracellular PMCA enzymes have also been identified in other
organisms. For example, a gene encoding a vacuolar PMCA-type pump (PMC1) has been cloned from S.
cerevisiae(28) , and intracellular PMCA-like activities
have been characterized in plants(55, 56) , although
in these cases the enzymes seem to be associated with the endoplasmic
reticulum.
Although PAT1 is a PMCA homolog and it colocalizes in the
cells with calmodulin, normally a regulator of PMCA activity, there is
no evidence that PAT1 activity is regulated by calmodulin. Sequence
alignment analysis of PAT1 with the highly conserved calmodulin-binding
domains of plasma membrane PMCA-type ATPases reveals that PAT1 (like
Pmc1p of S. cerevisiae) lacks this domain (Fig. 2). It
also lacks putative amphiphilic helices in the C-terminal region that
have been implicated in calmodulin binding(57) . In agreement
with the sequence analysis, biochemical characterization of
intracellular PMCA-type activity in Dictyostelium cells
indicates that the activity is unaffected by calmodulin supplementation
or by the addition of calmodulin
antagonists(19, 20, 22) . In mammalian cells,
however, the activity of PMCA enzymes can often be elevated by limited
proteolysis of the C-terminal regulatory domain to levels comparable to
Ca/calmodulin activation(32) , and this
process is thought to be a significant regulatory mechanism in vivo(58, 59) . If PAT1 is regulated by such a
process, it might obscure the calmodulin sensitivity of the enzyme in
biochemical assays. Therefore, at this time, we cannot rule out the
possibility that calmodulin regulates PAT1 activity by interacting with
as yet unidentified sequences on the enzyme.
Contractile vacuoles
are morphologically complex organelles found in many freshwater
protozoa and amoebas where they are thought to function in
osmoregulation(24, 51, 60) . These structures
accumulate water and ions by poorly understood mechanisms and discharge
their contents outside the cell by fusion with the plasma
membrane(23) . Based on the properties and intracellular
localization of a Ca ATPase in Dictyostelium, Milne and Coukell (20) proposed that
extrusion of excess Ca
from these cells might be
facilitated by the fusion of Ca
-sequestering vesicles
with the cell membrane. Subsequent studies revealed that the high
affinity Ca
ATPase associated with these vesicles
resides in a buoyant membrane fraction(21, 22) ,
probably a component of the contractile vacuole
system(23, 24) . In the present study, we show that
the putative Ca
ATPase, PAT1, colocalizes with bound
calmodulin to membranes of the contractile vacuole and that
Ca
-adapted cells overexpress PAT1 and possess
elevated intracellular Ca
uptake activity. Together,
these observations suggest that in D. discoideum the
contractile vacuole complex might play an important role in
Ca
homeostasis as well as in water regulation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89369[GenBank].