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INTRODUCTION |
Programmed cell death (PCD),1 a physiological
suicide process, is implicated in
the development and maintenance of an organism's integrity. Its most
common phenotype, apoptosis, is generally considered as the paradigm of
PCD in multicellular organisms. However, PCD is no longer confined to
situations initially termed as apoptosis. This is particularly so in
unicellular organisms where morphological diversity and multiple
mechanisms of PCD have been observed. The nature of executioners
involved in PCD has also broadened, with the discovery of
caspase-independent pathways and autophagic processes (1).
In the cellular slime mold, Dictyostelium discoideum, the
developmental cycle includes a multicellular phase that involves both
differentiation and morphogenesis (2, 3). Within the multicellular
structure that forms from the initial aggregation of starving
individual cells, prestalk cells undergo a PCD process that leads to
the formation of a stalk comprising dead vacuolated cells (4)
supporting a mass of viable spores. In an in vitro system
that permits differentiation without morphogenesis, cells committed to
PCD were unable to re-grow and showed specific morphological features,
including massive vacuolization and cytoplasmic and focal chromatin
condensations (5, 6). Study of this type of developmentally regulated
cell death in eukaryotes, which diverged early in the development of
life, might reveal underlying mechanisms of PCD that are conserved as
such, or slightly modified, in more complex organisms. The sequencing
of the genome of D. discoideum is almost completed (7).
Investigating the amebal genome shows that only a limited number of the
proteins involved in mammalian PCD have homologues in D. discoideum. In addition to the "apoptosis inducing factor" or
AIF and a putative paracaspase (8, 9), we have identified D. discoideum homologues of the apoptosis-linked gene 2 (ALG-2) and
of its binding partner, ALG2-interacting protein X,
Alix.2 In mammalian cells,
both proteins have been determined as being components of the apoptotic
machinery (10-12). The molecule, ALG-2, is a calcium-binding
protein of the penta-EF-hand family, which includes sorcin, peflin,
grancalcin, and calpain (13-17).
During the development stage induced by starvation in D. discoideum, calcium ions have been described as regulating cell
type differentiation (18-20). Even though a number of calcium-binding proteins have been characterized in this organism, among which are
CBP1-4, CAF1, CaM, and CalB (21-27), none so far has been assigned a
specific role as a mediator of calcium ion signaling during this phase
of differentiation. In view of the proposed involvement of ALG-2 in
apoptosis in higher eukaryotes, we decided to examine whether D. discoideum multicellular development was dependent upon this
calcium-binding protein.
In this paper, we describe the calcium-dependent
biochemical properties of the two D. discoideum homologues
of mammalian ALG-2 that we have identified and their interaction with
mammalian Alix. While this work was in progress, the two ALG-2
homologues were independently identified (28). Our report provides
original and complementary biochemical data pertaining to their
physiological role with respect to Alix and describes alg-2
knock-outs and their characterization during vegetative life and the
developmental cycle.
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EXPERIMENTAL PROCEDURES |
Materials--
Phenylmethylsulfonyl fluoride, leupeptin,
pepstatin, aprotinin, and IPTG were from Roche Diagnostics. TNS was
from Sigma-Aldrich. pMAL-C2, pQE-30, and pGEX-6P-2 expression vectors
were from New England Biolabs, Qiagen, and Amersham Biosciences, respectively.
Cell Culture Conditions and Differentiation--
All experiments
were done using wild-type developing D. discoideum KAx-3 and
JH10, a thymidine auxotroph (29), as parental strains. Cells were grown
at 21 °C in cultures shaken at 180 rpm in HL5 medium supplemented
when necessary with 7.5 µg/ml blasticidin, 20 µg/ml G418, or 100 µg/ml thymidine. Developmental phenotypes were studied after plating
cells on non-nutritive Na,KPi-buffered agar plates
(30).
Cloning and Sequencing the D. discoideum ALG-2 genes--
For
both alg2a and alg2b genes, the sequence of the
coding cDNA and the full-length genomic sequence including introns
were obtained after PCR experiments with forward primers
(5'-TCAAAAATGATGTCATATGGATAC and 5'-TTAAATGTACGGATACGGATATAC)
and reverse primers (5'-TTAAACTAAGCTAATAATATCATAAATGTG and
5'-TTAAACTAAAGCAATGATATCATAAAG) (from Oligo Express, Paris, France)
for Dd-ALG-2a and Dd-ALG-2b, respectively. Primers were designed
on the basis of relevant clones in the Dictyostelium cDNA data base (www.csm.biol.tsukuba.ac.jp/cDNAproject.html). The genomic and cDNA sequences have been deposited at GenBankTM under accession nos. AF358913 and AF358911 for Dd-alg-2a and
AF358914 and AF358912 for Dd-alg-2b, respectively.
Generation of Mutants and Plasmid Constructs--
The single
Dd-alg-2a and -2b null mutants were made in KAx-3
strain by inserting the blasticidin S resistance cassette
bsr (31) at position 294 (in Dd-alg-2b) and
position 376 (in Dd-alg-2a) of the genomic DNAs. To obtain a
double knock-out mutant, a Dd-alg-2b-deficient strain was
first generated in the JH10 strain by disruption of the coding sequence
with the THY1 gene-containing cassette (29). This was then
used to introduce the Dd-alg-2a gene knock-out construct containing the bsr cassette. Transformants were cloned by
plating cells on SM-agar plates in the presence of
Klebsiella aerogenes. Potential gene knock-out
clones were first analyzed by Western blot and confirmed by Southern blot.
The cDNA sequences (except for the initial methionine) coding for
full-length or truncated proteins ALG-2a, ALG-2a
Nt (amino acids
28-197), ALG-2aEF5 (amino acids 3-175), ALG-2b, and ALG-2bEF5 (amino acids 2-183) were subcloned in-frame with the maltose-binding protein (MBP), into the pMAL-c2 expression vector (MBP-ALG-2a and -2b)
or with the amino-terminal polyhistidine tag contained in the pQE-30
expression vector (His6-ALG-2b). In all cases, PCR amplification was used to create the appropriate subcloning sites and
the constructs were then verified by sequencing. Recombinant proteins
were expressed in Escherichia coli strain BL21 cells.
Purification of Recombinant Proteins--
BL21 bacteria
expressing the various recombinant Dd-ALG-2 proteins were grown
overnight at 37 °C in Luria Broth, diluted to 1/50 in Terrific
Broth, and grown at 37 °C to A600 = 0.6. IPTG was then added at a final concentration of 1 mM and growth
resumed for 3 h. Cells were then harvested by centrifugation at
10,000 × g for 15 min, and the bacterial pellets
frozen at -20 °C until use. The MBP-fused proteins were expressed
at high levels (about 50 mg/liter of bacterial suspension) and in
soluble forms. In contrast, His6-ALG-2b was expressed at a
high level but always as inclusion bodies. To purify recombinant
MBP-tagged proteins, the bacterial pellets were thawed in 25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM
EDTA (buffer A) containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 20 µg/ml pepstatin and leupeptin, and
0.5% Triton X-100. All steps were carried out at 4 °C.
Suspensions were sonicated for 3 min and centrifuged for 1 h at
200,000 × g. Supernatants were mixed with amylose
beads washed in buffer A and rotated on a wheel overnight at 4 °C.
After extensive washing with buffer A, MBP-tagged proteins were
batch-eluted with buffer A containing 10 mM maltose and 1 mM EDTA. The recombinant proteins were >98% pure (see
Fig. 2) without any additional purification step.
Mouse Alix--
N-terminal GST (glutathione
S-transferase)-tagged mouse Alix, cloned into vector
pGEX-6P-2, was expressed in BL21 bacteria and induced with 1 mM IPTG, then grown for another 15 h at 15 °C.
After purification of the fusion protein on glutathione-Sepharose 4B,
the GST tag was cleaved with PreScission protease (Amersham Biosciences) to obtain Alix. The protein appeared as a single band on
SDS-PAGE.
Antibodies and Western
Blots--
His6-ALG-2b was expressed in BL21
bacterial strain (see above). Purified inclusion bodies were dissolved
in 6 M urea, purified on nickel-nitrilotriacetic
acid-agarose (Qiagen) and extensively dialyzed against 10 mM Tris-Cl, pH 8.0. The preparation was checked for
homogeneity by SDS-PAGE, lyophilized, and injected into rabbits to
raise polyclonal antibodies. Western blot analysis showed that the
immune serum recognized both Dd-ALG-2a and -2b. To follow the
expression of the endogenous Dd-ALG-2 throughout development, protein
samples were prepared from developing amebae from the wild-type strain,
KAx-3, or single knock-out strains, Dd-alg-2a
and -2b. Protein samples were separated by
SDS-PAGE, transferred onto PVDF Immobilon-P membranes (Millipore) and
probed with the antiserum using the enhanced chemiluminescence system
(ECL; Amersham Biosciences) for detection.
Northern Blots--
Total RNA was isolated as described (32)
from vegetative amebae or cells that had been filter-developed on
non-nutritive Na/KPi-buffered agar plate for 4, 8, 12, 16, 20, or 24 h. At each time, 6 µg of total RNAs were separated by
electrophoresis on a formaldehyde-containing agarose gel, transferred
to a nylon blotting membrane, and hybridized with a specific
digoxigenin (DIG)-labeled RNA probe. DIG-labeled RNA probes were
generated according to the manufacturer's instructions (Roche
Molecular Biochemicals). The RNA probes for Dd-alg-2a and
-2b corresponded to their full-length sequence.
Hybridization was performed in DIG-Easy buffer at 50 °C. The
mRNAs were detected after binding of anti-DIG antibodies coupled to
alkaline phosphatase with CDP-StarTM as substrate.
Surface Plasmon Resonance Measurements and Data
Evaluation--
Real-time analysis of the
Ca2+-dependent interactions between the two
Dd-ALG-2 isoforms and mouse Alix was performed at 25 °C on a
BIAcoreTM instrument. Proteins were diluted to 10 µg/ml in 10 mM sodium acetate, pH 5.0, and coupled to the
carboxylmethylated dextran surface of a CM5 sensor chip using a
N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling mixture. The amount of immobilized protein was between 3000 and 4000 resonance units. Running buffer for protein interaction contained 150 mM NaCl, 10 mM HEPES, pH 7.5, 0.25% Tween 20, and varying amounts of Ca2+ and
circulated at a flow rate of 5 µl/min. To obtain blank sensorgrams, for subtraction of bulk refractive index background, equal volumes of
each protein were injected over a surface with immobilized MBP alone.
Regeneration of the surfaces was carried out by injecting 5 µl of 8 M urea after checking that binding isotherms were
unaffected by such a denaturation step.
Sensorgrams were analyzed by nonlinear least square curve fitting. A
single-site binding model was used for kinetic analysis of
interactions. The equation Rt = Req (1 
exp(ks(t t0))) was used for the association phase, where
Rt is the amount of ligand bound at time
t; Req is the amount of ligand bound
at equilibrium; t0 is the beginning of the
association phase; and ks = konC + koff,
where C is the concentration of injected analyte. The rate constants
kon and koff were
determined from the plot of ks versus C. The koff constant was also
obtained from the analysis of the dissociation phase using the equation
Rt = R0
exp(koff(t t0)). The parameter Rt is
the amount of ligand bound at time t, and R0 is the amount of ligand bound at time
t0 corresponding to the beginning of the
dissociation phase. The mean value obtained from a series of injections
was taken as dissociation constant koff. The
values of koff derived directly from
dissociation curves or from secondary association plots were in
agreement. The apparent dissociation constant,
KD, was calculated from the ratio of the two
kinetic constants
(koff/kon).
In the case where interactions approached equilibrium, the dissociation
constant KD was also determined directly from
equilibrium levels of the analyte binding using the equation Req/C = (Rmax Req)/KD. The parameter,
Rmax, was the maximal binding capacity of the
immobilized ligand and KD was derived from the slope
of the Req/C versus
Req plot.
Free Ca2+ and Ca2+-buffer systems were
calculated using the WEBMAXC (version 2.10) program (33) available at
www.stanford.edu/~cpatton/webmaxc2.htm.
Fluorescence Measurements--
Two types of fluorescence
experiments were performed to follow changes of protein conformation:
intrinsic fluorescence alteration and
2-(p-toluidino)naphthalene-6-sulfonate (TNS) fluorescence enhancement. All fluorescence measurements were recorded at 21 °C
with a Spex Fluoromax spectrofluorometer in a buffer made of 150 mM NaCl, 25 mM HEPES, pH 7.5. Intrinsic
fluorescence of the MBP-ALG2 isoforms (40 µg/ml, ~0.6
µM) was measured using excitation and emission
wavelengths set at 295 and 348 nm, respectively. In control
experiments, it was checked that the addition of Ca2+ or
EGTA had no effect on the shape of the fluorescence spectra of the
proteins and solely affected the fluorescence intensity. TNS
fluorescence was measured in 2.5 ml of buffer containing 100 µg of
the protein of interest to which 10 µM TNS was added.
Excitation and emission wavelengths were set at 325 and 427 nm,
respectively. Fluorescence variations were expressed as
F/F0 in percentages.
Subcellular Fractionation--
For subcellular fractionation,
1.5 × 109 cells were homogenized with a cell cracker
in 3 ml of buffer: 0.25 M sucrose, 0.5 mM EDTA,
25 mM MES-KOH, pH 6.5, and a mixture of protease
inhibitors. Unbroken cells and nuclei were recovered by first
centrifuging the homogenates at 1,000 × g for 5 min.
The postnuclear supernatant was then loaded onto a 21-ml 24% (v/v)
self-forming Percoll gradient in 0.11 M KCl, 0.5 mM EDTA, 10 mM HEPES, pH 7.5; spun for 1 h at 18,000 rpm in a Kontron TFT70 rotor; and eluted in 2-ml fractions.
Immunofluorescence--
Immunofluorescence observations were
made from D. discoideum cells carrying C-terminal
c-Myc-tagged constructs of ALG-2a and -2b under the control of Act15
promoter (pEXP4+ vector). Approximately 2 × 105 amebae were allowed to adhere onto coverslips for 10 min and fixed in a solution of 40 mM MES-Na, pH 6.5, 4%
paraformaldehyde for 10 min at 20 °C. They were then permeabilized
in 0.2% Triton X-100-40 mM MES-Na, pH 6.5, for 2 min.
After several washes in PBS, 0.5% bovine serum albumin, cells were
incubated with the primary anti-c-Myc antibody (Roche Molecular
Biochemicals, 4 mg/ml stock, dilution 1/500) for 1 h. The
secondary antibody (fluorescein isothiocyanate-conjugated goat
anti-mouse antibody, Jackson Immunoresearch) was added at a dilution of
1/200 after three washes in PBS plus 0.1% Tween 20. Finally the
samples were washed extensively in PBS-Tween 20, mounted on glass
slides, and observed in a Zeiss Axioplan microscope.
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RESULTS |
D. discoideum Possesses Two Homologues of Apoptosis-linked Gene 2 (alg-2)--
The ALG-2 protein, first described by Vito et
al. (11), belongs to the family of calcium-binding proteins with
penta-EF-hands. The D. discoideum expressed sequence tag
data base was searched for homology with murine ALG-2. We found several
clones corresponding to two distinct cDNA sequences. There were 10 members in a first group and 5 members in a second group with
significant probability scores. The full sequences of the two D. discoideum alg-2a and alg-2b genes were determined by
PCR experiments. The first gene, Dd-alg-2a, consisted of 4 exons interlaced with 3 short (217, 82, and 172 bp) and AT-rich introns
(97, 89, and 96% A+T, respectively). The short length and the biased
base composition are typical features of D. discoideum
introns (34). This gene encoded a 197-amino acid-long Dd-ALG-2a protein
with a calculated molecular mass of 22.3 kDa and a pI of 8.0. On the
D. discoideum genome, Dd-alg-2a was located
adjacent to Rho GDP-dissociation inhibitor (rdiA) gene
(GenBankTM accession no. AY044085). The second gene,
Dd-alg-2b, consisted of 2 exons (243 and 375 bp) interrupted
by a 134-bp intron (90% A+T). The intron border in
Dd-alg-2b corresponded exactly to the intron II border in
Dd-alg-2a. The gene encoded a 205-amino acid-long Dd-ALG-2b
protein with a calculated molecular mass of 23.3 kDa and an isoelectric
point of 5.3.
As shown in Fig. 1A, Dd-ALG-2a
and -2b were 64 (74) % identical (similar). A BLAST search of
GenBankTM data base (www.ncbi.nlm.nih.gov: 80/BLAST/) showed that both
Dd-ALG-2a and -2b were homologous to various other penta-EF-hand
proteins. The highest scores were obtained with Entamoeba
histolytica URE3-binding protein (35) and grainins (36), but also
mouse members of the penta EF-hand family, including ALG-2, peflin,
sorcin, grancalcin, and calpains (13-15, 17, 37-39). Dd-ALG-2a and
-2b both exhibited five putative EF-hand
Ca2+-binding sites. EF1, EF2, and EF3 were
identical to the EF-hand consensus pattern (ProSite PS00018). As for
all other penta-EF-hand proteins, the EF-hand scores of Dd-ALG-2a and
-2b were lower in EF4 and EF5. The structure of the two D. discoideum proteins was obtained by homology modeling and
threading (40) the amebal proteins onto the three-dimensional structure
of Ca2+-loaded mouse ALG-2 (amino acids 21-191) (41). A
quite remarkable superimposition characterized by high confidence
factors was obtained in particular at the level of the eight
-helices (Fig. 1, B and C). The main
difference between Dd-ALG-2a and -2b was found at the level of helix 7 contributing to EF4 and EF5. Helix 7 in Dd-ALG-2a closely resembles the
mouse counterpart, but is shorter on the N-terminal side ending EF4 in
Dd-ALG-2b. Furthermore, the amino acid side chains in the EF5-loop of
the D. discoideum proteins could only establish three
putative calcium coordination bonds. This will not favor calcium
binding at this site. Finally, the two amebal proteins differed from
the murine ALG-2 at the level of EF2. For Dd-ALG-2a and -2b, this is
100% identical to the canonical Ca2+-binding site but only
68% similar for the mouse protein.
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Fig. 1.
Multiple amino acid sequence alignment of
Dd-ALG-2a and -2b with the mouse protein and homology modeling of
Dd-ALG-2a and -2b. A, alignments were performed with
ClustalW (47) using the sequence for mouse ALG-2 (GenBankTM accession
no. P12815) and the D. discoideum sequences for ALG-2a
(GenBankTM accession nos. AF358913 and AF358911; this work) and ALG-2b
(GenBankTM accession nos. AF358914 and AF358912; this work).
Identities between the proteins are boxed, and homologies
are indicated by a gray background. Secondary
structure helices 1-8 are placed according to the
three-dimensional structure (41), and the five putative EF-hands are
indicated as text between the bars.
B and C, homology models for Dd-ALG-2a
(B) and Dd-ALG-2b (C) were calculated by Swiss
Model and visualized with Deep View Swiss-PDB Viewer (40) using the
published structure of mouse ALG-2 (1HQV) as a template (41).
Structures were colored according to secondary structure succession,
with a transition from blue to red from the most
N-terminal helix to the C-terminal end.
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Expression and Purification of MBP-ALG-2a/2b Constructs--
All
the constructs used to examine protein-protein interactions (Dd-ALG-2a,
Dd-ALG-2aEF5, Dd-ALG-2aNter, Dd-ALG-2b, and Dd-ALG-2bNter)
were produced in E. coli with an MBP tag, a strategy that
allowed us to obtain soluble proteins with a high yield. Proteins
purified on amylose columns by affinity were always more than 98% pure
in one step, and they were used without any further purification (Fig.
2, lanes 1-5). For
immunization purposes, a polyhistidine (His6) tag was
preferred to the MBP tag. The protein was produced as inclusion bodies.
His6-ALG-2b was solubilized in 8 M urea,
purified on a nickel-nitrilotriacetic acid-agarose column (Fig. 2,
lane 6), and lyophilized before injection into rabbits.
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Fig. 2.
Purification of MBP-ALG-2a/b constructs.
Recombinant proteins used for interaction experiments and antibody
production were analyzed by SDS-PAGE. Lane 1, MBP-ALG-2a;
lane 2, MBP-ALG-2a EF5; lane 3, MBP-ALG-2a
Nter; lane 4, MBP-ALG-2b; lane 5, MBP-ALG-2b
EF5; lane 6, His6-ALG-2b.
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Ca2+ Induces Conformational Changes of MBP-ALG-2a and
-2b--
To detect conformational changes because of calcium binding
to Dd-ALG-2a and -2b, the intrinsic fluorescence spectra of both MBP-tagged proteins were recorded in the presence of either EGTA or
Ca2+. In both cases, maximal excitation and emission
occurred at 283 and 348 nm, respectively. The presence of calcium ions
resulted in a rapid increase in the fluorescence emission intensity of MBP-ALG-2a. This could be reversed by addition of an excess of EGTA
(Fig. 3A). Identical data (not
shown) were obtained with MBP-ALG-2b. These results reveal for both
proteins conformational changes sensed by proximal tryptophan(s). In a
control experiment, fluorescence intensity of MBP on its own was
unaffected by the addition of calcium ions (data not shown).
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Fig. 3.
Ca2+-induced conformational
changes and exposure of hydrophobic regions in Dd-ALG-2a and -2b
proteins. Kinetics of fluorescence variations using the above
recombinant proteins were recorded as described under "Experimental
Procedures." A, intrinsic fluorescence variations of
MBP-ALG-2a; B, TNS fluorescence variations of MBP-ALG-2a.
Additions of Ca2+ and EGTA are indicated by the
white or black arrowheads,
respectively. C, Intrinsic ( ,  ) and TNS fluorescence
( ,  ) variations were recorded as Ca2+ increments were
progressively added to the solution for Dd-ALG-2a (, ) and
Dd-ALG-2b (, ). Data normalized to maximal fluorescence
amplitude are plotted as a function of Ca2+
concentration.
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The lipophilic fluorescent probe, TNS, was used to evaluate the
exposure of ALG-2 hydrophobic regions upon Ca2+-induced
change of conformation (37). In the absence of calcium ions, the
fluorescence of TNS alone was very weak. Addition of MBP-ALG-2a led to
only a small increase. Addition of calcium ions resulted in a large
increase in TNS fluorescence and indicates that TNS is bound to newly
exposed hydrophobic region(s) of MBP-ALG-2a (Fig. 3B).
Addition of EGTA fully reversed the fluorescence induced by the calcium
ions. Similar results were obtained for MBP-ALG-2b (data not shown).
The affinity of MBP-ALG-2a and -2b for calcium was estimated from
intrinsic and TNS fluorescence experiments, where the variations of
fluorescence intensity were recorded as a function of calcium ion
concentration. Intrinsic and TNS fluorescence responses exhibited a
similar trend and gave an apparent KD for
Ca2+ in the 15-40 µM range for MBP-ALG-2a
and in the 300-600 µM range for MBP-ALG-2b (Fig.
3C). These data indicate that the observed conformational
changes and exposure of hydrophobic stretches are likely to be related
to binding of calcium ions at the same sites. The affinity of
MBP-ALG-2b for calcium was roughly 10-20 times lower than that of
Dd-ALG-2a. These values are noticeably higher than the affinity (6 µM) for Ca2+ reported for mouse ALG-2 (37).
Magnesium ions did not replace calcium ions in inducing conformational
changes. Adding 2 mM Mg2+ to the assay buffer
did not affect the titration curves. These observations indicate that
the binding sites are specific to calcium ions.
MBP-ALG-2a and -2b Associate in the Presence of Calcium
Ions--
The ability of Dd-ALG-2a and -2b to form dimers was studied
using surface plasmon resonance spectroscopy. The results (Fig. 4A) indicate that MBP-ALG-2a
in the analyte binds efficiently to immobilized MBP-ALG-2a in the
presence of Ca2+. This does not occur in the presence of
EGTA. Furthermore, the value of koff measured
during the dissociation phase increased by a factor of ~5 when the
calcium ions were chelated with EGTA. Sensorgrams recorded at different
calcium ion concentrations were used to determine the calcium ion
concentration for oligomerization of MBP-ALG-2a. A plateau in the
values of the resonance units at equilibrium
(Req) was observed for calcium ion
concentrations higher than 100 µM. Half-maximum binding
was obtained at a concentration of 13 ± 4 µM
(n = 7) (Fig. 4B). Such Ca2+
concentrations agree with those needed in MBP-ALG-2a (see above) to
induce a conformational change and to expose hydrophobic regions.
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Fig. 4.
Analysis by surface plasmon resonance
spectroscopy of Ca2+-dependent Dd-ALG-2a
interactions. A, MBP-ALG-2a was immobilized on a sensor
chip as described under "Experimental Procedures." 70 µl of
analyte (1.52 µM MBP-ALG-2a) in interaction buffer
containing 200 µM Ca2+ (trace
1) or 200 µM EGTA (trace
2) were injected and association recorded as a function of
time. The dissociation phase (trace 1) was then
recorded while flushing the sensor chip with association buffer without
added protein. The addition of 1 mM EGTA to the
Ca2+-containing buffer is indicated by a solid
arrowhead. B, interactions were determined with
running buffers containing free Ca2+ concentrations between
20 and 330 µM. Req values were
then determined as described under "Experimental Procedures" from
the association phases of the different sensorgrams and plotted as a
function of the Ca2+ concentration. Further sensorgrams
were recorded at a fixed Ca2+ concentration of 100 µM and various protein concentrations. C,
concentration dependence of ks values for
binding Dd-ALG-2a to immobilized MBP-ALG-2a () or MBP-ALG-2b
(). The slope of the curve gives the kon
value. D, Scatchard type plot analysis of the MBP-ALG-2a
data. Req/C values (bound/total) were
plotted as a function of Req (bound). The
reciprocal of the curve slope yields the dissociation constant
KD.
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The kinetic parameters of the MBP-ALG-2a dimerization were determined
by recording sensorgrams at different protein concentrations with a
fixed saturating Ca2+ concentration (100 µM).
The association phases from a representative series of experiments
performed with six MBP-ALG-2a concentrations (0.07-1.53
µM) were determined from a nonlinear least squares fit to
give a value for ks at each concentration. A
plot of ks as a function of MBP-ALG-2a
concentration gave the association rate, kon = 1840 M1·s1 and
koff = 1.9 × 103
s1 (Fig. 4C). Analysis of the dissociation
phases gave a value of koff = 2.2 ± 0.5 × 103 s1, n = 7. Thus, KD determined from the
(koff/kon) ratio was
1.0 ± 0.1 µM (n = 7). As the
binding of MBP-ALG-2a is close to equilibrium, the affinity of the
interaction could be determined from a
Req/C versus
Req plot. A representative plot gave a
KD value of 0.86 ± 0.05 µM
(n = 7). This is consistent with the
KD value derived from the kinetic analysis of the
data (Fig. 4D) described above.
In contrast to these results, similar homotypic experiments with
MBP-ALG-2b at Ca2+ concentrations (and up to 5 mM) for which Ca2+ binding to MBP-ALG-2b
occurred, showed no association between immobilized and soluble
proteins. Nevertheless, when immobilized on the sensor chip, MBP-ALG-2b
in the presence of Ca2+ was able to form heterodimers with
MBP-ALG-2a. From the plot of ks
versus MBP-ALG-2a concentration, an apparent equilibrium dissociation constant, KD, was calculated from the
ratio koff/kon, and was
2.0 µM for the MBP-ALG-2a/MBP-ALG-2b interaction (Fig.
4C). The dissociation constant derived from a
Req/C versus Req plot was 1.7 ± 0.2 µM
(n = 6) for that interaction (data not shown). These
values are similar to those found for the homotypic MBP-ALG-2a
interaction. Surprisingly, in a mirror experiment, MBP-ALG-2b in the
analyte was unable to interact with immobilized MBP-ALG-2a.
ALG-2 proteins deleted from EF-hand 5 that mediates dimerization in
mouse (41), or from their hydrophobic N terminus were used in
interaction experiments. MBP-ALG-2a on the sensor chip was unable to
interact with MBP-ALG-2a Nter. On the other hand, immobilized
MBP-ALG-2aEF5 bound MBP-ALG-2a in the analyte with an affinity
constant of 1.1 µM. A strict dependence on calcium ion
concentration was observed. MBP-ALG-2aEF5 could not homodimerize, nor associate with MBP-ALG-2b and MBP-ALG-2bEF5.
Dd-ALG-2a Interacts with Murine Alix in a
Ca2+-dependent Manner--
The primary
sequence data and homology modeling obtained for Dd-ALG-2a and -2b
clearly showed that they belong to the penta EF-hand family. The strong
homology with murine ALG-2 suggests that the functional interaction
could well be conserved in a cross-species assay. To examine whether
the amebal proteins represent genuine orthologues of mammalian ALG-2
proteins, we explored their capacity to bind mouse Alix, a protein
previously shown to directly interact with the ALG-2 protein (10, 12).
Mouse Alix was immobilized on the sensor chip, and MBP-ALG-2a or -2b
were added in the analyte fluid. MBP-ALG-2a was found to bind with
increasing concentration, and protein-protein interaction was dependent
on the calcium ion concentration (Fig.
5). From the plot of
ks versus C (Fig. 5, inset), we calculated that mouse Alix was able to bind
MBP-ALG-2a with an apparent affinity constant of 0.6 µM.
No interaction was detected when MBP-ALG-2a was immobilized on the
sensor chip, and with Alix present in the analyte. Mouse Alix bound
MBP-ALG-2aEF5 with an apparent affinity constant of 1.2 µM (Fig. 5, inset), but no interaction with
Alix was observed for MBP-ALG-2aNter. For MBP-ALG-2b, or its
truncated form MBP-ALG-2bEF5 in the analyte fluid, there was no
interaction with immobilized Alix. This was independent of the presence
or absence of calcium ions.
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Fig. 5.
Analysis by surface plasmon resonance
spectroscopy of the interactions between mouse Alix and Dd-ALG-2a.
Mouse Alix was immobilized on the sensor chip as described under
"Experimental Procedures." 70 µl of analyte were injected in the
running buffer containing various MBP-ALG-2a concentrations:
spectra 1-5, 0.38, 0.51, 0.76, 1.52, and 3.05 µM at a saturating Ca2+ concentration of 100 µM. Inset, ks values
determined from the association phases as detailed under
"Experimental Procedures" were plotted as a function of analyte
protein concentration for MBP-ALG-2a () or MBP-ALG-2aEF5
().
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Intracellular Localization of Dd-ALG-2a and -2b--
Protein
localization was determined by subcellular fractionation and
immunocytochemistry. Immunofluorescent staining of overexpressed Dd-ALG-2a was found to be associated with both the cytosol and membrane/cytoskeleton (Fig.
6A). Dd-ALG-2b showed a
similar localization together with some nuclear enrichment (Fig.
6A). After subcellular fractionation of a post-nuclear
extract on a Percoll gradient (Fig. 6B), Dd-ALG-2a/-2b were
mainly recovered in the soluble cytosolic fraction (tubes 9-12). It
was also found broadly distributed in deeper fractions (tubes 4-7),
which did not coincide with the endolysosomal compartment as determined
by the acid phosphatase distribution (Fig. 6B,
dashed line).
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Fig. 6.
Subcellular distribution of Dd-ALG-2a
and -2b proteins. Cells overexpressing c-Myc constructs
of Dd-ALG-2a or Dd-ALG-2b (panel A) under Act15 promoter
were incubated with a primary anti-c-Myc antibody and stained with a
fluorescein-labeled secondary antibody. Plasma membrane and nuclear
labeling are indicated by white and black
arrowheads, respectively. Parent KAx-3 cells were broken and
subcellular compartments separated on a 24% Percoll gradient
(panel B). Eluted fractions were assayed by Western blot for
the presence of Dd-ALG-2a/2b proteins and for lysosomal acid
phosphatase (dashed line).
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Expression of Dd-ALG-2a and -2b mRNAs and Proteins during
Differentiation--
The levels of Dd-ALG-2a and -2b mRNA
transcripts were developmentally regulated. The mRNAs were present
in vegetative and early developing cells (Fig.
7A), in agreement with the
transcriptional profile published by Van Driessche et al.
(42). To assess whether protein levels were similarly regulated during
the course of differentiation, Dd-ALG-2a and -2b were revealed on
Western blots using polyclonal antibodies raised against the
recombinant His6-ALG-2b protein. Three bands in the 20-kDa
region were detected in the parent strain (Fig. 7B), which
were absent in
alg-2a/alg-2b.
From the patterns obtained on Western blots of
Dd-alg-2a (where only Dd-ALG-2b was expressed)
and Dd-alg-2b (where only Dd-ALG-2a was
expressed), the upper band could be attributed to Dd-ALG-2a and the two
lower bands to Dd-ALG-2b. The size and relative intensity of these two
bands were unchanged no matter what protease inhibitor mixture was used
during sample preparation. Translation of the gene sequences gave
theoretical molecular masses of 22.3 and 23.2 kDa for Dd-ALG-2a and
-2b, respectively. The reasons why Dd-ALG-2b migrated slightly faster
than Dd-ALG-2a, despite a higher molecular mass, and exhibited two
protein bands are not clear, but are probably not the result of a
calcium-dependent migration artifact as for calmodulin or
grancalcin (43).
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Fig. 7.
Developmental expression of Dd-alg-2a and -2b
mRNAs and Dd-ALG-2a and -2b proteins. A, Northern
blot analyses of Dd-alg-2a and Dd-alg-2b
mRNAs were performed on 6 µg of total RNA for each condition of a
development time course (every 4 h over a 24-h period). Equivalent
loading on the gel and transfer to the membrane was controlled by
staining ribosomal RNAs with ethidium bromide (data not shown).
B, total protein samples were prepared from vegetative
amebae of various strains, separated by SDS-PAGE, blotted on an
Immobilon membrane, and revealed using a polyclonal antibody raised
against His6-ALG-2b. Lane 1, parent strain
KAx-3; lane 2, Dd-alg-2a;
lane 3, Dd-alg-2b; lane
4,
Dd-alg-2a/alg-2b.
C, protein samples from Dd-alg-2b
(upper panel) and
Dd-alg-2a (lower panel)
cells obtained every 4 h during development were analyzed by
Western blot. After immunodetection, the membranes were stained with
Coomassie Blue to assess equivalent protein loads along the time course
(data not shown).
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Antibodies raised against Dd-ALG-2b were used to follow the expression
of Dd-alg2a and -2b during development in the
single null-mutants (alg-2b and
alg-2a). The amount of both proteins remained
fairly constant throughout development (Fig. 7C), with only
a slight decrease after 12 h of development.
Development of Dd-alg-2 Simple and Double
Knock-outs--
Disruption of Dd-ALG-2 genes either
individually in Dd-alg-2a or
Dd-alg-2b mutants or in combination in
Dd-alg-2a/2b mutant
resulted in strains that grew normally in axenic medium or on bacterial
lawns. Although Dd-alg-2b-null mutants have a stalk base
thicker than normal (data not shown), there was no major
differentiation defect in the single knockouts or the double knockout
Dd-alg-2a/2b that
produced mature fructifications and spores with identical kinetics and
proportioning than the parent stain.
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DISCUSSION |
The experiments reported here were aimed at obtaining a better
understanding of the role of ALG-2 calciproteins and to establish whether the apoptotic function described in mammals had a functional counterpart in D. discoideum PCD. We have biochemically
characterized two ALG-2 isoforms from D. discoideum and
described their interaction both with calcium ions and the mouse
protein Alix. The latter is one of the physiological binding partners
of ALG-2 protein(s) in mammals.
Besides the calciproteins with 4 EF-hands identified in D. discoideum (21-27), we describe two new Ca2+-binding
proteins with 5 putative EF-hands, Dd-ALG-2a and -2b. During this work,
these proteins have been independently identified by others (28). On
the basis of their high sequence identity/homology, both proteins
clearly belong to the family that includes grancalcin, calpain, sorcin,
and peflin in other organisms (16). The structural homology of the
above amebal proteins with mouse ALG-2 is very striking, and both
molecules superimpose remarkably well on the mouse ALG-2 structure
(41).
Dd-ALG-2a and -2b exhibited the general biochemical properties shared
by all members of the family, i.e. Ca2+-induced
conformational changes and exposure of hydrophobic domains. Their
10-fold difference in affinity toward calcium ions is unlikely to be
related only to differences at the level of the EF-hands, because these
regions of the proteins are similar in sequence alignments and homology
modeling. The amino acid sequences of Dd-ALG-2a and -2b are noticeably
different in their N terminus, with the insertion of a 10-amino acid
hydrophobic sequence in Dd-ALG-2b. The N-terminal domain might be able
to regulate the affinity for Ca2+ through hydrophobic
interactions with the cleft in the molecule.
Homo- and heterotypic interactions of the two amebal ALG-2 isoforms
were measured by a surface plasmon resonance approach. In such
experiments, MBP-ALG-2a was able to interact with itself and with the
MBP-ALG-2b form, and the presence of calcium was necessary for the
interaction. Different aggregation states of the partners in solution
and on the sensor chip may account for the non-commutativity of the
interactions. Indeed, we know from gel filtration experiments that
MBP-ALG-2a exists in solution as a monomer-multimer mixture, whereas
MBP-ALG-2b exists mainly as a dimer (data not shown).
Truncated forms of Dd-ALG-2a/2b proteins in their N-terminal (amino
acids 1-27) and C-terminal (8 helix) extremities were generated to
determine the relative importance of these domains for dimerization.
MBP-ALG-2aEF5 is able to form a complex with MBP-ALG-2a, but no
homodimer was detected. This is a strong indication that one 7-8
helix pair is necessary and sufficient to stabilize an
MBP-ALG-2aEF5/MBP-ALG-2a dimer. No MBP-ALG-2a/MBP-ALG-2aNter dimers were observed, despite the presence of the two pairs of 7-8 helices. This indicates that the N terminus participates in
the dimer formation. We cannot exclude that shortening the distance
between the MBP-tag and EF-hand 1 in MBP-ALG-2aNter may grossly
affect the overall structure of the protein and hence its capacity to interact.
In surface plasmon resonance experiments, Dd-ALG-2a in solution is able
to bind to immobilized Alix from mouse and the interaction is strictly
Ca2+-dependent, two arguments that the amebal
protein is indeed a genuine functional homologue of mammalian ALG-2.
Dd-ALG-2b was unable to interact with Alix, whatever the calcium
concentration. Both Alix and Dd-ALG-2b are thus binding partners of
Dd-ALG-2a. In view of its longer hydrophobic N-terminal sequence,
Dd-ALG-2b could be closer to a peflin, which, in mammals, regulates the ALG-2/Alix interaction by competing for ALG-2 (14, 15). However, the
strictly calcium-dependent interaction between Dd-ALG-2a
and Dd-ALG-2b is not the mirror image of mammalian ALG-2/peflin
interaction that is observed even in the absence of Ca2+
(14). The moderate affinity observed for the
Ca2+-dependent complex between mouse Alix and
Dd-ALG-2a is not surprising, considering that we are using recombinant
proteins from heterologous sources. Furthermore, an additional level of
regulation is likely to occur in vivo, such as interaction
with partners, in particular the protein SETA (44, 45) or
phosphorylation (46).
Only a 2-fold reduction in the binding affinity between the
Dd-ALG-2aEF5 and Alix was noticed. On the other hand, deletion of
the N-terminal sequence of Dd-ALG-2a completely abolished its ability
to interact with Alix. This points again to the importance of the
N-terminal stretch of Dd-ALG-2a for interaction with partners. Whether
the hydrophobic N terminus is directly used for the interaction with
Alix or is necessary for the right structuring of ALG-2a to bind to
Alix will be examined with truncated proteins with their MBP tag removed.
Immunofluorescence and subcellular fractionation experiments indicate
that Dd-ALG-2a and -2b are essentially cytosolic, with some association
with the plasma membrane/cytoskeleton and with the nucleus. The latter
localization is more prominent for Dd-ALG-2b.
Western blot analysis indicate that the level of ALG-2a/2b proteins is
stable during development. Ohkouchi et al. (28) established by in situ hybridization that Dd-ALG2a was
enriched in the prespore region and that Dd-ALG-2b was enriched in the
prestalk region. The reported analysis of the cell type-specific
transcriptional profile provided an opposite trend. Dd-ALG-2a mRNA,
followed by its representative clone SSG263, was maximal in stalk
cells, whereas Dd-ALG-2b mRNA, represented by clone SSB886, was
maximal in prespores (42).
Simple and double knock-outs of Dd-alg-2a/2b produced no
obvious developmental phenotype, thus excluding an indispensable role
for Dd-ALG-2a/2b in the calcium response during differentiation, as
also reported for other calciproteins in D. discoideum
(21-27). Nevertheless, an alix-null mutant has a strong
defect in development2 and Dd-ALG-2a, by binding Alix, may
participate in the overall regulation of that pathway.
,
TOP
ABSTRACT
INTRODUCTION
-D-thiogalactopyranoside;
TNS, 2-(p-toluidino)naphthalene-6-sulfonate;
ALG-2, apoptosis-linked gene 2;
Alix, apoptosis-linked gene 2-interacting
protein X;
MBP, maltose-binding protein;
MBP-ALG-2a, maltose-binding
protein-tagged Dd-ALG-2a;
MBP-ALG-2b, maltose-binding
protein-tagged Dd-ALG-2b;
His6-ALG-2b, polyhistidine-tagged
Dd-ALG-2b;
PBS, phosphate-buffered saline;
DIG, digoxigenin;
MES, 4-morpholineethanesulfonic acid.
