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Keywords:

  • calmodulin binding protein;
  • cell adhesion molecule;
  • contractile vacuoles;
  • Dictyostelium discoideum ;
  • unconventional protein transport

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The Ca2+-dependent cell–cell adhesion molecule DdCAD-1, encoded by the cadA gene of Dictyostelium discoideum, is synthesized at the onset of development as a soluble protein and then transported to the plasma membrane by contractile vacuoles. Calmodulin associates with contractile vacuoles in a Ca2+-dependent manner, and co-localizes with DdCAD-1 on the surface of contractile vacuoles. Bioinformatics analysis revealed multiple calmodulin-binding motifs in DdCAD-1. Co-immunoprecipitation and pull-down studies showed that only Ca2+-bound calmodulin was able to bind DdCAD-1. Structural integrity of DdCAD-1, but not the native conformation, was required for its interaction with calmodulin. To investigate the role of calmodulin in the import of DdCAD-1 into contractile vacuoles, an in vitro import assay consisting of contractile vacuoles derived from cadA cells and recombinant proteins was employed. Prior stripping of the bound calmodulin from contractile vacuoles by EGTA impaired import of DdCAD-1, which was restored by addition of exogenous calmodulin. The calmodulin antagonists W-7 and compound 48/80 blocked the binding of calmodulin onto stripped contractile vacuoles, and inhibited the import of DdCAD-1. Together, the data show that calmodulin forms a complex with DdCAD-1 and promotes the docking and import of DdCAD-1 into contractile vacuoles.


Abbreviations
CaM

calmodulin

DdCAD-1

Dictyostelium discoideum Ca2+-dependent cell adhesion molecule-1

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In eukaryotic cells, an increasing number of soluble cytoplasmic proteins are known to be secreted by non-classical transport mechanisms [1, 2]. Interleukin 1β [3] and galectin-1 [4] were the first proteins shown to be secreted by unconventional secretory pathways. Other secreted soluble proteins include cytokines and growth factors, and molecules with important signalling roles in inflammation, angiogenesis, cell differentiation and proliferation [2]. Although most of these proteins have well-established intracellular and extracellular roles, molecular details of their export mechanisms remain poorly understood.

Recent studies have shown that cells utilize several strategies to target soluble proteins for secretion, including plasma membrane transporters, the endosomal pathway, formation of exosomes, flip-flop mechanisms and contractile vacuoles [2, 5]. The Ca2+-dependent cell adhesion molecule DdCAD-1, which is encoded by the cadA gene in the social amoeba Dictyostelium discoideum, is synthesized at the onset of development as a soluble protein [6-8], and then transported to the plasma membrane by contractile vacuoles [9, 10]. Contractile vacuoles are the osmoregulatory organelles of free-living amoebae and protozoa, controlling their intracellular water balance by accumulating and expelling excess water from the cell, thus allowing cells to survive under hypotonic stress [11]. DdCAD-1 is made up of two Ig-like domains connected by a short linker sequence [6]. Mutational analysis has shown that import of DdCAD-1 depends on the integrity of the protein [10]. Genetic analysis and biochemical studies indicate that the unconventional DdCAD-1 transport process involves at least four distinct steps: (a) binding of DdCAD-1 to contractile vacuoles, (b) translocation of DdCAD-1 into the lumen of contractile vacuoles, (c) association of DdCAD-1 with an ‘anchoring’ protein on the luminal side of the vacuole, and (d) diffusion of DdCAD-1 from the vacuole membrane to the cell surface upon fusion of the contractile vacuole with the plasma membrane [5, 9, 10].

The ubiquitous Ca2+ regulator, calmodulin (CaM), is present on the surface of contractile vacuole in high abundance [9, 12, 13]. CaM is a highly conserved Ca2+-binding protein that has been found in all eukaryotic cells examined so far [14], and CaM from eukaryotic micro-organisms is functionally interchangeable with CaM from mammals [15]. Many proteins bind to CaM via their CaM-binding motifs, which are not conserved sequences but typically consist of hydrophobic residues located in specific positions relative to other amino acids [16]. Bioinformatics analysis shows that DdCAD-1 contains several putative CaM-binding motifs, suggesting that CaM may interact with DdCAD-1. Several reports have demonstrated that CaM is present on endosomal and lysosomal membranes [17, 18] and plays a role in the endosome-mediated transport system [19]. It is therefore of interest to determine whether CaM plays a role in the unconventional pathway involved in the transport of DdCAD-1.

In this paper, we demonstrate that DdCAD-1 is a CaM-binding protein and that their interaction is functionally important for import of DdCAD-1 into contractile vacuoles. CaM binds DdCAD-1 in a Ca2+-dependent manner to form a complex that promotes docking of DdCAD-1 on to the vacuole membrane, and CaM associated with contractile vacuoles facilitates translocation of DdCAD-1 into the vacuole lumen.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In vitro interactions between CaM and DdCAD-1

DdCAD-1 is transported from the cytoplasm to the cell surface by contractile vacuoles [5, 9, 10]. CaM is associated with the cytoplasmic surface of the contractile vacuole and serves as a marker of the contractile vacuole network [13]. Double immunofluorescence staining showed that DdCAD-1 co-localized with CaM on the surface of contractile vacuoles (Fig. S1). Their close proximity suggests that CaM may participate in the import of DdCAD-1 into contractile vacuoles. To test this hypothesis, bioinformatics analysis was performed, and the results showed that DdCAD-1 contains five 1-5-10, five 1-8-14 and two 1-5-8-14 Ca2+-dependent putative CaM-binding motifs [16] (Fig. S2). Three of these motifs are found in the N-terminal domain, two in the C-terminal domain, and seven spanning the inter-domain region of DdCAD-1. These observations suggest potential interactions between DdCAD-1 and CaM.

To investigate whether CaM and DdCAD-1 undergo interactions and play a role in DdCAD-1 transport, DdCAD-1 and CaM recombinant proteins were produced in Escherichia coli (Fig. 1A). The Ca2+-bound form of CaM migrated faster in SDS gel than the Ca2+-free form, similar to previous reports [20]. Both recombinant CaM and DdCAD-1 showed excellent immunoreactivity with their respective antibodies. When various amounts of DdCAD-1 were immobilized on nitrocellulose membrane for far-western blot analysis, dose-dependent binding of CaM to DdCAD-1 was observed (Fig. 1B). To determine which of the two Ig-like domains of DdCAD-1 is involved in CaM binding, recombinant N- and C-terminal domains of DdCAD-1 were prepared and subjected to far-western blot analysis. CaM failed to bind to either of them (Fig. 1B), suggesting that structural integrity of DdCAD-1 is required for its interaction with CaM.

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Figure 1. Binding interactions between recombinant DdCAD-1 and CaM. (A) Gel profiles of purified recombinant proteins. (a) Schematic drawings showing the His6-tagged DdCAD-1 and His6-tagged CaM fusion proteins expressed in E. coli. (b) Coomassie Blue staining of recombinant DdCAD-1 and Ca2+-bound and Ca2+-free CaM separated by 12% SDS/PAGE. (c) Protein blots were probed with rabbit antibodies raised against DdCAD-1 or monoclonal antibody directed against CaM. (B) Far-western blots containing various amounts of (a) His6-tagged DdCAD-1, the His6-tagged N-terminal domain of DdCAD-1 (b) and the His6-tagged C-terminal domain of DdCAD-1 (c). Proteins blots were incubated with 20 μg·mL−1 His6-tagged CaM in 50 mm MES buffer, 1 mm CaCl2 for 1 h at 22 °C. After washing, the blots were probed with anti-CaM monoclonal antibody. Western blots showing the recombinant DdCAD-1, N- and C-terminal domains of DdCAD-1 (a′, b′ and c′) immobilized on the nitrocellulose membrane are shown on the right. (C) Binding of CaM to wild-type and mutant DdCAD-1. Various amounts of (a) His6-tagged DdCAD-1 and (b) the His6-tagged penta mutant DdCAD-1 were separated on polyacrylamide gel and transferred to a nitrocellulose membrane. The amounts of protein used are shown at the top of the panel. The protein blots were incubated with 20 μg·mL−1 His6-tagged CaM for 1 h at room temperature, followed by incubation with anti-CaM monoclonal antibody. (D) Binding of DdCAD-1 to CaM depends on CaM conformation. Ca2+-bound and Ca2+-free His6-tagged CaM was immobilized on nitrocellulose membrane after SDS/PAGE and subjected to far-western blot analysis. The membrane was incubated with 20 μg·mL−1 His6-tagged DdCAD-1, and then probed with rabbit antibodies against DdCAD-1 (a). Binding of DdCAD-1 was not observed. In order to determine the presence of His6-tagged CaM on the membrane, the blots were probed with anti-CaM monoclonal antibody (b).

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In order to test whether the conformation of DdCAD-1 plays a role in its interaction with CaM, we performed binding assays using a His6-tagged mutant DdCAD-1 protein that contained five amino acid substitutions (N58A, E59A, D61A, D201A and N202A) [10]. Circular dichroism spectra revealed significant conformational changes in the His6-tagged penta mutant form of DdCAD-1 [10]. Far-western blot analysis showed that CaM was capable of binding to the mutant DdCAD-1, suggesting that the proper conformation of DdCAD-1 is not required for its interaction with CaM (Fig. 1C). In contrast, when blots of denatured CaM were probed with soluble His6-tagged DdCAD-1, binding of DdCAD-1 to either Ca2+-bound or Ca2+-free CaM was not observed (Fig. 1D). These results indicate that the proper three-dimensional conformation of CaM is required for its interaction with DdCAD-1.

CaM interacts with DdCAD-1 in a Ca2+-dependent manner

To examine the interactions between CaM and DdCAD-1 in their native form, pull-down experiments were performed using CaM-conjugated agarose. Wild-type AX4 cells and cadA transfectants expressing DdCAD-1–GFP [10] were solubilized using 1% Nonidet P-40 (Sigma, St Louis, MO, USA), and proteins were incubated with CaM affinity beads in the presence or absence of EGTA. Both endogenous DdCAD-1 and ectopically expressed DdCAD-1–GFP were pulled down successfully by the CaM-conjugated agarose beads (Fig. 2A,B). In contrast, neither were capable of binding CaM in the presence of EGTA, indicating that their interaction is Ca2+-dependent. Consistent with the far-western blot results, both the N- and C-terminal domains of DdCAD-1 failed to interact with CaM-conjugated beads in pull-down experiments (Fig. 2B).

image

Figure 2. Involvement of Ca2+ in the interaction between CaM and DdCAD-1. (A–C) CaM interacts with DdCAD-1 in a Ca2+-dependent manner. A pull-down experiment was performed using His6-tagged CaM-conjugated beads in the presence of absence of 5 mm EGTA. (A) DdCAD-1 present in wild-type AX4 cytosol (5 mg total protein) was pulled down by CaM beads in the absence of EGTA. (B) DdCAD-1–GFP in the cytosol of cadA::gfp transfected cadA cells (5 mg total protein) was pulled down by CaM in the absence of EGTA. (C) Direct interaction between immobilized recombinant His6-tagged S(I+II) mutant DdCAD-1 and soluble CaM. Far-western blots were performed in the presence of 50 mm MES buffer, 1 mm CaCl2 and 20 μg·mL−1 His6-tagged CaM. The blots were stained with anti-CaM monoclonal antibody. The blot was stripped and then re-probed with rabbit antibodies against DdCAD-1. (D) A pull-down experiment was performed by incubating His6-tagged CaM-conjugated beads with cytosol derived from cadA cells expressing His6-tagged calcium-binding site mutant (SI+II) DdCAD-1-GFP.

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Both CaM and DdCAD-1 are Ca2+-binding proteins. CaM consists of four Ca2+-binding sites, and CaM undergoes major conformational changes in the presence of Ca2+ [21], while DdCAD-1 contains three Ca2+-binding pockets [6]. In the N-terminal domain of DdCAD-1, site I (SI) involves the residues Asp39, Thr81, Asn84 and Phe41, and site II (SII) consists of Asp35, Asn38, Glu56 and Ser87. Site III (SIII) comprises Glu59 and Asp61 from the N-terminal domain and Thr179, Gln181 and Asn202 from the C-terminal domain. In order to test whether DdCAD-1 requires Ca2+ for its interaction with CaM, binding assays were performed using the DdCAD-1 Ca2+-binding site mutant S(I+II), which contains D35A, N38A and D39A substitutions to inactivate sites I and II [6, 10]. The results of binding studies show that the soluble CaM is capable of binding to the S(I+II) mutant of DdCAD-1 immobilized on nitrocellulose filters (Fig. 2C). These results were confirmed by pull-down assays using cadA transfectants expressing GFP-tagged S(I+II) DdCAD-1 mutant protein, indicating that Ca2+ binding is not required for DdCAD-1 to interact with CaM.

Formation of CaM–DdCAD-1 complexes in solution

To determine whether DdCAD-1 and CaM binding occurs in solution, a mixture of recombinant DdCAD-1 and CaM was added to cadA-null cells that had been developed for 3 h. Binding of DdCAD-1 to its anchoring protein on the cell surface brings together CaM molecules associated with it. To visualize CaM–DdCAD-1 complexes bound to the cell surface, cells were treated with antibodies against DdCAD-1 to induce clustering of DdCAD-1 bound to the cell surface, eventually leading to ‘cap’ formation [6]. To examine the presence of CaM in these caps, cells were fixed without permeabilization and then stained with antibodies against CaM. DdCAD-1 and CaM co-localized in the cap structures on the surface of cadA cells (Fig. 3A), indicating the presence of CaM–DdCAD-1 complexes on the cell membrane. As a negative control, only CaM was added to cells. Cap formation was not observed, although a small amount of CaM was bound to the cell membrane (Fig. 3B). Next, co-immunoprecipitation experiments were performed to detect cellular complexes of DdCAD-1 and CaM. Immunoblot results showed that the Ca2+-bound form of CaM co-precipitated with DdCAD-1 in the presence of DdCAD-1 polyclonal antibodies (Fig. 3C). These results confirmed that CaM and DdCAD-1 are capable of forming complexes inside the cell.

image

Figure 3. Co-capping and co-immunoprecipitation of DdCAD-1 and CaM. (A) Co-capping of DdCAD-1 with the CaM on the surface of cells. A mixture of DdCAD-1 and CaM (40 μg each) was added to cadA cells at 3 h of development. Samples were incubated at 22 °C for 30 min. Then anti-DdCAD-1 antibodies were added to induce DdCAD-1 clustering on the cell surface, followed by incubation with Alexa Fluor 568-conjugated goat anti-rabbit antibody (red) to induce ‘cap’ formation. After transferring the cells to a cover slip, they were fixed and stained with anti-CaM monoclonal antibody, followed by Alexa Fluor 488-conjugated goat anti-mouse antibody (green) to visualize the cap. Scale bar = 5 μm. (B) As a negative control, a similar capping experiment was performed in the presence of recombinant CaM alone. Scale bar = 5 μm. (C) Co-immunoprecipitation of DdCAD-1 and CaM was performed by incubating AX4 cell lysates (5 mg total protein) with anti-DdCAD-1 antibody-conjugated protein A beads for 18 h at 4 °C (a,c). As a control, immune precipitation was performed using non-immune rabbit IgG (b,d). Immune precipitates were subjected to SDS/PAGE, followed by western blot analysis using either anti-CaM monoclonal antibody (a,b) or rabbit antibodies against DdCAD-1 (c,d). In (c), DdCAD-1 is indicated by an arrowhead, and the open circle indicates the small chain of IgG in the immune precipitates.

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CaM promotes DdCAD-1 import into contractile vacuole

To investigate the potential role of CaM in the selective import of DdCAD-1 into contractile vacuoles, we first examined the binding of CaM to contractile vacuoles. Contractile vacuoles isolated from cadA cells were incubated with EGTA to release CaM bound to the vacuole surface. Recombinant CaM was then added to the stripped vacuoles. The binding of CaM to contractile vacuoles was much augmented by Ca2+, but inhibited by EGTA (Fig. 4A), suggesting that the interaction between CaM and the contractile vacuole is a Ca2+-dependent process.

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Figure 4. CaM promotes the import of DdCAD-1 into contractile vacuoles. (A) CaM binds to contractile vacuoles in a Ca2+-dependent manner. Purified contractile vacuoles were treated with EGTA to release the bound CaM and then incubated with His6-tagged CaM (2 μm) in TM buffer in the presence of various concentrations of Ca2+ or EGTA. Contractile vacuoles were washed twice, and samples were subjected to SDS/PAGE. Protein blots were probed using anti-CaM monoclonal antibody. (B) Effects of His6-tagged CaM on DdCAD-1 import. Contractile vacuoles were washed with 5 mm EGTA to remove the endogenous CaM bound to the vacuoles. (a) An import assay was performed in the presence of 2 μm His6-tagged DdCAD-1 and various concentrations of recombinant CaM. The imported His6-tagged DdCAD-1 was revealed after treating contractile vacuoles with proteinase K to remove proteins bound to the vacuole surface. As a control, a sample was disrupted with SDS to allow access of proteinase K to the luminal proteins. The samples were subjected to SDS/PAGE, and protein blots were probed with anti-DdCAD-1 antibody. (b) The intensity of gel bands shown in (a) was quantified using NIH imagej software and plotted against the various concentrations of CaM.

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The effects of recombinant CaM on DdCAD-1 import were assessed in the cell-free in vitro import assay [10]. Contractile vacuoles derived from cadA cells were washed with EGTA to remove bound CaM. Import assays were performed in the presence of His6-tagged DdCAD-1 and increasing concentrations of His6-tagged CaM. At the end of the incubation period, contractile vacuoles were washed and then subjected to proteinase K digestion. Analysis of samples prior to protease digestion showed the total amount of DdCAD-1 docked on the vacuole membrane and DdCAD-1 imported into the lumen, while the protease-treated samples represent the level of DdCAD-1 inside the lumen only. Western blot analysis showed that import of DdCAD-1 was much reduced in the absence of CaM. Addition of exogenous CaM promoted the import of DdCAD-1 in a dose-dependent manner, and the relative level of DdCAD-1 import was restored to the level of control (non-stripped) contractile vacuoles when using 2 μm His6-tagged CaM (Fig. 4B). An approximate threefold increase in DdCAD-1 import was observed for 10 μm CaM. When contractile vacuoles were treated with detergent to permeabilize the membrane to allow access of proteinase K to the imported DdCAD-1, the DdCAD-1 that was previously protected from protease attack in the vacuolar lumen was digested completely.

CaM promotes docking of DdCAD-1 on to the contractile vacuole membrane

The pharmacological reagents W-7 and compound 48/80 are potent inhibitors of CaM function [22-24]. They were used in in vitro reconstitution assays to examine the role of CaM in the import of DdCAD-1. Import assays were performed by incubating contractile vacuoles with His6-tagged DdCAD-1 and the antagonists W-7 and compound 48/80. Neither W-7 nor compound 48/80 had detectable effects on DdCAD-1 import up to a concentration of 250 μm (Fig. 5A). These results were unexpected because earlier experiments (shown in Fig. 4B) had shown that addition of CaM to EGTA-stripped contractile vacuoles led to a threefold increase in DdCAD-1 import (Fig. 4B). Taken together, the data suggested that the antagonists may inhibit the function of CaM prior to its association with the contractile vacuoles. To test whether the antagonists affected the binding of CaM to contractile vacuoles, CaM was pre-incubated with antagonists before adding to the EGTA-treated contractile vacuoles. In both cases, association of CaM with contractile vacuoles was inhibited (Fig. 5B, a,b). However, when CaM was incubated with EGTA-treated contractile vacuoles to allow binding prior to addition of W-7 or compound 48/80, neither reagent had any significant effect on the bound CaM (Fig. 5B, c,d).

image

Figure 5. CaM facilitates docking and translocation of DdCAD-1 in the import process. (A) Lack of effects of CaM antagonists W-7 (a) and compound 48/80 (b) on DdCAD-1 import into intact contractile vacuoles. Import assays were performed by incubating the contractile vacuoles with His6-tagged DdCAD-1 and a CaM antagonist without prior removal of CaM associated with the vacuole surface. Samples were subjected to SDS/PAGE, and protein blots were probed with anti-DdCAD-1 antibody. (B) Effects of W-7 and compound 48/80 on the binding of exogenous CaM to contractile vacuoles. Contractile vacuoles were first stripped of CaM by incubation with 5 mm EGTA. His6-tagged CaM (2 μm) and CaCl2 (1 mm) were mixed with W-7 (a) or compound 48/80 (b) for 30 min at 4 °C before adding to the CaM-free contractile vacuoles. Samples were incubated for 1 h at 4 °C, and washed twice with TM buffer to remove any unbound His6-tagged CaM. In separate experiments, His6-tagged CaM was added to EGTA-treated contractile vacuoles, incubated for 1 h at 4 °C, and washed to remove unbound material before addition of W-7 (c) or compound 48/80 (d). Samples were incubated for another 1 h at 4 °C and then washed twice with TM buffer. Both sets of samples were subjected to SDS/PAGE, and protein blots were probed with anti-CaM monoclonal antibody. (C) Inhibition of DdCAD-1 import into stripped contractile vacuoles by CaM antagonists. EGTA-treated contractile vacuoles were pre-incubated with His6-tagged CaM (2 μm) and either W-7 (a) or compound 48/80 (b) for 60 min before addition of recombinant DdCAD-1 in import assays. Samples were washed twice with TM buffer and subjected to SDS/PAGE, and protein blots were probed with anti-DdCAD-1 antibody.

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As CaM is capable of forming a complex with DdCAD-1 in solution (Fig. 3), it is possible that CaM facilitates docking of DdCAD-1 on contractile vacuoles. If this is the case, the antagonists may inhibit docking of the CaM–DdCAD-1 complex and consequently block DdCAD-1 import. To test this hypothesis, EGTA-treated contractile vacuoles were incubated with a mixture of His6-tagged CaM and antagonists for 60 min before addition of recombinant DdCAD-1 in import assays. DdCAD-1 docking and import into contractile vacuoles were impaired by both W-7 and compound 48/80 in a dose-dependent manner. Import of DdCAD-1 was inhibited more severely than DdCAD-1 docking and reduced to essentially background level when 250 μm of either W-7 or compound 48/80 was used (Fig. 5C).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The cell-adhesion protein DdCAD-1 is imported into contractile vacuoles through either V-H+-ATPases or invagination of the vacuolar membrane in a Ca2+- and conformation-dependent manner, and then transported to the plasma membrane [9, 10]. In this paper, we evaluated the role of CaM in membrane targeting of DdCAD-1 via this unconventional protein transport pathway. CaM is present in high abundance in Dictyostelium cells. Binding of Ca2+-bound CaM to DdCAD-1 occurs in the cytoplasm, and CaM promotes docking and import of DdCAD-1 into contractile vacuoles as shown in steps 1 and 2 of the DdCAD-1 transport pathway in Fig. 6.

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Figure 6. Schematic diagram showing the unconventional transport pathway of cytoplasmic DdCAD-1 to the plasma membrane by contractile vacuoles. The transport of DdCAD-1 involves at least five distinct steps: (1) formation of a complex of DdCAD-1 with Ca2+-bound CaM, (2) docking of the CaM–DdCAD-1 complex on the contractile vacuole, (3) translocation of DdCAD-1 into the lumen via either membrane invagination or V-H+-ATPases, (4) binding of DdCAD-1 to the anchoring protein on the luminal side of the contractile membrane, and (5) fusion of the contractile vacuole to the plasma membrane, where unbound DdCAD-1 is released into the medium and the bound DdCAD-1 diffuses laterally to become part of the plasma membrane.

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Calmodulin is a highly conserved multi-functional protein that is present in many subcellular locations. In Dictyostelium cells, approximately one-third of the cellular CaM is associated with the surface of the contractile vacuole network [12]. The binding of CaM to vacuolar membranes, in Dictyostelium as well as in yeast, is dependent on Ca2+ [22, 25]. The co-localization of DdCAD-1 with CaM on the surface of contractile vacuoles [9, 10] suggests that they may interact inside the cell. The results of the co-capping and co-immunoprecipitation experiments confirm that DdCAD-1 and CaM are capable of forming complexes, but do not rule out participation of other cellular components. Direct interaction between DdCAD-1 and CaM was demonstrated by far-western blot assays using purified recombinant proteins. However, it remains to be determined whether the in vitro interaction observed in these assays is the same as the in vivo interaction.

Although both the Ca2+-bound and Ca2+-free forms of CaM are known to bind cellular proteins [15, 21, 23], the interaction between DdCAD-1 and CaM is dependent on the native state of CaM and the availability of Ca2+. Intracellular DdCAD-1 may exist in the Ca2+-free form as its Ca2+-binding affinity is relatively low [6]. However, contractile vacuoles are highly efficient Ca2+ stores [26], and the release of Ca2+ from contractile vacuoles may serve as an attractive cue to both DdCAD-1 and CaM.

CaM acts as a Ca2+ sensor in the regulation of many cellular processes through its interaction with a variety of cellular proteins [22, 27]. Structural analysis has defined several CaM-binding motifs, which are mostly harbored within a short stretch of amino acids [16, 28, 29]. Bioinformatic analysis of the primary sequence of DdCAD-1 revealed the presence of multiple Ca2+-dependent CaM-binding motifs. Of particular interest are the overlapping motifs in the hinge region between the N- and C-terminal domains (Fig. S2). It is likely that the hinge region is involved in CaM binding as studies of deletion mutants of DdCAD-1 indicate that both the N- and C-terminal domains of DdCAD-1 are required for binding to CaM. NMR structural analysis shows that Ca2+ binding has little influence on the conformation of the hinge region of DdCAD-1 [6], consistent with the observation that the Ca2+-binding site mutant S(I+II) shows negligible effects on its interaction with CaM.

In vitro assays using a reconstituted import system have shown that exogenous CaM facilitates docking of cytoplasmic DdCAD-1 onto the contractile vacuole membrane, as well as the import of DdCAD-1 into contractile vacuoles in a dose-dependent manner. Both docking and import of DdCAD-1 are compromised in the absence of exogenous CaM. These results suggest that CaM may act as a carrier for DdCAD-1, and that the complex docks on the contractile vacuole membrane through interactions between CaM and a high-affinity CaM-binding component on the vacuolar membrane, which has yet to be identified. As W-7 and compound 48/80 block binding of CaM to contractile vacuoles, these antagonists may compete for the site(s) involved in CaM docking. Solution structures of Ca2+-bound CaM complexed with W-7 have revealed that one molecule of W-7 binds to each of the two domains of CaM and completely shields the key hydrophobic sites [24], which are crucial for binding of CaM target proteins such as myosin light-chain kinases [28] and CaM kinase IIa [29]. CaM has been shown to associate with unconventional myosin that is present in great abundance on the contractile vacuole network of Dictyostelium [12]. Therefore, W-7 may prevent binding of CaM to unconventional myosin on contractile vacuoles by shielding the key hydrophobic sites on CaM. Hydrophobic interactions are strong interactions, which may account for the inability of W-7 and compound 48/80 to displace CaM already bound to the contractile vacuole membrane and the lack of effect of the antagonists on DdCAD-1 import when non-stripped contractile vacuoles were used.

Both W-7 and compound 48/80 inhibited the import of DdCAD-1 more severely than docking of the CaM–DdCAD-1 complex. Exactly how CaM participates in the import of DdCAD-1 into contractile vacuoles is not known. We have shown previously that DdCAD-1 may be imported into the contractile vacuole through invagination of the vacuolar membrane (Fig. 6) [10]. In Saccharomyces cerevisiae, micro-autophagic uptake of soluble cytoplasmic proteins occurs via an autophagic tube, which is the result of a highly specialized invagination of the vacuolar membrane [30]. Interestingly, CaM is required for micro-autophagy, and invagination of the micro-autophagic vacuolar membrane is sensitive to CaM antagonists [23]. Moreover, CaM is involved in Ca2+-dependent protein translocation in the inner envelope of chloroplasts [27] and in post-docking events during late endosome–lysosome fusion in yeast [22].

DdCAD-1 is a Ca2+-binding protein that consists of three Ca2+-binding pockets [6, 8, 31]. However, Ca2+ binding is not required for DdCAD-1 in its interaction with CaM because the DdCAD-1 mutant S(I+II), which has lost its ability to bind Ca2+, is still capable of binding CaM. On the other hand, DdCAD-1 import into contractile vacuoles requires Ca2+ [10]. Therefore, subtle conformational changes induced by binding of Ca2+ to DdCAD-1, while bound to CaM, may somehow facilitate the import of DdCAD-1 into contractile vacuoles through membrane invagination (Fig. 6) [10]. DdCAD-1 is released from the internalized vesicles as they burst in the hypotonic environment of the lumen [10]. In addition, translocation of DdCAD-1 may involve a membrane transporter. CaM has been shown to interact with components of V-ATPases in yeast [25]. Contractile vacuoles in Dictyostelium are enriched with the proton pump V-H+-ATPases [32]. It is therefore possible that the DdCAD-1–CaM complex interacts with V-H+-ATPases, followed by translocation of DdCAD-1 into the lumen. Inside the lumen of the contractile vacuole, DdCAD-1 may associate with an anchoring protein in the vacuolar membrane. Upon fusion of the contractile vacuole with the plasma membrane, the bound DdCAD-1 diffuses laterally to become part of the plasma membrane, while the unbound protein is secreted into the medium [5, 9].

Multiple unconventional protein transport pathways have been proposed for the targeting of soluble proteins for surface expression and secretion [1-4]. DdCAD-1 is transported by contractile vacuoles. However, acyl CoA-binding protein, which is required for the terminal differentiation of pre-spore cells, is externalized through an unconventional protein secretion pathway that involves the Golgi reassembly stacking protein GRASP [33]. A combination of genetic and biochemical approaches in future studies is required to obtain a better understanding of these and other unconventional protein transport pathways in cells.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Construction and expression of His6-tagged wild-type and mutant DdCAD-1 proteins and His6-tagged CaM

His6-tagged wild-type DdCAD-1 (His6-WT) and His6-tagged DdCAD-1 N- and C-terminal domains (His6-N and His6-C) were expressed and purified as described previously [6]. The His6-tagged Ca2+-binding site mutant S(I+II) (D35A, N38A, D39A) and the His6-tagged penta mutant (N58A, E59A, D61A, D201A and N202A) were created using site-directed mutagenesis to substitute three or five residues with alanine as described previously [10]. The gene encoding D. discoideum CaM was sub-cloned into the pET-M vector [31] for expression. The primers used to create the CaM expression vector were 5′-CGCGGATCCCTGGTGCCACGCGGCGTTGCATCACAAGAAAGTTTAACTGAAGAACAAATTGCTG-3′ and 5′-CCCAAGCTTTTAATTTCTAACAATCATCATTTTAACAAATTCGTCATAGTTAACTTGACCATCGCC-3′.

All constructs were sequenced to confirm sequence fidelity. Protein expression was performed in E. coli strain BL21(DE3) and His-tagged proteins were purified using Ni-NTA resin (Qiagen, Valencia, CA, USA).

Isolation of contractile vacuoles and cytosol

Contractile vacuoles were isolated as described previously [9] with minor modifications [10]. Cells (2 × 109) were developed for 6 h in suspension, and then homogenized in 12 mL of TM buffer (10 mm Tris/HCl, pH 7.5, 2 mm MgCl2) at room temperature. Samples (5 mL each) were layered on top of discontinuous (28% and 48% w/w, 4 mL each) sucrose density gradients, and then centrifuged for 1 h at 100 000 g at 4 °C. Contractile vacuoles enriched at the interface were collected, and aliquots were stored at −70 °C. Cytosol derived from cadA-null cells was obtained after centrifugation of the cell lysate at 100 000 g, and aliquots were stored at −70 °C. Protein concentration was determined using the bicinchoninic acid assay kit (Pierce Chemical Co., Rockford, IL, USA). Proteolytic digestion of contractile vacuoles was performed by incubating samples (containing approximately 100 μg protein) with 0.01 mg·mL−1 proteinase K in the presence or absence of 0.05% SDS at 37 °C for 1 h. After addition of 2 mm phenylmethanesulfonyl fluoride, samples were boiled for 10 min and subjected to SDS/PAGE and western blot analysis.

In vitro reconstitution of DdCAD-1 import into contractile vacuoles

Contractile vacuoles and cytosol fractions derived from cadA-null cells were mixed at a 1 : 1 ratio in a reaction sample (total 600 μL) to give a final concentration of 2 mg·mL−1. His6-tagged DdCAD-1 and the various mutant forms of DdCAD-1 (2 μm) were added to the reaction mixture and incubated for 1 h at room temperature. The contractile vacuoles were pelleted by centrifugation at 20 000 g for 10 min at 4 °C, and washed twice with TM buffer. Samples were subjected to SDS/PAGE and western blot analysis. The amount of recombinant protein associated with the contractile vacuoles included protein bound to the cell surface (docking) and protein inside the vacuole lumen (import). To estimate the amount of protein imported into contractile vacuoles, samples were subjected to digestion with proteinase K (10 μg·mL−1) for 1 h at 37 °C. Addition of 0.1% SDS resulted in digestion of the protein imported into the vacuolar lumen.

For assays containing recombinant CaM together with the CaM antagonists W-7 (Sigma, St Louis, MO, USA) or compound 48/80 (Sigma), contractile vacuoles were first washed in the presence of 5 mm EGTA to remove the CaM associated with contractile vacuoles, and then the import assay was performed in the presence or absence of the antagonists.

Far-western blot analysis

DdCAD-1 or CaM recombinant proteins were separated by SDS/PAGE and transferred onto nitrocellulose membranes (Amersham Biosciences, Little Chalfont, UK). The membrane subsequently was incubated with 20 μg·mL−1 His6-tagged CaM or His6-tagged DdCAD-1 in 50 mm MES buffer (pH 6.4) containing 1 mm CaCl2 for 1 h at room temperature. For control experiments, the CaCl2 was replaced by 5 mm EGTA. After washing twice for 5 min each at room temperature with 1× Tris-buffered saline plus 0.1% Tween-20, the blots were incubated with either monoclonal antibody against CaM (Sigma) or rabbit antibodies raised against DdCAD-1 [10], followed by horseradish peroxidase-conjugated goat antibody against either mouse or rabbit IgG (Bio-Rad, Hercules, CA, USA).

Pull-down assays using CaM-conjugated agarose

Cell samples (each containing 5 mg protein) derived from AX4 cells or transfectants were solubilized using 1% Nonidet P-40 in the presence of 5 mm CaCl2. In control samples, CaCl2 was replaced by 5 mm EGTA. These samples were incubated with CaM-conjugated agarose (Stratagene, La Jolla, CA, USA) pre-incubated with 1% BSA for 16 h at 4 °C. The beads were collected by centrifugation at 500 g for 3 min at 4 °C and washed three times with 50 mm MES buffer (pH 6.4). Bound proteins were analyzed by SDS/PAGE followed by western blot analysis using an antibody raised against DdCAD-1.

Antibody-induced cap formation

cadA-null cells (2 × 107 cells·mL−1) were developed in 17 mm phosphate buffer, and collected after 3 h. Recombinant DdCAD-1 (40 μg) and CaM (40 μg) were added to 1.2 × 106 cells suspended in 50 mm MES buffer, pH 6.3, to a final volume of 300 μL. Samples were incubated at room temperature for 30 min. Antibody against DdCAD-1 (1 : 100 dilution) was added to these cells and incubated for 30 min at room temperature. After washing, Alexa Fluor 568-conjugated goat antibody against rabbit IgG was added at 1 : 400 dilution, and the cell sample was rotated at room temperature for another 30 min. Next, 300 μL of cells were deposited on a positively charged cover slip (Fisher Scientific, Ottawa, Canada) and allowed to attach for 15 min. The cover slips were washed gently with MCG buffer (50 mm MES, pH 6.4, 0.2 mm CaCl2, 2 mm MgCl2), fixed with 3.7% formaldehyde, and labelled with mouse monoclonal antibody against CaM (6D4; Sigma) and Alexa Fluor 488-conjugated goat secondary antibody against mouse IgG, and mounted in DAKO Fluorescent mounting medium (DakoCytomation, Glostrup, Denmark) for fluorescence microscopy.

Co-immunoprecipitation assays

AX4 cell samples containing 5 mg of total protein were solubilized in the presence of 1% Nonidet P-40 in immune precipitation buffer (10 mm Tris/HCl, pH 7.6, 100 mm NaCl, 1 mm sodium orthovanadate and 1 mm phenylmethanesulfonyl fluoride). Protein A–Sepharose beads (50 μL of a 50% slurry) were incubated with rabbit antibody against DdCAD-1 (5 μg) or control rabbit IgG (5 μg) in a final volume of 100 μL of the same buffer for 1 h at 4 °C. The antibody-coupled protein A–Sepharose beads were incubated with 900 μL of solubilized cellular proteins for 18 h at 4 °C on a vertical rotator at approximately 60 rpm. After extensive washing with 10 mm Tris/HCl, pH 7.6, electrophoresis sample buffer was added to the beads and boiled for 5 min. Samples were subjected to SDS/PAGE and western blot analysis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Drs David Isenman (Department of Biochemistry) and Mitsu Ikura (Department of Medical Biophysics) for advice and invaluable suggestions, Gong Chen and Chunxia Yang (Siu laboratory) for discussion, and Xiangfu Wu for technical support. This work was supported by Canadian Institutes of Health Research operating grant FRN-6140. S.S. and S.K.B. were recipients of an Ontario Graduate Scholarship.

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  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
febs12203-sup-0001-FigS1-S2.zipapplication/ZIP356K

Fig. S1. Confocal micrographs showing the association of DdCAD-1–GFP with the contractile vacuole network.

Fig. S2. List of Ca2+-dependent calmodulin binding motifs present in DdCAD-1.

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