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- Experimental procedures
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In eukaryotic cells, an increasing number of soluble cytoplasmic proteins are known to be secreted by non-classical transport mechanisms [1, 2]. Interleukin 1β  and galectin-1  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 . 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 . DdCAD-1 is made up of two Ig-like domains connected by a short linker sequence . Mutational analysis has shown that import of DdCAD-1 depends on the integrity of the protein . 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 , and CaM from eukaryotic micro-organisms is functionally interchangeable with CaM from mammals . 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 . 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 . 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.
- Top of page
- Experimental procedures
- 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.
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 . 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 . However, contractile vacuoles are highly efficient Ca2+ stores , 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 , 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 , which are crucial for binding of CaM target proteins such as myosin light-chain kinases  and CaM kinase IIa . CaM has been shown to associate with unconventional myosin that is present in great abundance on the contractile vacuole network of Dictyostelium . 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) . 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 . Interestingly, CaM is required for micro-autophagy, and invagination of the micro-autophagic vacuolar membrane is sensitive to CaM antagonists . Moreover, CaM is involved in Ca2+-dependent protein translocation in the inner envelope of chloroplasts  and in post-docking events during late endosome–lysosome fusion in yeast .
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+ . 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) . DdCAD-1 is released from the internalized vesicles as they burst in the hypotonic environment of the lumen . In addition, translocation of DdCAD-1 may involve a membrane transporter. CaM has been shown to interact with components of V-ATPases in yeast . Contractile vacuoles in Dictyostelium are enriched with the proton pump V-H+-ATPases . 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 . 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.