Cell Adhesion Molecule DdCAD-1 Is Imported into Contractile Vacuoles by Membrane Invagination in a Ca2+- and Conformation-dependent Manner

The cadA gene in Dictyostelium encodes a Ca2+-dependent cell adhesion molecule DdCAD-1 that contains two β-sandwich domains. DdCAD-1 is synthesized in the cytoplasm as a soluble protein and then transported by contractile vacuoles to the plasma membrane for surface presentation or secretion. DdCAD-1-green fluorescent protein (GFP) fusion protein was expressed in cadA-null cells for further investigation of this unconventional protein transport pathway. Both morphological and biochemical characterizations showed that DdCAD-1-GFP was imported into contractile vacuoles. Time-lapse microscopy of transfectants revealed the transient appearance of DdCAD-1-GFP-filled vesicular structures in the lumen of contractile vacuoles, suggesting that DdCAD-1 could be imported by invagination of contractile vacuole membrane. To assess the structural requirements in this transport process, the N-terminal and C-terminal domains of DdCAD-1 were expressed separately in cells as GFP fusion proteins. Both fusion proteins failed to enter the contractile vacuole, suggesting that the integrity of DdCAD-1 is required for import. Such a requirement was also observed in in vitro reconstitution assays using His6-tagged fusion proteins and purified contractile vacuoles. Import of DdCAD-1 was compromised when two of its three Ca2+-binding sites were mutated, indicating a role for Ca2+ in the import process. Spectral analysis showed that mutations in the Ca2+-binding sites resulted in subtle conformational changes. Indeed, proteins with altered conformation failed to enter the contractile vacuole, suggesting that the import signal is somehow integrated in the three-dimensional structure of DdCAD-1. The cadA gene in Dictyostelium encodes a Ca2+-dependent cell adhesion molecule DdCAD-1 that contains two β-sandwich domains. DdCAD-1 is synthesized in the cytoplasm as a soluble protein and then transported by contractile vacuoles to the plasma membrane for surface presentation or secretion. DdCAD-1-green fluorescent protein (GFP) fusion protein was expressed in cadA-null cells for further investigation of this unconventional protein transport pathway. Both morphological and biochemical characterizations showed that DdCAD-1-GFP was imported into contractile vacuoles. Time-lapse microscopy of transfectants revealed the transient appearance of DdCAD-1-GFP-filled vesicular structures in the lumen of contractile vacuoles, suggesting that DdCAD-1 could be imported by invagination of contractile vacuole membrane. To assess the structural requirements in this transport process, the N-terminal and C-terminal domains of DdCAD-1 were expressed separately in cells as GFP fusion proteins. Both fusion proteins failed to enter the contractile vacuole, suggesting that the integrity of DdCAD-1 is required for import. Such a requirement was also observed in in vitro reconstitution assays using His6-tagged fusion proteins and purified contractile vacuoles. Import of DdCAD-1 was compromised when two of its three Ca2+-binding sites were mutated, indicating a role for Ca2+ in the import process. Spectral analysis showed that mutations in the Ca2+-binding sites resulted in subtle conformational changes. Indeed, proteins with altered conformation failed to enter the contractile vacuole, suggesting that the import signal is somehow integrated in the three-dimensional structure of DdCAD-1. IntroductionIn eukaryotes, soluble secretory proteins are typically transported through the classical ER 3The abbreviations used are: ERendoplasmic reticulumFGFfibroblast growth factorGFPgreen fluorescence proteinNN-terminal domainCC-terminal domainmAbmonoclonal antibodyMes4-morpholineethanesulfonic acid. -Golgi transport pathway (1.Palade G.E. Science. 1975; 189: 347-358Crossref PubMed Scopus (2320) Google Scholar, 2.Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). However, it has become evident in recent years that a growing number of soluble proteins synthesized in the cytoplasm are targeted for secretion (3.Nickel W. Rabouille C. Nat. Rev. Mol. Cell Biol. 2009; 10: 148-155Crossref PubMed Scopus (517) Google Scholar, 4.Nickel W. Seedorf M. Annu. Rev. Cell Dev. Biol. 2008; 24: 287-308Crossref PubMed Scopus (204) Google Scholar). This group of secretory proteins usually shares several common features, including the lack of conventional signal peptides, the absence of post-translational modifications, and the presence of free cysteines (5.Cleves A.E. Curr. Biol. 1997; 7: R318-R320Abstract Full Text Full Text PDF PubMed Google Scholar, 6.Nickel W. Eur. J. Biochem. 2003; 270: 2109-2119Crossref PubMed Scopus (519) Google Scholar). Many of these proteins play key roles in diverse biological processes. Among them are galectins (7.Pohlschröder M. Hartmann E. Hand N.J. Dilks K. Haddad A. Annu. Rev. Microbiol. 2005; 59: 91-111Crossref PubMed Scopus (97) Google Scholar, 8.Cleves A.E. Cooper D.N. Barondes S.H. Kelly R.B. J. Cell Biol. 1996; 133: 1017-1026Crossref PubMed Scopus (185) Google Scholar), interleukin-1β (9.Orci L. Tagaya M. Amherdt M. Perrelet A. Donaldson J.G. Lippincott-Schwartz J. Klausner R.D. Rothman J.E. Cell. 1991; 64: 1183-1195Abstract Full Text PDF PubMed Scopus (352) Google Scholar), thioredoxin (10.Rubartelli A. Bajetto A. Allavena G. Wollman E. Sitia R. J. Biol. Chem. 1992; 267: 24161-24164Abstract Full Text PDF PubMed Google Scholar), macrophage migration inhibitory factor (11.Flieger O. Engling A. Bucala R. Lue H. Nickel W. Bernhagen J. FEBS Lett. 2003; 551: 78-86Crossref PubMed Scopus (178) Google Scholar), and fibroblast growth factor 1 and 2 (FGF-1 and FGF-2) (12.Engling A. Backhaus R. Stegmayer C. Zehe C. Seelenmeyer C. Kehlenbach A. Schwappach B. Wegehingel S. Nickel W. J. Cell Sci. 2002; 115: 3619-3631Crossref PubMed Scopus (69) Google Scholar, 13.Mignatti P. Morimoto T. Rifkin D.B. J. Cell Physiol. 1992; 151: 81-93Crossref PubMed Scopus (405) Google Scholar). Several viral proteins, such as HIV-Tat and herpes simplex VP22, are also known to be secreted via ER-Golgi-independent routes (14.Denny P.W. Gokool S. Russell D.G. Field M.C. Smith D.F. J. Biol. Chem. 2000; 275: 11017-11025Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 15.Elliott G. O'Hare P. Cell. 1997; 88: 223-233Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar, 16.Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (443) Google Scholar).Multiple unconventional transport mechanisms have been discovered, and cells can utilize one or more pathways to target soluble proteins for secretion. The interleukin-1β export mechanism involves intracellular vesicles in its secretory pathway (17.Andrei C. Dazzi C. Lotti L. Torrisi M.R. Chimini G. Rubartelli A. Mol. Biol. Cell. 1999; 10: 1463-1475Crossref PubMed Scopus (387) Google Scholar, 18.Rubartelli A. Cozzolino F. Talio M. Sitia R. EMBO J. 1990; 9: 1503-1510Crossref PubMed Scopus (646) Google Scholar), whereas the muscle lectin galectin-1 is externalized by the shedding of membrane vesicles (19.Cooper D.N. Barondes S.H. J. Cell Biol. 1990; 110: 1681-1691Crossref PubMed Scopus (406) Google Scholar) or through a protein transporter (20.Seelenmeyer C. Wegehingel S. Tews I. Künzler M. Aebi M. Nickel W. J. Cell Biol. 2005; 171: 373-381Crossref PubMed Scopus (79) Google Scholar). Secretion of the growth factor FGF-2 is also likely mediated by a plasma membrane-resident transporter (21.Schäfer T. Zentgraf H. Zehe C. Brügger B. Bernhagen J. Nickel W. J. Biol. Chem. 2004; 279: 6244-6251Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Additionally, cell surface counter receptors are essential components in the export machinery of galectin-1 because they can provide an extracellular trap mechanism (20.Seelenmeyer C. Wegehingel S. Tews I. Künzler M. Aebi M. Nickel W. J. Cell Biol. 2005; 171: 373-381Crossref PubMed Scopus (79) Google Scholar).Several soluble proteins expressed by the social amoeba Dictyostelium discoideum have been found to be targeted for secretion by unconventional pathways (22.Siu C.H. Harris T.J. Wang J. Wong E. Semin. Cell Dev. Biol. 2004; 15: 633-641Crossref PubMed Scopus (39) Google Scholar, 23.Sriskanthadevan S. Ivanov I. Yang C. Siu C.H. Recent Research Developments in Cell Biology.in: Pandalai S.G. Vol. 3. Research Signpost, Trivandrum2007: 9-21Google Scholar). At the onset of development, amoeboid cells undergo chemotactic migration to form large aggregates of ∼105 cells, which eventually culminate in the formation of fruiting bodies (24.Aubry L. Firtel R. Annu. Rev. Cell Dev. Biol. 1999; 15: 469-517Crossref PubMed Scopus (134) Google Scholar). The endogenous soluble lectin discoidin-I, which is externalized to facilitate cell-substratum adhesion (25.Crowley T.E. Nellen W. Gomer R.H. Firtel R.A. Cell. 1985; 43: 633-641Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 26.Springer W.R. Cooper D.N. Barondes S.H. Cell. 1984; 39: 557-564Abstract Full Text PDF PubMed Scopus (134) Google Scholar), is targeted for secretion through vesicular structures (27.Barondes S.H. Haywood-Reid P.L. Cooper D.N. J. Cell Biol. 1985; 100: 1825-1833Crossref PubMed Scopus (39) Google Scholar). Multicellularity during Dictyostelium development is maintained by the expression of several cell adhesion molecules (22.Siu C.H. Harris T.J. Wang J. Wong E. Semin. Cell Dev. Biol. 2004; 15: 633-641Crossref PubMed Scopus (39) Google Scholar). One of them is the Ca2+-dependent cell adhesion molecule DdCAD-1 that is encoded by the cadA gene. DdCAD-1 lacks the classical signal peptide and is synthesized as a soluble protein in the cytoplasm (28.Brar S.K. Siu C.H. J. Biol. Chem. 1993; 268: 24902-24909Abstract Full Text PDF PubMed Google Scholar, 29.Wong E.F. Brar S.K. Sesaki H. Yang C. Siu C.H. J. Biol. Chem. 1996; 271: 16399-16408Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 30.Yang C. Brar S.K. Desbarats L. Siu C.H. Differentiation. 1997; 61: 275-284Crossref PubMed Scopus (25) Google Scholar). It is imported into contractile vacuoles for transport to the plasma membrane (31.Sesaki H. Wong E.F. Siu C.H. J. Cell Biol. 1997; 138: 939-951Crossref PubMed Scopus (56) Google Scholar). DdCAD-1 contains two distinct domains with β-sandwich architecture. Although the N-terminal domain is involved in homophilic binding, the C-terminal domain tethers the protein to a membrane anchor, thus allowing it to function as a cell adhesion molecule on the cell surface (32.Lin Z. Sriskanthadevan S. Huang H. Siu C.H. Yang D. Nat. Struct. Mol. Biol. 2006; 13: 1016-1022Crossref PubMed Scopus (28) Google Scholar). Both DdCAD-1 and discoidin-I have been found to be enriched in contractile vacuoles (31.Sesaki H. Wong E.F. Siu C.H. J. Cell Biol. 1997; 138: 939-951Crossref PubMed Scopus (56) Google Scholar), the osmoregulatory organelles that regulate water balance during the growth and the initial stages of development (33.Gerisch G. Heuser J. Clarke M. Cell Biol. Int. 2002; 26: 845-852Crossref PubMed Scopus (46) Google Scholar, 34.Heuser J. Zhu Q. Clarke M. J. Cell Biol. 1993; 121: 1311-1327Crossref PubMed Scopus (179) Google Scholar). As they fuse with the plasma membrane, the contents of the contractile vacuoles are released into the medium.In this study, we investigated the structural requirements for the import of DdCAD-1 into contractile vacuoles. Using transfectants that express DdCAD-1-GFP fusion proteins, we observe that DdCAD-1 is imported through the invagination of contractile vacuole membranes. However, the whole protein is required because neither the N-terminal domain nor the C-terminal domain alone is sufficient for transport into the contractile vacuole. Additionally, the import mechanism is dependent on Ca2+ and proper protein conformation, suggesting that the secretory targeting motif of DdCAD-1 lies in its three-dimensional structure rather than in a linear stretch of amino acids.DISCUSSIONIn Dictyostelium, the contractile vacuoles serve as the major vehicle for the transport of DdCAD-1 to the plasma membrane for either secretion or cell surface presentation. Our in vivo analysis using time-lapse microscopy has revealed transient membrane budding into the lumen of contractile vacuoles. DdCAD-1 docked on the contractile vacuoles is mobilized to fill these invaginations, which are then pinched off to become vesicles inside the lumen. DdCAD-1 is released upon the burst of these vesicles. In many ways, the export of DdCAD-1 via contractile vacuole is similar to the budding of vesicles in yeast vacuoles (41.Müller O. Sattler T. M. H. H. A. J. Cell Biol. 2000; 151: PubMed Scopus Google Scholar) or the formation of bodies in L. G. J. PubMed Scopus Google Scholar). In the structures facilitate that of proteins and into vesicles inside the vacuole (41.Müller O. Sattler T. M. H. H. A. J. Cell Biol. 2000; 151: PubMed Scopus Google Scholar). In the bodies are the vehicle for the transport of interleukin-1β inside the L. G. J. PubMed Scopus Google Scholar). However, vesicles and are transient structures in the lumen of contractile vacuoles in Dictyostelium cells, and structures are It is that the water in the contractile vacuole a that can the of vesicles they off into the further the structural mechanisms involved in DdCAD-1 import into contractile vacuoles, domains were into cadA-null Both in vivo and in vitro that neither the N-terminal domain nor the C-terminal domain of DdCAD-1 alone is sufficient for import into the contractile vacuole. the of to contractile vacuoles is by the presence by either or the import signal is likely integrated in the three-dimensional structure of analysis has revealed a role for Ca2+ because the import of DdCAD-1 is by that the Ca2+-binding of DdCAD-1 also its import. Ca2+ the of DdCAD-1 to contractile vacuoles as as its into the lumen. DdCAD-1 is a Ca2+-binding However, the Ca2+-binding of DdCAD-1 is (32.Lin Z. Sriskanthadevan S. Huang H. Siu C.H. Yang D. Nat. Struct. Mol. Biol. 2006; 13: 1016-1022Crossref PubMed Scopus (28) Google Scholar). the intracellular free Ca2+ is to be in cells S. K. I. J. Cell Sci. 1996; PubMed Google Scholar), of the intracellular DdCAD-1 in the the contractile vacuoles are enriched in Ca2+ J. A. J. Cell Sci. 1999; PubMed Google Scholar, and they a Ca2+ D. D.F. C. A. A. Dev. Biol. 2006; PubMed Scopus Google Scholar). It is that the of Ca2+ contractile vacuoles serve as an to DdCAD-1. the surface of is known to the targeting of proteins with S. D. Nature. 2005; PubMed Scopus Google Scholar, T. G.E. J. J. A. S. Science. 2008; PubMed Scopus Google Scholar). The Ca2+ the on the surface of DdCAD-1. The as as the with and facilitate the of DdCAD-1 to the contractile vacuole membrane. have that Ca2+-binding membrane proteins, such as with via Ca2+ Nat. Struct. Biol. PubMed Scopus Google Scholar). Such a the of the and N-terminal fusion neither the C-terminal domain nor the N-terminal domain with DdCAD-1 for to the contractile vacuole, it is likely that DdCAD-1 to a in the of on the membrane. In to Ca2+ an of the Ca2+ is with the surface of contractile vacuoles Q. Clarke M. J. Cell Biol. 1992; 347-358Crossref PubMed Scopus Google Scholar, Q. T. Clarke M. J. Cell Sci. 1993; Google Scholar). is also found on and C. S. 1996; 24: PubMed Google Scholar, Field J. Cell Sci. Google Scholar) and is known to play a role in the transport J. Cell Biol. 2000; PubMed Scopus Google Scholar). analysis that DdCAD-1 contains a for in the C-terminal suggesting that the form of serve as a for DdCAD-1. Indeed, DdCAD-1 and has been in whereas of function by to a in DdCAD-1 to contractile vacuoles. T. and DdCAD-1 contains three Ca2+-binding and the structures of DdCAD-1 have that of Ca2+ to more in the Ca2+-binding sites and the whole structure of DdCAD-1 (32.Lin Z. Sriskanthadevan S. Huang H. Siu C.H. Yang D. Nat. Struct. Mol. Biol. 2006; 13: 1016-1022Crossref PubMed Scopus (28) Google Scholar). analysis of the of the Ca2+-binding conformational in that is in the structure and of with in in similar to observed for the and The as as mutations in either the or Ca2+-binding in the of DdCAD-1 import into contractile vacuoles, a role for the three-dimensional structure in DdCAD-1 It is that the Ca2+-binding sites serve a Although by Ca2+ to the surface can the of DdCAD-1 to contractile vacuoles, conformational by Ca2+ regulate its import into the and cells, several unconventional pathways are known to utilize to import or export soluble proteins Cell Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus (39) Google Scholar). components of are transported in form the cytoplasm into the C. 2003; PubMed Scopus Google Scholar, W. 2005; PubMed Scopus Google Scholar). pathways that can the pathway K. M. EMBO J. 26: PubMed Scopus Google Scholar, Sci. 1992; PubMed Scopus Google Scholar), the pathway of S. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar), the targeting pathway of J. J. Cell Biol. 1997; PubMed Scopus Google Scholar), and the of proteins, such as FGF-2 and the plasma membrane in cells (20.Seelenmeyer C. Wegehingel S. Tews I. Künzler M. Aebi M. Nickel W. J. Cell Biol. 2005; 171: 373-381Crossref PubMed Scopus (79) Google Scholar, T. Zentgraf H. Zehe C. Brügger B. Bernhagen J. Nickel W. J. Biol. Chem. 2004; 279: 6244-6251Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Among the and pathways targeting whereas the to a signal for protein transport G. B. Science. 1996; 271: PubMed Scopus Google Scholar, W. R. Science. 2005; PubMed Scopus Google Scholar). has been targeting motif in the of and the import signal is likely integrated in its three-dimensional export of soluble proteins more than one unconventional pathway (3.Nickel W. Rabouille C. Nat. Rev. Mol. Cell Biol. 2009; 10: 148-155Crossref PubMed Scopus (517) Google Scholar, 4.Nickel W. Seedorf M. Annu. Rev. Cell Dev. Biol. 2008; 24: 287-308Crossref PubMed Scopus (204) Google Scholar). Our in vitro that the of DdCAD-1 the contractile vacuole membrane a membrane transporter (31.Sesaki H. Wong E.F. Siu C.H. J. Cell Biol. 1997; 138: 939-951Crossref PubMed Scopus (56) Google Scholar). and an the import of suggesting the of in the contractile vacuole membrane. the import of DdCAD-1 is by a of I. R. A. Annu. Rev. Biochem. PubMed Google Scholar, H. M. PubMed Scopus Google Scholar), a role for the in the import Among secreted proteins in Dictyostelium, the protein which is required for the of cells, is also externalized through an unconventional protein secretion pathway. this pathway involves and a membrane transporter C. D. G. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of the transporter for to be It is evident that unconventional pathways are by cells to target soluble proteins for surface expression and secretion. The of and biochemical in Dictyostelium a for the of these IntroductionIn eukaryotes, soluble secretory proteins are typically transported through the classical ER 3The abbreviations used are: ERendoplasmic reticulumFGFfibroblast growth factorGFPgreen fluorescence proteinNN-terminal domainCC-terminal domainmAbmonoclonal antibodyMes4-morpholineethanesulfonic acid. -Golgi transport pathway (1.Palade G.E. Science. 1975; 189: 347-358Crossref PubMed Scopus (2320) Google Scholar, 2.Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). However, it has become evident in recent years that a growing number of soluble proteins synthesized in the cytoplasm are targeted for secretion (3.Nickel W. Rabouille C. Nat. Rev. Mol. Cell Biol. 2009; 10: 148-155Crossref PubMed Scopus (517) Google Scholar, 4.Nickel W. Seedorf M. Annu. Rev. Cell Dev. Biol. 2008; 24: 287-308Crossref PubMed Scopus (204) Google Scholar). This group of secretory proteins usually shares several common features, including the lack of conventional signal peptides, the absence of post-translational modifications, and the presence of free cysteines (5.Cleves A.E. Curr. Biol. 1997; 7: R318-R320Abstract Full Text Full Text PDF PubMed Google Scholar, 6.Nickel W. Eur. J. Biochem. 2003; 270: 2109-2119Crossref PubMed Scopus (519) Google Scholar). Many of these proteins play key roles in diverse biological processes. Among them are galectins (7.Pohlschröder M. Hartmann E. Hand N.J. Dilks K. Haddad A. Annu. Rev. Microbiol. 2005; 59: 91-111Crossref PubMed Scopus (97) Google Scholar, 8.Cleves A.E. Cooper D.N. Barondes S.H. Kelly R.B. J. Cell Biol. 1996; 133: 1017-1026Crossref PubMed Scopus (185) Google Scholar), interleukin-1β (9.Orci L. Tagaya M. Amherdt M. Perrelet A. Donaldson J.G. Lippincott-Schwartz J. Klausner R.D. Rothman J.E. Cell. 1991; 64: 1183-1195Abstract Full Text PDF PubMed Scopus (352) Google Scholar), thioredoxin (10.Rubartelli A. Bajetto A. Allavena G. Wollman E. Sitia R. J. Biol. Chem. 1992; 267: 24161-24164Abstract Full Text PDF PubMed Google Scholar), macrophage migration inhibitory factor (11.Flieger O. Engling A. Bucala R. Lue H. Nickel W. Bernhagen J. FEBS Lett. 2003; 551: 78-86Crossref PubMed Scopus (178) Google Scholar), and fibroblast growth factor 1 and 2 (FGF-1 and FGF-2) (12.Engling A. Backhaus R. Stegmayer C. Zehe C. Seelenmeyer C. Kehlenbach A. Schwappach B. Wegehingel S. Nickel W. J. Cell Sci. 2002; 115: 3619-3631Crossref PubMed Scopus (69) Google Scholar, 13.Mignatti P. Morimoto T. Rifkin D.B. J. Cell Physiol. 1992; 151: 81-93Crossref PubMed Scopus (405) Google Scholar). Several viral proteins, such as HIV-Tat and herpes simplex VP22, are also known to be secreted via ER-Golgi-independent routes (14.Denny P.W. Gokool S. Russell D.G. Field M.C. Smith D.F. J. Biol. Chem. 2000; 275: 11017-11025Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 15.Elliott G. O'Hare P. Cell. 1997; 88: 223-233Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar, 16.Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (443) Google Scholar).Multiple unconventional transport mechanisms have been discovered, and cells can utilize one or more pathways to target soluble proteins for secretion. The interleukin-1β export mechanism involves intracellular vesicles in its secretory pathway (17.Andrei C. Dazzi C. Lotti L. Torrisi M.R. Chimini G. Rubartelli A. Mol. Biol. Cell. 1999; 10: 1463-1475Crossref PubMed Scopus (387) Google Scholar, 18.Rubartelli A. Cozzolino F. Talio M. Sitia R. EMBO J. 1990; 9: 1503-1510Crossref PubMed Scopus (646) Google Scholar), whereas the muscle lectin galectin-1 is externalized by the shedding of membrane vesicles (19.Cooper D.N. Barondes S.H. J. Cell Biol. 1990; 110: 1681-1691Crossref PubMed Scopus (406) Google Scholar) or through a protein transporter (20.Seelenmeyer C. Wegehingel S. Tews I. Künzler M. Aebi M. Nickel W. J. Cell Biol. 2005; 171: 373-381Crossref PubMed Scopus (79) Google Scholar). Secretion of the growth factor FGF-2 is also likely mediated by a plasma membrane-resident transporter (21.Schäfer T. Zentgraf H. Zehe C. Brügger B. Bernhagen J. Nickel W. J. Biol. Chem. 2004; 279: 6244-6251Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Additionally, cell surface counter receptors are essential components in the export machinery of galectin-1 because they can provide an extracellular trap mechanism (20.Seelenmeyer C. Wegehingel S. Tews I. Künzler M. Aebi M. Nickel W. J. Cell Biol. 2005; 171: 373-381Crossref PubMed Scopus (79) Google Scholar).Several soluble proteins expressed by the social amoeba Dictyostelium discoideum have been found to be targeted for secretion by unconventional pathways (22.Siu C.H. Harris T.J. Wang J. Wong E. Semin. Cell Dev. Biol. 2004; 15: 633-641Crossref PubMed Scopus (39) Google Scholar, 23.Sriskanthadevan S. Ivanov I. Yang C. Siu C.H. Recent Research Developments in Cell Biology.in: Pandalai S.G. Vol. 3. Research Signpost, Trivandrum2007: 9-21Google Scholar). At the onset of development, amoeboid cells undergo chemotactic migration to form large aggregates of ∼105 cells, which eventually culminate in the formation of fruiting bodies (24.Aubry L. Firtel R. Annu. Rev. Cell Dev. Biol. 1999; 15: 469-517Crossref PubMed Scopus (134) Google Scholar). The endogenous soluble lectin discoidin-I, which is externalized to facilitate cell-substratum adhesion (25.Crowley T.E. Nellen W. Gomer R.H. Firtel R.A. Cell. 1985; 43: 633-641Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 26.Springer W.R. Cooper D.N. Barondes S.H. Cell. 1984; 39: 557-564Abstract Full Text PDF PubMed Scopus (134) Google Scholar), is targeted for secretion through vesicular structures (27.Barondes S.H. Haywood-Reid P.L. Cooper D.N. J. Cell Biol. 1985; 100: 1825-1833Crossref PubMed Scopus (39) Google Scholar). Multicellularity during Dictyostelium development is maintained by the expression of several cell adhesion molecules (22.Siu C.H. Harris T.J. Wang J. Wong E. Semin. Cell Dev. Biol. 2004; 15: 633-641Crossref PubMed Scopus (39) Google Scholar). One of them is the Ca2+-dependent cell adhesion molecule DdCAD-1 that is encoded by the cadA gene. DdCAD-1 lacks the classical signal peptide and is synthesized as a soluble protein in the cytoplasm (28.Brar S.K. Siu C.H. J. Biol. Chem. 1993; 268: 24902-24909Abstract Full Text PDF PubMed Google Scholar, 29.Wong E.F. Brar S.K. Sesaki H. Yang C. Siu C.H. J. Biol. Chem. 1996; 271: 16399-16408Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 30.Yang C. Brar S.K. Desbarats L. Siu C.H. Differentiation. 1997; 61: 275-284Crossref PubMed Scopus (25) Google Scholar). It is imported into contractile vacuoles for transport to the plasma membrane (31.Sesaki H. Wong E.F. Siu C.H. J. Cell Biol. 1997; 138: 939-951Crossref PubMed Scopus (56) Google Scholar). DdCAD-1 contains two distinct domains with β-sandwich architecture. Although the N-terminal domain is involved in homophilic binding, the C-terminal domain tethers the protein to a membrane anchor, thus allowing it to function as a cell adhesion molecule on the cell surface (32.Lin Z. Sriskanthadevan S. Huang H. Siu C.H. Yang D. Nat. Struct. Mol. Biol. 2006; 13: 1016-1022Crossref PubMed Scopus (28) Google Scholar). Both DdCAD-1 and discoidin-I have been found to be enriched in contractile vacuoles (31.Sesaki H. Wong E.F. Siu C.H. J. Cell Biol. 1997; 138: 939-951Crossref PubMed Scopus (56) Google Scholar), the osmoregulatory organelles that regulate water balance during the growth and the initial stages of development (33.Gerisch G. Heuser J. Clarke M. Cell Biol. Int. 2002; 26: 845-852Crossref PubMed Scopus (46) Google Scholar, 34.Heuser J. Zhu Q. Clarke M. J. Cell Biol. 1993; 121: 1311-1327Crossref PubMed Scopus (179) Google Scholar). As they fuse with the plasma membrane, the contents of the contractile vacuoles are released into the medium.In this study, we investigated the structural requirements for the import of DdCAD-1 into contractile vacuoles. Using transfectants that express DdCAD-1-GFP fusion proteins, we observe that DdCAD-1 is imported through the invagination of contractile vacuole membranes. However, the whole protein is required because neither the N-terminal domain nor the C-terminal domain alone is sufficient for transport into the contractile vacuole. Additionally, the import mechanism is dependent on Ca2+ and proper protein conformation, suggesting that the secretory targeting motif of DdCAD-1 lies in its three-dimensional structure rather than in a linear stretch of amino

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