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First published online 14 February 2006
doi: 10.1242/jcs.02808
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Research Article |
1 Université de Genève, Centre Médical Universitaire, Département de Physiologie Cellulaire et Métabolisme, CH-1211 Genève 4, Switzerland
2 IFR 128 BioSciences Lyon-Gerland, Institut de Biologie et Chimie des Protéines, UMR5086–CNRS/Université Lyon I, 7 Passage du Vercors, 69367 Lyon CEDEX 07, France
* Author for correspondence (e-mail: f.letourneur{at}ibcp.fr)
Accepted 24 November 2005
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Summary |
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-adaptin AP-1 subunit in a yeast protein-protein interaction assay. For the first time we report the existence of an indirect transport pathway from the plasma membrane to the CV via endosomes. Upon internalization, the small fraction of CsA-Rh50 present at the cell surface was first concentrated in endosomes distinct from early and late p80-positive endosomes and then slowly transported to the CV. Together our results suggest the existence of an AP-1-dependent selective transport to the contractile vacuole in Dictyostelium.
Key words: Contractile vacuole, Endosomes, Sorting signals, Clathrin-adaptor, Dictyostelium discoideum
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Introduction |
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; Heuser et al., 1993; Nolta and Steck, 1994; Gerisch et al., 2002). The structure, function and biochemical composition of the CV are reminiscent of the tubulo-vesicular structure seen in acid-secreting gastric parietal cells (reviewed by Yao and Forte, 2003).
Not much is known about the biogenesis of the CV in Dictyostelium. CV-resident proteins enter the secretory pathway and are transported through the Golgi apparatus before arriving at the CV. Indeed O-glycosylated proteins, a post-translational modification acquired in the Golgi apparatus, have been identified in CV proteins (Gabriel et al., 1999). Further insights into the traffic of proteins to the CV came from the study of cells deficient for coat proteins involved in vesicular transport. One of the best-characterized vesicular coat proteins is clathrin, which, in association with adaptor-protein (AP) complexes, participates in multiple transport steps (Hirst and Robinson, 1998; Smith and Pearse, 1999). In mammalian cells four different AP complexes have been identified (AP-1 to AP-4), which share a similar composition (Boehm et al., 2001). In addition to clathrin recruitment, AP complexes are also implicated in the sorting of cargo proteins transported by clathrin-coated vesicles. The AP-1 complex contains ß1- and -adaptin large chains (80 and 130 kDa respectively), a µ1A or µ1B medium chain (50 kDa) and a 
1A or 1B small chain (20 kDa) (Scales et al., 2000). It ensures transport between the trans-Golgi network (TGN) and late endosomes/lysosomes, but it has also been implicated in several other steps of intracellular transport in the endocytic pathway, in particular in the retrograde transport from endosomes to the TGN of mannose 6-phosphate receptors in mammalian cells, chitin synthase III and syntaxin Tlg1p in yeast cells (Zizioli et al., 1999; Meyer et al., 2000; Valdivia et al., 2002), the transport of the transferrin receptor from apical to basolateral membranes in epithelial cells (Futter et al., 1998), the recycling of the low-density lipoprotein receptor and the transferrin receptor to the basolateral membrane (Gan et al., 2002), and the transport from the TGN to the basolateral membrane of many membrane proteins (Folsch et al., 1999; Folsch et al., 2001).
Clathrin-deficient Dictyostelium cells show dramatic morphological and functional defects of the CV (O'Halloran and Anderson, 1992; Wang et al., 2003). Deletion of the apm1 gene encoding the medium subunit of the clathrin adaptor-protein AP-1 also results in the absence of a characteristic CV (Lefkir et al., 2003). Together these results suggest that clathrin-coated vesicles, and more precisely AP-1 clathrin-coated vesicles, are involved in the biogenesis of the CV. In Dictyostelium cells, endosomes and the CV share common proteins such as the proton ATPase (Fok et al., 1993; Heuser et al., 1993; Adessi et al., 1995). However endocytosed fluid phase does not get access to the CV (Gabriel et al., 1999), and no exchange of material between these two compartments has yet been documented. Moreover, despite the close contact between the CV bladder and the plasma membrane during water expulsion, no direct protein exchange occurs between the CV and the plasma membrane either (Gabriel et al., 1999).
Only a few Dictyostelium proteins specifically localized in the CV have been identified, including drainin (Becker et al., 1999), calmodulin (Zhu and Clarke, 1992), rab 11 (Harris et al., 2001) and Rh50 (Benghezal et al., 2001). Rh50 is the Dictyostelium homolog of the mammalian rhesus protein. It is an integral membrane protein only found in the CV. There must exist a precise sorting mechanism to ensure the sorting and transport of such proteins to the CV, but this question has not been addressed so far. The targeting to the CV has only been studied for drainin, a cytosolic protein associated with the CV membrane (Becker et al., 1999). The specific localization of this cytosolic protein to the vacuolar membrane is dependent on the presence of a stretch of eight hydrophobic amino acids in its C-terminal half. No transport or CV retention signals have been identified so far for integral membrane proteins.
In this study, we characterized for the first time a signal responsible for the transport of Rh50 to the CV. Making use of chimeric proteins, we showed that acidic-based signals present in the last C-terminal domain of Rh50 are necessary and sufficient for efficient transport to the CV. A fraction of the chimeric proteins can be transported to the cell surface and it is then redirected to the CV through endosomal compartments. We thus provide for the first time evidence for a connection between the plasma membrane, endosomal compartments and the CV network. In addition, we demonstrated a direct interaction between CV-specific acidic signals and the subunit of the AP-1 clathrin-adaptor complex. Our results suggest that AP-1 is directly implicated in the transport of Rh50 chimera from intracellular compartments to the CV.
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). In contrast to the proton ATPase, which is found both in the CV and in endosomes (Fok et al., 1993; Heuser et al., 1993; Adessi et al., 1995), Rh50 is strictly restricted to the CV compartment. This suggests the existence of a highly specific intracellular sorting machinery involved in targeting to the CV, and of targeting signals responsible for the localization of Rh50 in the CV. To determine which domain is responsible for the localization of Rh50 on CV, we expressed a fusion protein composed of the contact site A (CsA) extracellular domain, the transmembrane domain of the integral membrane protein P29F8 (Barth et al., 1994) and the 91 residues forming the Rh50 C-terminal cytoplasmic domain (Fig. 1A). Similar constructs were previously used to study the role of other cytoplasmic sequences in intracellular targeting in Dictyostelium (Gabriel et al., 1999; Ravanel et al., 2001).
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). By contrast, the addition of the last 91 residues of Rh50 was sufficient to ensure the localization of CsA-Rh50 to the CV as demonstrated by the colocalization of the chimeric protein with endogenous Rh50 or with calmodulin, another CV resident protein (Zhu and Clarke, 1992) (Fig. 1B). In addition, as observed for endogenous Rh50 (Benghezal et al., 2001), CsA-Rh50 was excluded from early and late endosomal vacuoles characterized by the presence of the p80 endosomal marker (Ravanel et al., 2001). Note that, whereas the majority of CsA-Rh50 was found in the CV, a small amount was targeted to the plasma membrane (Fig. 1B).
The C-terminal domain of Rh50 exhibits potential sorting signals
The fact that the Rh50 C-terminal cytoplasmic domain was sufficient for CV targeting suggested that this domain displays sorting signals. Indeed, sequence analysis revealed the presence of three putative sorting signals, a diaromatic motif (F448W449) and two acidic motifs (478DDEEE482, 516DDEEED521) (Fig. 2).
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Diaromatic motifs have been shown to influence endosomal sorting of several mammalian and viral membrane proteins (reviewed by Bonifacino and Traub, 2003), including the cation-dependent mannose 6P receptor (CD-MPR) (Schweizer et al., 1997), the human mannose receptor (MR) (Schweizer et al., 2000), and envelope glycoprotein (Env) of the human immunodeficiency virus type 1 (HIV-1) (Blot et al., 2003). Interestingly, the Rh50 C-terminal domain presents an additional Y located five residues from the diaromatic motif (F448W449) that could be part of a putative internalization signal (YxxxxF, Fig. 2A) as reported for both CD-MPR and MR (Johnson et al., 1990; Schweizer et al., 2000).
The two Rh50 acidic motifs (designated here Rh50a and Rh50b) belong to another family of sorting signals characterized by clusters of acidic residues often presenting sites for phosphorylation by casein kinase 2 (CKII) (reviewed by Bonifacino and Traub, 2003). In contrast to the membrane-proximal acidic signal of Rh50 (Rh50a), the Rh50b acidic motif shows a consensus CKII phosphorylation site (SxxE, Fig. 2B), although it is not known whether Rh50 is phosphorylated. In addition, an F residue is located directly downstream of the Rh50b motif. This sequence is reminiscent of the acidic-phenylalanine motifs (DE)2-3Fxx
(where is a hydrophobic residue) recently identified in two yeast epsin-like proteins, Ent3p and Ent5p, and shown to interact with -ear domains of GGA2 and AP-1 (Duncan et al., 2003).
Acidic signals are necessary and sufficient for CV targeting
To determine which signal was required for CV targeting, several mutants of the CsA-Rh50 chimera were constructed (Fig. 3). Constructs were stably expressed in Dictyostelium cells and the localization of the chimeras was analyzed by confocal microscopy after labeling with anti-CsA (green) and anti-calmodulin (red) antibodies (Fig. 4). A domain containing the diaromatic signal (pFL760) alone did not allow the transport of the CsA-Rh50 mutant to the CV. By contrast, chimeras harboring one (pFL759 and pFL786) or two acidic motifs (pFL761) were targeted to the CV, as demonstrated by the colocalization of the chimera with calmodulin.
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These results demonstrated that the presence of at least one acidic motif in the cytosolic domain of a chimeric transmembrane protein was sufficient to target it to the CV. To further establish the role of acidic signals in Rh50 targeting, acidic residues of the first acidic signal of Rh50 were mutated to alanine (pFL778) in the context of a construct truncated for the last acidic signal (pFL759). The resulting mutant was not transported to the CV but instead localized at the plasma membrane and in small vesicular structures distinct from the CV (Fig. 4). Mutations of the FW motif to alanine (pFL775) did not prevent transport of the CsA-Rh50 mutant to the CV. Together these results indicate that each acidic motif is necessary and sufficient for the targeting of the CsA-Rh50 chimera to the CV.
Finally, to exclude the possibility that other residues in the Rh50 cytoplasmic tail participate in CV targeting in addition to the acidic signals, the sequence SDDEEEFKQ (corresponding to the last C-terminal acidic signal) was fused to the CsA reporter protein with a spacer sequence of nine amino acids (composed of SG repeats). The presence of the acidic signal (pFL905) allowed transport of the chimera to the CV (Fig. 4). This result indicates that acidic motifs are sufficient for CV targeting, in the absence of any other motif. However the distance of the acidic domain from the transmembrane domain seems important because the direct fusion of the motif to the CsA reporter protein without a spacer sequence prevents CV targeting (data not shown).
Proteins destined to the CV are sorted in intracellular compartments
As mentioned above, targeting of CsA-Rh50 to the CV is very efficient, but a small portion of the chimeric protein is present at the cell surface. This offered us the possibility of following more precisely the intracellular transport of the CsA-Rh50 chimera. For this, cells were first incubated with the anti-CsA antibody for 5 minutes at 4°C, washed and then warmed up for different periods of time to allow intracellular transport to occur. To monitor the amount of CsA-Rh50 left on the cell surface after internalization and to identify the plasma membrane unambiguously, cells were then transferred back at 4°C and incubated prior to fixation or permeabilization with an anti-mouse secondary antibody coupled to Alexa Fluor 647 (blue). Cells were then fixed, permeabilized to gain access to internalized chimeras and incubated with an anti-mouse secondary antibody coupled to Alexa Fluor 568 (red) to detect the presence of internalized CsA-Rh50 proteins. In addition, to characterize the CV, cells were colabeled with anti-Rh50 (green). Note that this antiserum only recognized the endogenous Rh50 protein but not the CsA-Rh50 chimera (pFL759) (data not shown).
The CsA-Rh50 chimera present at the cell surface was rapidly internalized and accumulated in punctuate intracellular structures as early as five minutes after warming up the cells (Fig. 5). After 60 minutes of internalization, in addition to these initial endosomal structures, CsA-Rh50 localized on the CV network as observed by colabeling with anti-Rh50 (Fig. 5) and anti-calmodulin antibodies (data not shown). Only very minor amounts of the CsA-Rh50 chimera colocalized with p80-positive endosomal vacuoles even after 2 hours of internalization (Fig. 6A). Note that the CV compartment was not observed upon staining with the anti-CsA antibody (Fig. 6A) because p80 endosomal vacuoles and the CV are not usually in the same cellular region (Ravanel et al., 2001). In Fig. 6A, we specifically selected pictures appropriate for the observation of p80 endosomal vacuoles but usually not for the detection of the CV network. Thus, the punctuate structures containing internalized CsA-Rh50 are distinct from p80-positive endosomal vacuoles and represent distinct endosomal structures. Using the same experimental procedure, no endocytosis was detected when cells expressed a transmembrane CsA protein with a short cytoplasmic domain (data not shown) (Ravanel et al., 2001).
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). After 5 minutes of internalization CsA-Rh50 did not colocalize with golvesin (C)-GFP (Fig. 6B). Even after longer internalization times, CsA-Rh50 was still excluded from the Golgi apparatus (data not shown). Together these results indicate that CsA-Rh50 mislocalized at the cell surface is constantly retrieved from the surface back to the CV. This transport involves transit through an endosomal compartment distinct from p80 endosomal vacuoles and from the Golgi complex. However, confocal microscopy studies with antibodies to known lysosomal proteins did not convincingly allow us to further establish the nature of this compartment.
To gain further insights concerning the site where sorting to the CV occurs, we analyzed the transport of CsA-Rh50 proteins where sorting motifs have been mutated. CsA-Rh50 (pFL759) carries a putative diaromatic signal and an acidic signal, and internalization experiments were repeated with cells that express constructs exhibiting only one potential sorting motif (see Fig. 3). Mutation of the FW signal to alanine residues (pFL775) did not affect transport of the chimera to the CV (Fig. 7A) indicating that the acidic cluster is sufficient for the sorting of Rh50 to the CV. By contrast, mutation of the acidic cluster to alanine (pFL778) resulted in a loss of CV targeting (Fig. 7B). However it is noticeable that this mutated chimeric protein still gained access to a punctuate endosomal compartment with an internalization rate comparable to pFL759 and pFL775 (data not shown). Apparently, transport of pFL778 to the endosomes still proceeded, but a further step of intracellular sorting was abrogated in this mutant. This suggests that the acidic motif is recognized in an intracellular compartment, and further supports the notion that transport from the cell surface to the CV occurs through at least one endosomal compartment and not by a direct exchange between the plasma membrane and the CV.
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AP-1-subunit of the AP-1 clathrin adaptor complex as previously reported for Ent3p (Duncan et al., 2003). To test this hypothesis, we performed yeast two-hybrid interaction assays between the -ear domain of AP-1 and Ent3p or Rh50 C-terminal domain (Fig. 8). Both Ent3 and Rh50 proteins interacted with the -ear domain of AP-1 in this assay whereas no interaction was detected with another subunit of AP-1, µ1, involved in the recognition of tyrosine-based sorting signals. A mutation of one acidic cluster to alanine residues led to a reduction of the interaction between the -ear domain and Rh50 suggesting that the two acidic clusters of Rh50 acted synergistically in the optimal recruitment of . Recently the crystal structure of the human 1-ear domain has revealed a cluster of basic residues required for the recruitment of accessory proteins, -synergin and Rabaptin 5 (Nogi et al., 2002). To test whether this cluster was also involved in the binding of Rh50 C-terminal domain, residues R871 and K873 in Dictyostelium -ear (equivalent of human 1-ear residues R795 and K797) were mutated to glutamine. As observed in Fig. 8, both mutations inhibited the interaction with both Rh50 and Ent3p proteins in this yeast two-hybrid assay. Note that the interaction between GGA (another -ear-containing protein) and Rh50 was not tested here because BLAST searches failed to detect any GGA proteins in Dictyostelium (data not shown). Thus it seems likely that AP-1 coated vesicles are implicated in the transport of Rh50 to the CV, by virtue of a direct interaction between the Rh50 cytosolic acidic motifs and the basic residues of the -AP1 ear domain.
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Rh50 acidic clusters and the AP-1 vesicular coat
Acidic clusters have been previously shown to function as sorting signals in different transport pathways, namely in endosomes to TGN, endosomes to multivesicular bodies, and plasma membrane to endosomes (reviewed by Bonifacino and Traub, 2003). It is not clear how one family of signals can direct proteins to different intracellular compartments. The amino acid sequence flanking the acidic clusters could be an important factor. For instance, the retrieval of the prohormone-processing enzyme furin from endosomes to the TGN depends on the phosphorylation of the CKII sites close to the acidic signal (Jones et al., 1995). Furthermore these signals and their flanking regions can be recognized by different sets of molecules. Some acidic signals specifically interact with a protein termed PACS-1 (Wan et al., 1998), which is essential for the endosome to TGN transport pathway, whereas the yeast Ent3p acidic-phenylalanine signal interacts with -ear containing proteins, GGA and -adaptin (Duncan et al., 2003).
Here we demonstrate that both acidic clusters of Rh50 can weakly interact with -adaptin as revealed by a yeast two-hybrid protein-protein interaction assay. The requirement for the presence of two acidic clusters to detect a significant interaction with -adaptin presumably reflects the weak affinity of each individual motif for -adaptin in vitro. This weak affinity could be sufficient in vivo because the presence of only one signal allows the transport of CsA-Rh50 to the CV. Despite several attempts, we failed to detect a direct interaction between AP-1 and the acidic signals in vivo (data not shown). This is not so surprising because the interaction between targeting signals and adaptor proteins is very transient and involves only a very small fraction of the protein at any given time. Such interactions have always proved difficult to detect even for the well-characterized tyrosine-based signals (Bonifacino and Traub, 2003). To our knowledge, Rh50 is the first cargo protein whose transport may be dependent upon the direct interaction of the AP-1 -ear domain and acidic signals. However we cannot exclude the fact that individual acidic signals are interacting with another protein such as PACS-1 (though Dictyostelium presents no proteins homologous to PACS-1), which would mediate the interaction with AP-1 and participate in the targeting of Rh50 to the CV.
The interaction between -adaptin and Rh50 is consistent with our previous study that demonstrated the role of AP-1 coat in the biogenesis of the CV (Lefkir et al., 2003). AP-1-coated vesicles could play an essential role in intracellular transport of membrane proteins to the CV, accounting for the fact that the loss of AP-1 results in the complete loss of a functional CV. In addition to AP-1, other factors might participate to the transport of Rh50 to the CV network. Further experiments will be required to fully address the role of AP-1 in the delivery of Rh50 to the CV network.
A new transport pathway between the plasma membrane and the CV
Previous studies have ruled out a direct exchange of membrane components between the CV and the plasma membrane during the transient fusion of the bladder with the plasma membrane, which allows water expulsion in hypo-osmotic conditions. Indeed components of the plasma membrane labeled with the fluorescent dye Cy3.5 failed to accumulate in the bladder during its periods of discharge and refilling (Gabriel et al., 1999). This result is in agreement with the observation that several plasma membrane proteins such as p25 and p80 are excluded from the CV (Ravanel et al., 2001) (data not shown). An indirect exchange between the plasma membrane and the CV via endosomes has also been excluded using a similar approach. After 10 minutes of internalization of cell surface proteins labeled with Cy3.5 into endosomes, no labeling could be detected in the CV (Gabriel et al., 1999). Although no longer internalization times were tested in these experiments owing to technical limitations, the fact that p80, a marker of early and late endosomes, is excluded from the CV might appear to strengthen the notion that there is no communication between endocytic and CV compartments. However the existence of proteins present in both the CV and endosomes suggests a partial, difficult-to-study link between these two compartments.
We noted that a small amount of CsA-Rh50 is present at the cell surface rather than in the CV. This gave us the opportunity to further explore the possibility of the exchange of membrane constituents between the plasma membrane and the CV. The most striking result is that the surface pool of CsA-Rh50 protein is constitutively internalized and rapidly concentrated in a perinuclear compartment distinct from the Golgi apparatus. This compartment does not contain detectable amounts of p80 protein, a marker of early and late endosomal vacuoles (Ravanel et al., 2001). Therefore, in Dictyostelium cells newly internalized CsA-Rh50 is rapidly sequestered in endosomes distinct from p80-positive endosomal vacuoles.
What is the fate of proteins localized to this compartment? Confocal microscopy studies revealed that the chimera is detected in the CV only after 1 hour of internalization, the signal getting significantly stronger after 2 hours. Therefore the rapid accumulation of internalized proteins in endosomes strongly suggests that CsA-Rh50 proteins first transit through these endosomes, before reaching the CV. The sorting of CsA-Rh50 to the CV is likely to take place in the endosomes, where internalized CsA-Rh50 has a rather long residency time. This sorting process probably depends on the presence of acidic clusters, because mutations of the acidic clusters to alanine residues do not abolish access of CsA-Rh50 to these punctuate endosomal structures. Based on our yeast two-hybrid experiments, we propose that AP-1 could participate to the selection of Rh50 in this compartment. Whether this compartment is only an intermediate compartment between the plasma membrane and the CV or a more ubiquitous endosomal structure where sorting to different organelles could take place is currently under investigation. Interestingly, the CV network resembles the tubulo-vesicular structure seen in acid-secreting gastric parietal cells. In these cells, clathrin and AP-1 are thought to be involved in protein targeting to the tubulo-vesicular network (reviewed by Yao and Forte, 2003). Therefore, a role for AP-1 in many vesicular trafficking events seems to be supported in various organisms and is a genuine feature of the AP-1 clathrin-adaptor complex.
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) was grown at 22°C in HL5 medium and subcultured twice a week. Cells were not allowed to reach a density of more than 2x106 cells/ml. Antibodies used were a rabbit antiserum to the C-terminal cytosolic tail of Rh50 (controls showed that pFL759 was not recognized by this antiserum) (Benghezal et al., 2001), a rabbit antiserum to calmodulin (a kind gift from T. Soldati, University of Geneva, Switzerland) (Ulbricht and Soldati, 1999) and a mouse monoclonal antibody to CsA (mAb 41-71-21) (Bertholdt et al., 1985). Note that in our cell culture conditions (low density), endogenous CsA was not expressed and therefore this anti-CsA antibody only recognized CsA-Rh50 chimeras. To allow simultaneous labeling with an anti-p80 antibody and another mouse monoclonal antibody, the anti-p80 H161 antibody (Ravanel et al., 2001) was directly coupled to Alexa Fluor 488 (Molecular Probes, Leiden, The Netherlands) according to the manufacturer's instructions.
Plasmids and cell transfection
The cDNA encoding the contact site A (CsA) protein with the transmembrane domain of the integral protein P29F8 (Barth et al., 1994) in the expression vector pDCEV4 was kindly provided by G. Gerisch (Max-Planck-Institut für Biochemie, Martinsried, Germany). The sequence of the cytoplasmic domain of the CsA-Stop construct is KTRVSQNSG. To obtain fusion proteins of this short cytoplasmic domain with Rh50 (CsA-Rh50), a BstXI-XhoI fragment was replaced with the DNA encoding the 91 C-terminal residues of Rh50. All mutations and truncations described in Fig. 3 were produced by PCR using pairs of oligonucleotides containing either BstXI or XhoI sites at the 5' and 3' regions respectively. PCR fragments were digested by BstXI and XhoI, cloned into BstXI/XhoI sites of pDCEV4-CsA-Rh50 and sequenced (Genome express, Grenoble, France). Plasmids were linearized by ScaI and transfected in Dictyostelium by electroporation as described (Cornillon et al., 2000).
The golvesin (C)-GFP expression vector (Schneider et al., 2000) was a kind gift from A. Mueller-Taubenberger (Ludwig Maximillians University Munich, Muenchen, Germany). The neomycin-resistance gene present in this vector was substituted with the blasticidin-S-resistance gene. After electroporation, cells expressing golvesin (C)-GFP were selected with 10 µg/ml blasticin S.
Immunofluorescence microscopy
For immunofluorescence analysis, cells were applied on a glass coverslip for 2 hours, then fixed with 4% paraformaldehyde for 30 minutes, washed and permeabilized with methanol at –20°C for 2 minutes. Cells were incubated with the indicated antibodies for 30 minutes, and then stained with the corresponding fluorescent secondary antibodies for 30 minutes. Cells were observed by laser-scanning confocal microscopy (Zeiss LSM 510). For calmodulin staining, cells were directly fixed by incubation with methanol at –20°C for 10 minutes and treated as described above.
For internalization studies, cells were first incubated with the anti-CsA antibody for 5 minutes at 4°C, washed and then warmed up in culture medium for different periods of time. At each time point, cells were returned to 4°C and incubated with an anti-mouse secondary antibody coupled to Alexa Fluor 647 for 5 minutes. Cells were then fixed with 4% paraformaldehyde for 30 minutes, washed, permeabilized with –20°C methanol for 2 minutes, and incubated with an anti-mouse secondary antibody coupled to Alexa Fluor 568.
Testing protein-protein interaction by two-hybrid assay
Two-hydrid assays were carried out using the Matchmaker LexA two-hybrid system (Clontech Laboratories, Palo Alto, CA). The DNA sequence encoding the AP-1 -ear domain (residues 664 to 895) was fused to the DNA binding protein LexA in the expression vector pEG202. Point mutations in -ear (R871Q and K873Q) were made by PCR, sequenced and cloned into pEG202. To test the ability of the Rh50 cytoplasmic domain to interact with the AP-1 -ear domain, the DNA sequence encoding the last 91 residues of Rh50 was fused to the B42 activation domain in the vector pJG4-5 containing the inducible GAL1 promoter. For specificity controls, Ent3p (full-length) and p80 (51 amino acid residues of the cytoplasmic loop of p80, ELEK to CLHA) DNA sequences were cloned into pJG4-5 and cotransfected in the reporter yeast with the AP-1 -ear-containing construct. Transformed EGY48 yeasts expressing the plasmid p80-LacZ were tested for their ability to grow on selective plates (synthetic complete medium without leucine and containing galactose) and give a blue color on Xgal-supplemented plates. For more accurate results, the ß-galactosidase activity was determined in liquid conditions for a fixed number of yeast cells using O-nitrophenyl-ß-D galactopyranoside as a substrate. The background activity in cells expressing only the B42 activation domain fusion protein was subtracted.
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