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Vol. 16, Issue 8, 3642-3658, August 2005
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* Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, D-39118 Magdeburg, Germany;
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854
Submitted January 28, 2005;
Revised April 19, 2005;
Accepted May 23, 2005
Monitoring Editor: Howard Riezman
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
Additionally, this complex machinery is connected with the cortical actin cytoskeleton, which may support vesicle formation by different means (Qualmann et al., 2000; Qualmann and Kessels, 2002). Syndapins, a family of proteins also referred to as PACSINs, were suggested to be molecular links between membrane trafficking and cortical cytoskeleton dynamics, because syndapins associated with both dynamin and N-WASP, a potent stimulator of the Arp2/3 complex actin polymerization machinery (Qualmann et al., 1999; Kessels and Qualmann, 2004). Both functions are supported by in vivo data, the dynamin-binding syndapin Src homology 3 (SH3) domain is a potent inhibitor of receptor-mediated endocytosis and overexpression of full-length syndapin induces numerous filopodia, actin-rich protrusions of the plasma membrane, in an Arp2/3-complex dependent manner (Qualmann and Kelly, 2000). Local actin polymerization has been observed at endocytic sites (Merrifield et al., 2002) and coincides with the transient recruitment of the Arp2/3 complex and N-WASP (Merrifield et al., 2004). Dominant-negative experiments and in vivo reconstitutions strongly suggested that the role of N-WASP in endocytosis involves the syndapin association and that syndapin-mediated actin polymerization supports clathrin-coated vesicle detachment and movement away from the plasma membrane (Kessels and Qualmann, 2002).
After endocytic internalization, most membrane proteins and lipids return to the plasma membrane after passing through one or several endosomal compartments, such as the sorting endosome and the endocytic recycling compartment (ERC) (Maxfield and McGraw, 2004). Transport from the ERC, which is a long-lived compartment, also requires the formation of transport vesicles. The machinery needed for the formation of such vesicles or tubules from the ERC is only beginning to be elucidated. The ERC may use mechanisms similar to those used for the formation of transport vesicles from other organelles. In particular, clathrin and dynamin can be found on endosomes (Stoorvogel et al., 1996; van Dam et al., 2002), implying that they may serve functions there similar to their well studied roles at the plasma membrane.
Proteins that specifically regulate transport from the ERC include the small GTPase Rab11 (Chen et al., 1998; Ren et al., 1998) and EHD1 (Lin et al., 2001). In mammals, EHD1 belongs to a family of four highly related proteins (EHD1–4) (Mintz et al., 1999; Pohl et al., 2000) that share a very similar domain structure with an N-terminal P-loop–containing nucleotide binding domain, a central region predicted to form coiled coils and a C-terminal Eps15-homology (EH) domain. The Caenorhabditis elegans homologue RME-1 was identified in a genetic screen for mutants defective in the receptor-mediated endocytosis of yolk protein. Closer examination of rme-1 mutants indicated that a block in endocytic recycling was the primary defect (Grant et al., 2001). The expression of a mRme-1/EHD1 G429R mutant, designed by analogy to a dominant C. elegans mutant (G459R), resulted in the redistribution of the ERC in mammalian cells and slowed the recycling of transferrin receptors (Lin et al., 2001), of major histocompatibility complex class I molecules (Caplan et al., 2002), the cystic fibrosis transmembrane conductance regulator (Picciano et al., 2003) and 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (Park et al., 2004) back to the cell surface. Depletion of mRme-1/EHD1 by RNA interference confirmed the role of mRme-1/EHD1 in mammalian cell protein recycling to the cell surface (Naslavsky et al., 2004). Here, we identify EHD proteins as differential interaction partners of syndapins. We examine the molecular requirements for these interactions as well as means of regulation. Furthermore, we demonstrate that the ability of mRme-1/EHD1 to form protein complexes through EH domain/asparagine proline phenylalanine (NPF) interactions is crucial for the recycling process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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SH3, and the short and long splice variants of syndapin II and for a maltose-binding protein (MBP)-fusion protein of syndapin I SH3 were described previously (Qualmann et al., 1999; Qualmann and Kelly, 2000). Further GST-fusion proteins, such as syndapin I SH3 domain (aa 378–441), syndapin II SH3 domain (aa 422–488), and the NPF motif regions of syndapin I (aa 336–386), syndapin II-s (aa 338–387), and syndapin II-l (aa 338–428) were generated by PCR on appropriate templates and cloning into pGEX vectors. Mammalian expression constructs encoding green fluorescent protein (GFP) or FLAG-tagged syndapins and fragments thereof were generated by subcloning into pEGFP-C1 (BD Biosciences Clontech, Palo Alto, CA) and pCMV-Tag2B (Stratagene, La Jolla, CA), respectively. Xpress-tagged syndapin II-l and syndapin II-l SH3 were described in Qualmann and Kelly (2000). For yeast two-hybrid analyses, syndapin I full-length and syndapin I SH3 were inserted into the pGBTK7 vector. The plasmid encoding mitochondria-targeted full-length syndapin I was described in Kessels and Qualmann (2002). Point mutations in the NPF motifs of syndapin I and II-l, resulting in the exchange of phenylalanine to valine, were introduced by site-directed mutagenesis via PCR. Syndapin III was amplified by PCR from a rat retina library (Seidenbecher et al., 2004) and cloned into EcoRI-SalI sites of pGEX-5X-1 (Amersham Biosciences, Piscataway, NJ).
GST-mRme-1/EHD1 full-length and GST-EH domain (aa 408–534) expression constructs were created by PCR with full-length mRme-1/EHD1 as template and cloned into pGEX-2T. Full-length FLAG-mRme-1/EHD1, EGFP-mRme-1/EHD1 wild-type, G65R, and G429R constructs were described previously (Lin et al., 2001). The W485A mutation was engineered into the full-length EGFP-mRme-1/EHD1 construct using appropriate mutation primers according to manufacturer's instructions (QuikChange XL; Stratagene).
Mouse EHD2, EHD3, and EHD4 were amplified from expressed sequence tag clones (RZPD) IMAGp998G059315Q3, IMAGp998H2413737Q3, and IMAGp998A058521Q3, respectively, and subcloned into pCMV-Tag2 (Stratagene), pEGFP-C (BD Biosciences Clontech), and pGEX (Amersham Biosciences) vectors. The partial yeast two-hybrid clone of EHD3 (aa 367–535) was subcloned into pGEX-4T2. The EH domain of mRme-1/EHD1 was subcloned into pEGFP-C1 (BD Biosciences Clontech) and into a derivative of our mitochondrial targeting vector (Kessels and Qualmann, 2002), Mito-GFP vector, that was generated by inserting the green fluorescent protein (GFP) sequence between the FLAG-tag and the multiple cloning site. Constructs encoding for GST-Eps15 EH (encompassing all three EH domains) and GST-Intersectin EH (encompassing EH domains a and b) were from Brian Kay (Argonne National Laboratory, Chicago, IL). The bacterial expression vector permitting the expression of C. elegans Rme-1 as a GST-fusion protein has been described previously (Lee et al., 2005). All PCR-amplified DNAs were verified for integrity by sequencing.
GST- and MBP-fusion proteins were expressed and purified as described previously (Qualmann et al., 1999; Kessels et al., 2000).
Antibodies
Rabbit anti-syndapin I and anti-syndapin II antibodies, guinea pig anti-syndapin II antibodies (antisera 2521, 2704, and P339) and anti-GST antibodies were described previously (Qualmann et al., 1999; Qualmann and Kelly, 2000).
Polyclonal anti-syndapin I antibodies were raised in guinea pig (Pineda Antikörper-Service, Berlin, Germany) against a purified GST-fusion protein of amino acid residues 1–382 of rat syndapin I. Antibodies were affinity purified on GST and MBP-syndapin I (aa 1–382) blotted to nitrocellulose membranes.
Polyclonal anti-mRme/EHD antibodies were raised in rabbit (Covance Immunological Services, Princeton, NJ) against a synthetic peptide (CADLPPHLVPPSKRRHE), corresponding to the extreme C terminus of the mouse Rme-1/EHD1 protein, conjugated to keyhole limpet hemocyanin. Antisera raised against this peptide were affinity purified against immobilized antigenic peptide (Sulfolink kit) according to the manufacturer's instructions (Pierce Chemical, Rockford, IL). Due to high sequence conservation, the antibodies also recognize the EHD protein isoforms 3 and 4 relatively well (Figure S1) and work for several species, such as mouse, rat, and human.
Monoclonal anti-FLAG (M2) and anti-synaptophysin antibodies were from Sigma (St. Louis, MO), monoclonal anti-GFP (B34) antibodies were from BAbCO (Richmond, CA), monoclonal anti-TGN38 antibodies were from BD Transduction Laboratories (Lexington, KY), and monoclonal anti-Xpress antibodies were from Invitrogen (Carlsbad, CA).
Secondary antibodies used include goat anti-mouse peroxidase and goat anti-rabbit peroxidase from Dianova (Hamburg, Germany); goat anti-guinea pig peroxidase and fluorescein isothiocyanate (FITC) goat anti-guinea pig from MP Biomedicals (Irvine, CA); and Alexa Fluor 488 goat anti-mouse, Alexa Fluor 568 goat anti-mouse, Alexa Fluor 568 goat anti-rabbit, Alexa Fluor 647 goat anti-mouse, and Alexa Fluor 647 goat anti-rabbit from Molecular Probes (Eugene, OR).
Blot Overlay and Coprecipitation Assays
Coprecipitations of rat brain proteins with immobilized GST-fusion proteins were performed with rat brain extracts containing 10 mM (Figure 1E) or 150 mM NaCl according to Qualmann and Kelly (2000) and Qualmann et al. (2004). Bound proteins were separated on SDS-PAGE, blotted to nitrocellulose, and probed with anti-syndapin I (2704) or anti-EHD antibodies.
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; Kessels and Qualmann, 2002).
Blot overlay experiments were performed with recombinant GST-mRme-1/EHD1 EH domain and GST on extracts from HEK293 cells transfected with GFP or GFP-syndapin II-l according to the procedure described previously (Kessels and Qualmann, 2002).
Tissue Fractionation
Tissue fractionation was carried out essentially as described in Qualmann et al. (2004). In brief, rat brain cortices and hippocampi were homogenized in 320 mM sucrose, 5 mM HEPES, pH 7.4. The homogenate was centrifuged at 1000 x g to remove cell debris and nuclei, and the resulting low-speed supernatant (S1) was recentrifuged at 12,000 x g for 15 min. While the obtained supernatant S2 was further fractionated by centrifugation at 100,000 x g for 1 h, yielding a microsomal pellet (PM) and a ultrahigh-speed supernatant (SM), the resulting pellet P2 (crude membrane fraction) was loaded onto a sucrose step gradient (0.85/1.0/1.2 M). Myelin, light membranes, and synaptosomes were isolated at the different sucrose interfaces. The mitochondria- and heavy-membrane–containing fraction (mitochondria) was obtained as pellet. Synaptosomal membranes were isolated after the osmotic lysis of synaptosomes in 1 mM Tris/HCl, pH 8.1, for 30 min by centrifugation at 33,000 x g for 30 min. The samples were analyzed by SDS-PAGE and immunoblotting.
Coimmunoprecipitation from Rat Brain Extract
Rat brain extracts were prepared as described previously (Qualmann et al., 2004) and precleared by incubation with protein A agarose (Santa Cruz Biotechnology, Santa Cruz, CA) in 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Equal amounts of affinity-purified guinea pig anti-syndapin I antibodies or unrelated guinea pig immunoglobulins G (IgG) were immobilized onto protein A agarose in 5% BSA in PBS. After several washes with immunoprecipitation (IP) buffer (10 mM HEPES, 1 mM EGTA, 0.1 mM MgCl2, 50 mM NaCl, 1% Triton X-100, pH 7.4), 1 mg of protein of rat brain extract was added. Beads were incubated for 3 h at 4°C, washed with IP buffer, and eluted with SDS sample buffer. Eluates and supernatants were separated on SDS-PAGE and analyzed by immunoblotting.
Preparation of Cell Extracts and Coimmunoprecipitation
For immunoprecipitations of epitope-tagged proteins, HEK293 cells were transiently cotransfected with different GFP- and FLAG-tagged constructs, grown for additional 40 h, harvested, and lysed in IP buffer containing 100 mM NaCl. Insoluble material was removed by centrifugation.
Anti-FLAG antibodies or unrelated mouse IgG (Santa Cruz Biotechnology) were coupled to protein G-Sepharose beads (Amersham Biosciences) at 4°C for 5 h. In some experiments, antibodies were subsequently covalently linked to beads by dimethyl-pimelimidate-dihydrochloride (Fluka Chemical, Ronkonkoma, NY) for 45 min at room temperature (Schneider et al., 1982), and HEK293 cell lysates were preincubated at room temperature for 10 min with AMP, ADP
S, ATP
S, or ATP (all from Sigma) and MgCl2 (final concentration each 5 mM). Lysates were incubated with the antibody-coated beads overnight at 4°C. The beads were washed with IP buffer, and eluted immunoprecipitates were subjected to immunoblot analysis.
Yeast Two-Hybrid Analyses
Y2H-screenings were performed using the GAL4-based Matchmaker yeast two-hybrid system 3 (BD Biosciences Clontech) with full-length syndapin I as a bait. Both a rat brain cDNA library and a pretransformed mouse brain library (BD Biosciences Clontech) were screened. Prey plasmids were isolated, retransformed into yeast, and mated with yeast strains transformed with BD-syndapin I, BD-syndapin I SH3, and with the pGBTK7 vector encoding for the BD domain alone. The diploids were subsequently assayed for the activation of reporter genes, as described in Kessels and Qualmann (2002).
Cell Culture and Immunofluorescence Microscopy
HEK293, HeLa, and COS-7 cells were maintained in DMEM containing 10% fetal bovine serum. Primary hippocampal cultures were prepared and grown as described previously (Kessels et al., 2001; Qualmann et al., 2004).
Primary neurons 15 days in vitro (DIV) were transfected using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were processed for immunofluorescence as described previously (Qualmann et al., 2004). For the evaluation of the specificity and affinity of the guinea pig anti-syndapin I antibodies, COS-7 cells were transiently transfected using the FuGENE reagent (Roche Diagnostics). For mitochondrial targeting experiments, HeLa cells were transfected with Polyfect reagent (QIAGEN, Valencia, CA).
Cells were fixed and processed for immunofluorescence according to Kessels et al. (2001). For mitochondrial staining, cells were incubated with Mito-Tracker Red CMXRos (Molecular Probes) as described previously (Kessels and Qualmann, 2002). Images were recorded digitally using a Leica TCS SP2 AOBS confocal microscope or a Leica DMRD fluorescence microscope and a Zeiss Axioplan 2 microscope both equipped with a charge-coupled device camera 2.1.1 from Diagnostic Instruments (Sterling Heights, MI) and processed in MetaVue and Adobe Photoshop.
Transferrin Internalization Assay
COS-7 cells were subjected to transferrin uptake assays 48 h after transfection as described previously (Qualmann and Kelly, 2000; Kessels and Qualmann, 2002). COS-7 cells treated with BioPorter according to the manufacturer's instructions (Gene Therapy Systems, San Diego, CA) to introduce immunoreagents were subjected to endocytosis assays 5 h after start of treatment. To be able to see putative dose responses, cells were split in three categories differing in the extent of uptake of immunoreagent by the BioPorter method as described by Kessels and Qualmann (2002). The categories can easily be distinguished as follows: weak (gray values as measured in MetaVue 60–119), medium (gray values 120–230), and strong uptake (>90% of cytosol area in saturation, i.e., gray value 255) of the respective immunoreagent. The gray value average for coverslip background was 53. All images were taken at an exposure time of 1000 ms. The percentages of transfected cells showing no detectable uptake of transferrin, significantly reduced transferrin signals, and normal levels of internalized transferrin and standard deviations were calculated by scoring and counting cells in independent experiments as described previously (Qualmann and Kelly, 2000; Kessels et al., 2001; Kessels and Qualmann, 2002). The categories used to evaluate endocytosis can be classified as follows: block, no endosomal structures labeled by transferrin are observable at 1000-ms exposure time; reduced, endosomal signal observable but 
70% intensity of untransfected cells average. This objectivity of our category definitions allows several independent investigators to work in parallel.
Transferrin Recycling Assay
HeLa cells were transfected using Polyfect transfection reagent (QIAGEN) for 24 h. After a 30-min starvation in DMEM containing 20 mM HEPES and 0.2% BSA, pH 7.5, cells were pulsed with 20 µg/ml transferrin-Alexa Fluor 488 or -Alexa Fluor 568 (Molecular Probes) for 10 min at 37°C. Cells were washed twice with ice-cold PBS (containing 0.5 mM CaCl2 and 0.5 mM MgCl2) and labeled transferrin was chased for 20 min at 37°C in DMEM with 10% fetal calf serum, 2 mM glutamine, and 1 mg/ml unlabeled holo-transferrin (Sigma). The cells were washed with CaCl2- and MgCl2-containing PBS and subsequently fixed and processed as described above. All transfected cells on several coverslips were identified by GFP fluorescence or by immunolabeling and scored for remaining transferrin fluorescence. Only cells with a readily detectable transferrin signal were considered positive for transferrin and counted. This corresponded to maximal gray values for endosomal areas (of 5 pixels in diameter) of <40%, as measured via the information palette of Adobe Photoshop. In most cases, endosomal signal intensity maxima reached values close to 0%, which look white (saturation of signal) for the eye (exposure times 2000 ms for transferrin-Alexa Fluor 568 and 5000 ms for transferrin-Alexa Fluor 488). This stringent definition of recycling defects led to a value of 
50% of recycling-inhibited cells upon overexpression of the mRme/EHD1 G429R mutant shown to introduce a prominent block in recycling (Lin et al., 2001) and to 20–25% of affected cells in control experiments of different kinds. The objectivity of our category settings allowed us to perform the scoring of the cells by as many as three different investigators. Their results were averaged and subjected to statistical significance calculations using Fisher's protected least significant difference (PLSD) and Dunnett's tests (StatView program; SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Because syndapins contain several additional protein–protein interaction modules such as predicted N-terminal coiled coil domains, NPF motifs, and a variety of consensus sites for different kinases, syndapins may act as multidomain scaffolding proteins integrating several different cellular functions. The three syndapin isoforms expressed in mammals show a highly conserved domain structure (Figure 1A). At the amino acid level, conservation between the isoforms is also very high. This is especially true for the C-terminal SH3 domains (Kessels and Qualmann, 2004). An exception is the NPF motif-containing regions N-terminal of the SH3 domains. They differ both in amino acid composition and in number of the NPF motifs. NPF motifs are absent in syndapin III (Figure 1A).
In yeast two-hybrid screens with a syndapin I full-length bait (Figure 1A) using different brain cDNA libraries, we obtained cDNA clones that encoded fragments of EHD3, a member of the EHD protein family implicated in endocytic recycling. The fragments encompassed part of the putative coiled coil domain and the C-terminal EH domain (Figure 1B). Analyses of reporter gene activities in yeast strains coexpressing the syndapin I bait yielded robust growth on drop-out plates of different stringency (Figure 1C) and clear positive signals in -galactosidase assays (Figure 1D). The interaction was independent of the syndapin SH3 domain (Figure 1, C and D). We verified the interaction by affinity purification experiments. Both a C-terminal fragment and full-length EHD3 fused to GST specifically precipitated endogenous syndapin I from rat brain cytosol (Figure 1E).
EH domains are present in a variety of proteins involved in membrane trafficking processes. Thus, we asked whether the syndapin interaction is restricted to EHD3, or whether it also occurs with multiple members of the EHD protein family or whether the interaction is promiscuous for many EH domain-containing proteins (Figure 1F). Syndapin I was successfully coprecipitated from rat brain extracts by immobilized GST-fusion proteins of full-length mouse Rme-1/EHD1 and of the only member of the EHD protein family in C. elegans, ceRme-1 (Figure 1F). The EH domain of mouse Rme-1/EHD1 alone was sufficient for the interaction. Interestingly, the EH domains of both Eps15 and intersectin failed to interact with syndapin I, although all three EH domains of Eps15 and the two EH domains of intersectin were used (Figure 1F). Syndapin I thus exhibits specificity for EH domains found in members of the EHD protein family.
EHD Proteins Are Differential Binding Partners of the Syndapin Family
We next evaluated whether the identified interaction with EHD proteins is a specialty of syndapin I. Coprecipitation analyses demonstrated that syndapin I, syndapin II-s, and syndapin II-l but not syndapin III interact with endogenous EHD proteins. The strongest interaction was observed with syndapin II-l (Figure 2, A and B).
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SH3, but not by the SH3 domain alone (Figure 2D). The interaction was further narrowed down to the non-SH3 part encompassing the NPF motifs (Sdp I-NPF) (Figure 2, C and D). Similar results were obtained with the more ubiquitously expressed syndapin II isoform. The interaction was independent of the syndapin II SH3 domain. The NPF regions alone of both syndapin II-s and syndapin II-l were sufficient to precipitate EHD proteins (Figure 2, E and F). Similar data were obtained using GFP-mRme-1/EHD1 overexpressed in HEK293 cells (our unpublished data). The sequence conservation of the region of syndapin proteins encompassing the NPF motifs is relatively high between syndapin I and II (Figure 2G). Interestingly, the additional amino acid insert present in the syndapin II-l splice variant contains a third NPF motif. In contrast, syndapin III lacks NPFs (Figures 1A and 2G).
The observed differential associations of syndapins (Figure 2B) prompted us to examine putative specificities for different EHD proteins as well. We therefore cloned all four mouse EHD isoforms, which are highly conserved and despite the phylogenetic distance also show a high homology with the C. elegans ortholog (Figure 2H). Immobilized GST-fusion proteins of syndapin I (Figure 2J), syndapin II-s (Figure 2K), and syndapin II-l (Figure 2L), but not GST alone (our unpublished data) efficiently precipitated GFP-mRme-1/EHD1, GFP-EHD3, and GFP-EHD4 from HEK293 cell extracts. The amount of GFP-EHD2 within the precipitates of all three syndapins was low (Figures 2, J–L). Thus, syndapins strongly bound only to three of the four EHD proteins, the relatively widely distributed mRme-1/EHD1, which our phylogenetic analyses suggest to be closest to the EHD ancestor (our unpublished data), EHD3, an isoform highly expressed in brain but also occurring in other tissues and the heart-enriched EHD4 (Pohl et al., 2000; Galperin et al., 2002). EHD1, 3, and 4 show a good expression overlap with the syndapin isoforms I and II and their splice variants. In contrast, the isoform not effectively bound by syndapins, EHD2, is highly expressed in muscle tissues (Pohl et al., 2000) where expression of syndapin I and II is very low and not detectable, respectively (Qualmann and Kelly, 2000).
Syndapin NPF Motifs Are Crucial for the Association with the EH Domain of EHD Proteins and the Interaction Is Direct
Our in vitro analyses clearly demonstrated that regions of syndapins that contain NPF motifs are sufficient for the interaction with EHD proteins (Figure 2). To directly prove that the NPF motifs are crucial and to identify which of the several NPF motifs is responsible for the interaction, we mutated all NPF motifs in syndapin I and II individually and in combination to asparagine proline valine (NPV) and expressed all constructs in HEK293 cells. Immobilized GST-fusion protein of the EH domain of mRme-1/EHD1 only precipitated wild-type syndapin I. Mutating the two NPFs of syndapin I individually or in combination abolished the interaction (Figure 3, A and B), indicating that the NPF motifs are crucial for the interaction with mRme-1/EHD1 and that both NPFs are required in combination.
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For syndapin II-l, we observed that mutating one of the three NPFs still permitted some association with the EH domain of mRme-1/EHD1. The F421V mutation showed the least and the F366V mutation showed the strongest negative effect (Figure 3, C and D). Mutating two NPF motifs abolished the interaction with the mRme-1/EHD1 EH domain completely irrespective of the combination of mutations. Consistently, the syndapin II-l (F366V, F409V, F421V) triple mutant showed no interaction (Figure 3, C and D).
The presence of at least two syndapin NPF motifs is also important for EH domain binding in the full-length context of EHD. Wild-type syndapin II-l was almost quantitatively precipitated from the cell extracts by full-length mRme-1/EHD1 but binding was almost completely abolished in mutants with a single NPF to NPV amino acid exchange. Only for syndapin II-l F421V some binding to full-length mRme-1/EHD1 was observed (Figure 3, E and F). Thus, the interaction is highly dependent on the presence of multiple NPF motifs within syndapins.
Our analyses of the binding interfaces showed that both NPF motifs and EH domains are required and sufficient for the interaction. Because these motifs are known to interact, this suggested that the interaction between syndapins and EHD proteins may be direct. To prove this, we overlaid lysates from HEK293 cells containing GFP-syndapin II-l or GFP as a control (Figure 3G) with a GST-EH domain probe. In the lane with the overexpressed GFP-syndapin II-l, the GST-EH probe readily detected a 90-kDa band that was absent in the GFP control lane (Figure 3I) and corresponded well with the band of GFP-syndapin II-l obtained by anti-GFP immunoblotting (Figure 3G). Additionally, several weaker bands were revealed by the GST-EH domain at 60, 120, and 170 kDa in the lane with GFP-syndapin II-l. Because these were absent from the HEK293 cell lysate containing GFP alone and were not detected by anti-GFP antibodies, they cannot represent endogenous proteins or GFP fusion proteins, but instead are likely to represent syndapin II-l proteins released from GFP by proteolysis. The molecular masses that we observed fit with syndapin II-l monomers (60 kDa), syndapin II-l dimers (120 kDa), and syndapin II-l trimers (170 kDa). In contrast, GST alone used as a control probe did not yield any signal (Figure 3H). These blot overlay studies formally demonstrate a specific and direct interaction of syndapins with the EH domain of EHD proteins.
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50 kDa) in brain extracts (Figure 4A, lanes 1 and 2) and also detected recombinant MBP-syndapin I SH3 (lane 3) with high-affinity. In immunofluorescence examinations, they detected FLAG-tagged mito-syndapin I expressed in COS-7 cells (Figure 4D) as specifically and efficiently as anti-FLAG antibodies applied in parallel (Figure 4B). In primary hippocampal neurons kept in culture for 2 days, syndapin I was localized to the soma and to neurites and displayed accumulations at actin-rich growth cones (Figure 4G, arrow). The distribution of anti-EHD immunosignal was very similar (Figure 4E) and overlapped well with that of syndapin I (Figure 4F).
As described previously (Qualmann et al., 1999), in mature neurons, syndapin I adopts a more punctate synaptic distribution in addition to the neuritic localization, as seen best in low-density cultures at lower magnification (Figure 4J). Anti-EHD immunosignals (Figure 4H) also were obtained throughout neurites and showed, in part, strong accumulations at puncta that were always also immunopositive for syndapin (Figure 4J, arrowheads). These were often at sites where neurites contacted one another (Figure 4, H–J). At higher magnification, we observed a very exact spatial overlap of anti-syndapin I and anti-EHD immunosignals at sites that are likely to represent synapses (Figure 4, K–M).
Analyses of endogenous syndapin II in cultured neurons were precluded by low syndapin II expression in brain (Qualmann and Kelly, 2000). We thus slightly overexpressed syndapin II-l (Figure 4P) together with GFP-mRme-1/EHD1 (Figure 4N) in primary hippocampal neurons. The merge of both images demonstrates the observed very high spatial overlap of both proteins in dot-like structures of a single transfected cell that often protrude from the neurites and may represent synaptic sites (Figure 4O). Both proteins also colocalized in cell somata (our unpublished data). Because both proteins were coexpressed in single, isolated cells within the neuronal cultures and showed a colocalization within these cells, it can be firmly concluded that the two proteins coexist in puncta representing the same synaptic compartment, i.e., it can be excluded that the two proteins are separated by the synaptic cleft between a pre- and a postsynaptic neuron. Because these puncta were observed in several neurites originating from the cell, we can furthermore conclude that syndapin and EHD1 coexist in the dendritic compartment and that the puncta thus represent postsynapses. A putative presynaptic localization of EHD1 remains to be confirmed by immunoelectron microscopy.
In nonneuronal cells, such as HeLa cells, FLAG-mRme-1/EHD1 (Figure 4Q) and GFP-syndapin II-l (Figure 4S) also showed a strong spatial overlap, especially at structures that looked tubular and vesicular (Figure 4R).
Our immunofluorescence data suggest that syndapins and EHD proteins both localize to several cellular membrane compartments. Biochemical subcellular fractionation analyses and preparations of different membrane and synaptic compartments from brain homogenates indeed revealed that EHD proteins are especially abundant in membrane-associated fractions but low in fractions that contain more soluble proteins (Figure 5A). Strong anti-EHD immunosignals were seen in the crude membrane fraction P2, whereas in the corresponding supernatant S2, the signal was very low. The fractionation of S2 showed that this material mostly reflected microsomal pellet material. When the crude membrane fraction P2 was analyzed further, it became obvious that EHD immunoreactivity was enriched strongest in the light membrane-enriched fraction and was especially low in the fraction containing mitochondria and heavy membranes. Our preparations of synaptosomes and synaptic membranes were positive for anti-EHD immunosignals (Figure 5A).
Syndapin I showed a distribution very similar to that of EHD proteins (Figure 5B). Both the material obtained in S2 and in the crude membrane fraction P2 fractionated further in a manner identical to that of EHD proteins. As with the EHD immunoreactivity, syndapin I accumulated in the microsomal pellet rather than in the high speed cytosol. Also similar to the anti-EHD pattern, syndapin I was readily detected in light membranes and synaptosomes but absent from the heavy membrane- and mitochondria-containing fraction (Figure 5B, mitochondria). Together with EHD, syndapin I was detectable in the preparation of synaptic membranes (Figure 5B). The anti-syndapin II immunosignal was extremely weak (our unpublished data). The low immunosignals obtained are in line with the fact that the expression level of the more ubiquitously expressed syndapin II isoform is low in brain (Qualmann and Kelly, 2000).
The similarity of the subcellular fractionation pattern of EHD proteins and syndapin I is highlighted best by comparison with other proteins, such as the synaptic vesicle protein synaptophysin (Figure 5C) and the trans-Golgi marker TGN38 (Figure 5D). Synaptophysin, in contrast to syndapin and EHD, cannot be detected in the SM fraction but for example strongly accumulates in synaptosomes and in the preparation of synaptic membranes (Figure 5C). TGN38 shows a very different pattern. Very little TGN38 was found in the synaptosome fraction. Also, TGN38 is abundant in S2 and relatively scarce in P2. No TGN38 immunoreactivity was detected in the synaptic membrane preparation (Figure 5D).
Syndapin I and Syndapin II-l Interact with EHD Proteins In Vivo
To address whether syndapins and EHD proteins interact in vivo, we coexpressed GFP-syndapin II-l (Figure 6, A–D) or GFP-syndapin I (Figure S2) together with FLAG-tagged mRme-1/EHD1 in HEK293 cells and subjected the lysates to coimmunoprecipitations. In both experimental series, FLAG-mRme-1/EHD1 was effectively and specifically immunoprecipitated by anti-FLAG antibodies (Figures 6B and S2), whereas in the control experiments FLAG-mRme-1/EHD1 remained in the supernatants (Figures 6A and S2). GFP-syndapin II-l was specifically coimmunoprecipitated with FLAG-mRme-1/EHD1 by anti-FLAG antibodies but not by the control IgGs (Figure 6D). GFP was not coimmunoprecipitated, demonstrating that indeed the syndapin part interacts with mRme-1/EHD1 (our unpublished data; compare Figure 10). Consistent with our in vitro data, GFP-syndapin I was also specifically coimmunoprecipitated with FLAG-mRme-1/EHD1 (Figure S2).
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The Strength of the EH Domain-mediated Interaction of mRme-1/EHD1 with Syndapin II Is Sufficient for a Recruitment of Syndapin to Intracellular Membranes In Vivo
Because EHD proteins were found to be predominantly membrane-associated (Figure 5), a property that is consistent with its subcellular localization (Figure 4) and proposed function (Lin et al., 2001; Caplan et al., 2002; Naslavsky et al., 2004), we next assayed whether EHD proteins would be able to recruit cytosolic syndapins to intracellular membranes. We constructed a mitochondrially targeted EH domain of mRme-1/EHD1 fused to GFP. This fusion protein was effectively targeted to the outer mitochondrial membranes of HeLa cells (Figure 7A), as shown by Mito-Tracker staining (Figure 7C). When full-length syndapin II-l was coexpressed, it adopted a mitochondrial localization pattern (Figure 7F) overlapping exactly with mito-GFP-EH–coated mitochondrial membranes (Figure 7D). Also syndapin II-l SH3 was very effectively recruited by mitochondrial membrane-associated EH domains of mRme-1/EHD1 (Figure 7, J–L). In both cases, mitochondria decorated with the EH domain became clustered, whereas this was not observed in cells overexpressing the mitochondrially targeted EH domain of mRme-1/EHD1 alone (Figure 7A). The successful in vivo reconstitution of the protein interaction at cellular membranes was based on a specific interaction of the EH domain of mRme-1/EHD1 with syndapin II-l. In control cells expressing only mito-GFP (Figure 7G), no such recruitment of syndapin II-l (Figure 7I) or syndapin II-l SH3 (our unpublished data) to mitochondria was observed, but both syndapin fusion proteins remained relatively evenly distributed within the cytosol. Consistent with our in vitro studies, these findings demonstrate that the NPF-containing N-terminal part of syndapin II-l is sufficient for the interaction in vivo and that the SH3 domain is not required. These experiments furthermore highlight the strength of the EHD/syndapin II interaction at membranes in vivo.
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) that were later corroborated by a generalized reduction in mRme1/EHD1 protein levels via RNA interference (RNAi) (Naslavsky et al., 2004). Because we found that syndapins and EHD proteins associate tightly in vivo, we sought to determine whether syndapins function with EHD proteins in receptor recycling. One method we applied to this problem was a general interference with the presence and/or function of syndapin complexes by the introduction of antibodies. The introduction of anti-syndapin antibodies raised against the C terminus of syndapin I, including the SH3 domain region into COS-7 cells, however, resulted in a strong dose-dependent interference with the uptake of transferrin (Figure 8), precluding analysis of transferrin recycling. At medium levels of immunoreagent uptake, about one-half of the cells were clearly affected. At higher levels, 50% of the cells showed a complete block in transferrin uptake. The effects were specific for the anti-syndapin antibodies because neither the uptake reagent (BioPorter) alone nor fluorescently labeled control IgGs caused any significant effects. Also the preimmune antibodies corresponding to the inhibiting anti-syndapin immunoreagent did not result in any inhibitory effects (Figure 8). These results indicate that syndapins are important for endocytic vesicle formation and that acute interferences with syndapin functions by the introduction of anti-syndapin antibodies cannot be compensated for by affected cells.
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; Kessels and Qualmann, 2002), these domains must not be involved in vesicle formation at the plasma membrane. Therefore, we turned to dominant-negative experiments to interfere specifically with the assembly of syndapin/EHD complexes. If such a particular syndapin interaction is required for recycling but not for internalization, then overexpression of the domains from either syndapins or an EHD protein mediating that interaction would be expected to inhibit recycling and trap endocytic cargo in the endosomal recycling compartment.
To be able to test this hypothesis, we first characterized all such reagents for putative defects prior to the recycling step. As already reported for full-length syndapin II-l (Kessels and Qualmann, 2002), overexpression of the NPF region of syndapin II-l and its corresponding NPF-to-NPV triple mutant did not cause any significant uptake defects (Figure S3). Furthermore, no significant endocytic internalization defects were observed upon overexpression of various mRme-1/EHD1 constructs. Only the G429R mutant showed a modest impairment of transferrin uptake, because only 70% of the cells showed wild-type internalization (Figure S3).
Thus, we next assayed receptor recycling using methods similar to those described by Lin et al. (2001). Untransfected cells preloaded with fluorescently labeled transferrin displayed very low levels of intracellular transferrin after 20 min of chase due to efficient transferrin recycling (see untransfected cells in Figure 9, A–C, and E–G). Only 20% of the untransfected cells showed a readily detectable endosomal labeling pattern at this time point (Figure 9D). In contrast, overexpression of the syndapin II-l region encompassing the NPF motifs as a GFP-fusion protein led to a massive impairment of transferrin recycling (44% transferrin positive; p < 0.0001) visible by the strong increase of fluorescent transferrin that remained within transfected cells after chase (Figure 9A). This value is more than twice as high as in untransfected and GFP-expressing control cells (Figure 9D). Overexpression of the corresponding triple NPF to NPV mutant (GFP-NPF***), which was unable to bind to EHD proteins (Figure 3, C–F), did not lead to recycling defects (Figure 9, B and D), indicating that the syndapin NPF motifs within the NPF motif-containing region of the protein are responsible for the observed block in recycling.
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Receptor Recycling Is Impaired by EH Domain Overexpression and Can Be Rescued by Syndapin Co-overexpression
Because the specific interference with EH domain functions by overexpression of its syndapin interaction interface led to impairments in receptor recycling (Figure 9, A–D), we next asked whether interfering with NPF/EH domain interactions from the opposite side of the association interface also would inhibit. Indeed, overexpression of a GFP-tagged EH domain inhibited recycling, as shown by the significantly increased transferrin retention in transfected cells (Figure 9E). Quantitation of the dominant-negative effects revealed that the impairment was almost as strong as the effect caused by the syndapin NPF motifs and highly significant. Forty-one percent of all GFP-EH domain-overexpressing cells showed a readily observable endosomal transferrin fluorescence compared with only 22% in GFP controls (Figure 9H).
We subsequently tried to rescue the GFP-EH domain effect by co-overexpression of full-length, i.e., presumably functional, syndapin. We first assayed whether overexpression of syndapin alone would have any negative effects on recycling of transferrin. Both Xpress- and FLAG-tagged syndapin II-l–overexpressing cells showed a transferrin recycling indistinguishable from that of untransfected or GFP-expressing cells (Figure 9, F and H). On double transfection of FLAG-syndapin II-l and mRme-1/EHD1 EH domain (Figure 9G), we observed a normal recycling, indicating a rescue of the EH domain induced phenotype. Only 28% of all double-transfected cells remained positive for fluorescent transferrin compared with 41% in the experiments with the EH domain alone (Figure 9H). Thus, 66% of the effect caused by overexpression of the EH domain was rescued by coexpression of the mRme-1/EHD1 binding partner syndapin II-l. The values obtained upon our rescue experiments did not significantly differ from control, as revealed by statistical analyses. These data indicate that complexes of syndapins and EHD proteins play an important role in receptor recycling.
EH Domain Interactions with Syndapins Seem to Be Controlled by the Nucleotide-binding Domain of mRme-1/EHD1 In Vitro and In Vivo
Our data show that interfering with NPF/EH domain interactions is sufficient to impair recycling, a phenotype previously attributed to EHD in total (Naslavsky et al., 2004). One of the endocytosis-deficient C. elegans RME-1 mutants carried a mutation in the nucleotide P-loop located within the N terminus of all EHD proteins (Grant et al., 2001), suggesting that either disrupted nucleotide binding leads to recycling impairments independent from EH domain functions or that nucleotide binding influences EH domain functions. We thus tested whether nucleotide binding could modulate EH domain interactions by performing a series of coimmunoprecipitation studies with cell lysates that were preincubated without nucleotide addition, with ATP, with AMP, with ATPS or ADPS (Figure 10, A and B). FLAG-mRme-1/EHD1 was immunoprecipitated equally well under all of these conditions (Figure 10B). GFP-syndapin II-l specifically coimmunoprecipitated with FLAG-mRme-1/EHD1 (Figure 10A). The coimmunoprecipitation was dependent on the syndapin part of the GFP-fusion protein because coexpressed GFP did not coimmunoprecipitate (Figure 10C) with FLAG-mRme1/EHD1 immunoprecipitated by anti-FLAG antibodies (Figure 10D). The amounts of GFP-syndapin II-l coimmunoprecipitated differed clearly under the five conditions applied (Figure 10). The addition of ATP or ATPS supported the interaction but ADPS and especially AMP suppressed it. Comparing several independent experiments, the amounts of GFP-syndapin II-l coimmunoprecipitated upon ATP or ATPS addition were always higher or comparable with those performed under similar conditions without addition of nucleotides, and much higher than those obtained after AMP or ADPS preincubation (Figure 10A). These coimmunoprecipitation experiments, however, did not exclude the possibility that the observed nucleotide effects were indirect or even EHD independent.
To demonstrate that nucleotide binding of mRme-1/EHD1 modulates the EH domain interaction with syndapin, we performed coprecipitation analyses with immobilized syndapin II-l and HEK293 cell lysates containing wild-type GFP-mRme-1/EHD1 and different mutants. GFP-mRme-1/EHD1 G65R represents a mutant in analogy to a dominant Rme-1 mutant isolated in mutational analyses of membrane trafficking in C. elegans (Grant et al., 2001). The G65R exchange is located in the nucleotide binding P-loop. We hypothesized that if the N-terminal nucleotide binding domain indeed influences syndapin's association with the C-terminal EH domain, then silencing the nucleotide binding domain by mutation may interfere with syndapin binding. As a control for interfering with EH domain-dependent syndapin binding, we additionally assayed a mutant of mRme-1/EHD1 with a disrupted EH domain (GFP-mRme-1/EHD1 W485A) that was constructed based on structural data for the Eps15 EH domain 2 (de Beer et al., 2000). Additionally, we included GFP-mRme-1/EHD1 G429R, the mammalian version of a second C. elegans Rme-1 dominant mutant. Overexpression of mRme-1/EHD1 G429R has been observed to block recycling in several recent studies (Lin et al., 2001; Caplan et al., 2002; Picciano et al., 2003; Park et al., 2004). The molecular mechanisms by which the G429R mutation brings about this effect are unknown.
We observed that the G65R mutant was strongly deficient for syndapin binding (Figure 10, E and F). The immunosignal obtained from the eluates was almost as weak as that of the EH domain mutant that served as a negative control and that lacked all specific syndapin binding activity. In contrast, the G429R mutant showed a strong syndapin binding and did not differ significantly from wild-type mRme-1/EHD1 under affinity purification conditions (Figure 10F).
EHD Mutants Defective in Syndapin Binding Do Not Act Dominant Negatively on Receptor Recycling
We have demonstrated that overexpression of the syndapin-binding EH domain of mRme-1/EHD1 alone has a dominant-negative effect on transferrin recycling (Figure 9). Lin et al. (2001) showed that overexpression of the G65R mutant did not cause any impairment. We hypothesized that the lack of G65R-induced recycling defects is due to the observed strongly decreased syndapin binding activity of G65R mutants (Figure 10, H and F). If this hypothesis is correct, then overexpression of the EH domain mutant (W485A) also should lack dominant-negative activity. Indeed, the W485A mutant did not cause an inhibition in recycling (Figure 10, I and K) compared with overexpression of the G65R mutant (Figure 10, H and K) or to untransfected cells (Figure 10K). In contrast, about one-half of all G429R-overexpressing cells showed significant remaining transferrin (Figure 10, J and K). Overexpression of a wild-type, i.e., presumably fully functional, mRme-1/EHD1, only caused extremely mild, if any, effects on transferrin recycling (Figure 10G). Quantitation of the effects elicited showed that only 30% of the mRme-1/EHD1–overexpressing cells were transferrin positive (Figure 10K). This is consistent with a previously reported lack of inhibition of transferrin recycling (Lin et al., 2001). Also, recycling of a major histocompatibility complexes component and AMPA receptors was rather enhanced than inhibited (Caplan et al., 2002; Park et al., 2004). In summary, our analysis of the different syndapin and mRme-1/EHD1 point mutants and deletion constructs revealed the critical importance of EH domain/NPF interactions for the receptor recycling process, because only proteins capable to interfere with these interactions disrupt this cellular process.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Our new data indicate that syndapins are parts of a more general mechanism in membrane trafficking, because they interact with EHD proteins that function in exit from the endosomal recycling compartment. Syndapin I, syndapin II-s, and syndapin II-l but not syndapin III interact with EHD proteins. The interaction is based on associations of the EH domain of EHD proteins with syndapin NPF motifs, as we demonstrated by yeast two-hybrid analysis, affinity purifications, and in vivo reconstitution of EHD/syndapin complexes. The interaction is direct, as proven by blot overlay studies. These findings are consistent with data Xu et al. (2004) reported while this manuscript was in preparation. They showed by biochemical methods that a C-terminal part of syndapin II that encompasses the region, which contains the NPF motifs, interacts with the EH domain of EHD1 in vitro (Xu et al., 2004). Our mutational analyses prove that the interaction indeed requires NPF motifs. Consistently, syndapin III, which does not exhibit NPF motifs, did not bind to EHD proteins. Additionally, our mutational analyses revealed that NPF 364-6 is the most important EHD-binding motif in syndapin II. This region is alternatively spliced and is absent in the short versions of syndapin II (Qualmann and Kelly, 2000). EHD proteins therefore not only represent the first differential in vivo binding partners for the three syndapin isoforms but also can differentiate between the long and short splice variants of syndapin II.
All syndapin NPF motifs except for one in syndapin I are preceded by one or even two (syndapin II NPF 364-6) serines. The positions and numbers of serines may correlate with the strength of interaction, as proposed for other EH domains (Salcini et al., 1997). Our data demonstrate that syndapin EH domain interactions are specific for EHD proteins. E
