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• W2011126063 abstract "The sensory and motor neuron-derived factor (SMDF) is a type III neuregulin that regulates development and proliferation of Schwann cells. Although SMDF has been shown to be a type II protein, the molecular determinants of membrane biogenesis, insertion, and topology remain elusive. Here we used heterologous expression of a yellow fluorescent protein-SMDF fusion protein along with a stepwise deletion strategy to show that the apolar/uncharged segment (Ile76-Val100) acts as an internal, uncleaved membrane insertion signal that defines the topology of the protein. Unexpectedly, removal of the transmembrane segment (TM) did not eliminate completely membrane association of C-terminal fragments. TM-deleted fusion proteins, bearing the amino acid segment (Ser283-Glu296) located downstream to the epidermal growth factor-like motif, strongly interacted with plasma membrane fractions. However, synthetic peptides patterned after this segment did not insert into artificial lipid vesicles, suggesting that membrane interaction of the SMDF C terminus may be the result of a post-translational modification. Subcellular localization studies demonstrated that the 40-kDa form, but not the 83-kDa form, of SMDF was segregated into lipid rafts. Deletion of the N-terminal TM did not affect the interaction of the protein with these lipid microdomains. In contrast, association with membrane rafts was abolished completely by truncation of the protein C terminus. Collectively, these findings are consistent with a topological model for SMDF in which the protein associates with the plasma membrane through both the TM and the C-terminal end domains resembling the topology of other type III neuregulins. The TM defines its characteristic type II membrane topology, whereas the C terminus is a newly recognized anchoring motif that determines its compartmentalization into lipid rafts. The differential localization of the 40- and 83-kDa forms of the neuregulin into rafts and non-raft domains implies a central role in the protein biological activity. The sensory and motor neuron-derived factor (SMDF) is a type III neuregulin that regulates development and proliferation of Schwann cells. Although SMDF has been shown to be a type II protein, the molecular determinants of membrane biogenesis, insertion, and topology remain elusive. Here we used heterologous expression of a yellow fluorescent protein-SMDF fusion protein along with a stepwise deletion strategy to show that the apolar/uncharged segment (Ile76-Val100) acts as an internal, uncleaved membrane insertion signal that defines the topology of the protein. Unexpectedly, removal of the transmembrane segment (TM) did not eliminate completely membrane association of C-terminal fragments. TM-deleted fusion proteins, bearing the amino acid segment (Ser283-Glu296) located downstream to the epidermal growth factor-like motif, strongly interacted with plasma membrane fractions. However, synthetic peptides patterned after this segment did not insert into artificial lipid vesicles, suggesting that membrane interaction of the SMDF C terminus may be the result of a post-translational modification. Subcellular localization studies demonstrated that the 40-kDa form, but not the 83-kDa form, of SMDF was segregated into lipid rafts. Deletion of the N-terminal TM did not affect the interaction of the protein with these lipid microdomains. In contrast, association with membrane rafts was abolished completely by truncation of the protein C terminus. Collectively, these findings are consistent with a topological model for SMDF in which the protein associates with the plasma membrane through both the TM and the C-terminal end domains resembling the topology of other type III neuregulins. The TM defines its characteristic type II membrane topology, whereas the C terminus is a newly recognized anchoring motif that determines its compartmentalization into lipid rafts. The differential localization of the 40- and 83-kDa forms of the neuregulin into rafts and non-raft domains implies a central role in the protein biological activity. Neuregulin-1 gene (NRG-1) 1The abbreviations used are: NRG-1neuregulin 1 gene familyDMPCdimyristoylphosphatidylcholineEGFepidermal growth factorFBSfetal bovine serumGFPgreen fluorescent proteinGSTglutathione S-transferasePBSphosphate-buffered salineSMDFsensory and motor neuron-derived factorTMtransmembrane segmentYFPyellow variant of green fluorescent protein products comprise a group of cell-cell signaling proteins that act as ligands for the same family of ErbB receptor tyrosine kinases (1Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Members of the neuregulin family are expressed in several tissues including nervous system and heart, where they are implicated in diverse cellular processes, such as cell proliferation, differentiation, and survival (2Buonano A. Fisbach G.D. Curr. Opin. Neurobiol. 2001; 11: 287-296Crossref PubMed Scopus (433) Google Scholar). neuregulin 1 gene family dimyristoylphosphatidylcholine epidermal growth factor fetal bovine serum green fluorescent protein glutathione S-transferase phosphate-buffered saline sensory and motor neuron-derived factor transmembrane segment yellow variant of green fluorescent protein The NRG-1 family comprises at least 14 different isoforms, each containing an EGF-like motif that is essential for receptor recognition. NGR-1 isoforms have been classified according to the structure of their N-terminal region. Thus, type I and type II isoforms (which include acetylcholine receptor-inducing activity and glial growth factor II) contain an Ig-like domain, whereas type III presents a cysteine-rich domain (2Buonano A. Fisbach G.D. Curr. Opin. Neurobiol. 2001; 11: 287-296Crossref PubMed Scopus (433) Google Scholar). In addition, most of the family members are membrane proteins that suffer a proteolytic cleavage to relieve a signaling domain essential for their functional activity (6Wang J.Y. Miller S.J. Falls D. J. Biol. Chem. 2001; 276: 2841-2851Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Among the NGR-1 family, the type III subfamily plays a role in the signaling that coordinates the interaction of peripheral nervous system with Schwann cells and muscles (3Wolpowitz D. Mason T.B.A. Dietrich P. Mendelsohn M. Talmage D.A. Role L.W. Neuron. 2000; 25: 79-91Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). The first type III NRG reported was SMDF, a neuregulin highly expressed in motor neurons and dorsal root ganglion neurons (4Ho W.H. Armanini M.P. Nuijens A. Philips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). At variance with other type III NRGs, SMDF has a type β3 EGF-like motif and is characterized by the absence of a second TM segment downstream of the EGF-like domain. Amino acid sequence and hydropathy analysis does not reveal the presence of a true TM segment (4Ho W.H. Armanini M.P. Nuijens A. Philips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). There is, however, an apolar/uncharged stretch of amino acids at the N-terminal domain that might act as a TM segment (4Ho W.H. Armanini M.P. Nuijens A. Philips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 5Schroering A. Carey J. J. Biol. Chem. 1998; 273: 30643-30650Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Indeed, SMDF has been proposed to be a group II membrane protein with a single TM segment that locates the N terminus to the cytosol, and exposes the EGF-like motif to the extracellular milieu (5Schroering A. Carey J. J. Biol. Chem. 1998; 273: 30643-30650Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The mitogenic activity of SMDF on neighboring cells may require the proteolytic release of the EGF-like motif, although the intact protein may also serve as a membrane-attached signal (5Schroering A. Carey J. J. Biol. Chem. 1998; 273: 30643-30650Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 6Wang J.Y. Miller S.J. Falls D. J. Biol. Chem. 2001; 276: 2841-2851Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In support of this notion, it has been reported that some members of the NRG-1 family are segregated into lipid rafts, membrane microdomains considered platforms for the selective delivery of proteins to specialized locations in neurons and epithelial cells (7Frenzel K.E. Falls D.L. J. Neurochem. 2001; 77: 1-12Crossref PubMed Scopus (37) Google Scholar). In this paper, we study the molecular determinants of SMDF topology. In addition, we investigate whether SMDF biogenesis involves compartmentalization in lipid rafts. The experimental approach uses an YFP-SMDF fusion protein that had the fluorescent protein fused to the N terminus of the NRG. We show that the apolar/uncharged region located at the N-terminal domain of the protein acts as an uncleaved, internal membrane insertion signal sequence, and report that the protein is additionally anchored to the plasma membrane through its C terminus, probably by an acylation-like post-translational modification. We also report that SMDF partitions into lipid rafts. Interestingly, only the 40 kDa form of SMDF was detected in rafts domains. Removal of the TM did not alter partitioning of the protein into membrane rafts. In contrast, segregation into these lipid microdomains was prevented by deletion of the C-terminal anchoring site. Taken together, our findings are consistent with a membrane topology model for this protein which resembles that proposed for other members of the type III NRG-1 gene subfamily, namely the protein is anchored to the cell membrane through two sites. Human dorsal root ganglion (Normal-NCI CGAP PNS1) cDNA library, primers, Dulbecco's modified Eagle's medium, fetal bovine serum, antibiotics, Optiprep, and pcDNA3.1 were obtained from Invitrogen. Pfu turbo DNA polymerase and BL21 codon plus Escherichia coli strain were from Stratagene. Horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG secondary antibodies, monoclonal anti-phosphotyrosine clone PT-66, and phosphatidylinositol-specific phospholipase C were obtained from Sigma. pEYFP-C1 and the anti-GFP antibody (Living Colors Aequorea victoria peptide antibody) were from CLONTECH. Streptavidin Alexa 546 was from Molecular Probes. Anti-HRGβ3 antibody was from Santa Cruz. Biotinylated anti-rabbit IgG was from Vector Laboratories. ECL Plus was fromAmersham Biosciences. SMDF-encoding cDNA was amplified from a human DRG library by PCR using Pfu turbo DNA polymerase and the EcoRI restriction site containing primers 5SMDF: CCT TGG AAT TCG ACG ATT TAT and 3SMDF: GTT AAT GTT CGA ATT CGA CAG GC. The amplified fragment was gel purified, EcoRI digested, and cloned into the pcDNA 3.1(+) plasmid. Direct and inverse oriented sequences were selected by restriction analysis and verified by automatic sequencing. To clone SMDF into the pEYFP-C1 plasmid, PCR amplification was performed using as template pcDNA-SMDF with the 5′-EcoRI restriction site containing primer 5.4SMDF: CAG GCC GAA TTC TGG AGG TGA GCC G and the 3′-SalI restriction site containing primer 3.1SMDF: GAT GCA GCA AGT CGA CAG CAG CAC C. The amplified product was digested with EcoRI and SalI and cloned directionally into the pEYFP-C1 plasmid. For the cloning in pGEX-4T1 a similar procedure was used, except the 5′-EcoRI restriction site containing primer 5.2SMDF: GCC TTC TTC TGA ATT CGA GCC GAT G was used. To obtain the pEYFP-SMDFΔ108–296 construct, pEYFP-SMDF was truncated at the BglII site. A similar strategy, but using the EspI site, was used to produce pEYFP-SMDFΔ1–64 and pEYFP-SMDFΔ65–296. All other deletions were obtained by one-step inverse PCR with the proofreading Pfu turbo DNA polymerase and two restriction digestions. Briefly, externally oriented primers with PAC1 unique restriction sites were used to amplify the whole construct except the region to be deleted. PCR products were digested (in the amplification buffer) with 20 units of DpnI for 2 h at 37 °C to remove the methylated plasmid templates, subsequently purified, and digested with PAC1. Purified digestions were re-ligated, transformed in DH5α, and selected for kanamycin resistance. More than 95% of the colonies contained the designed deletions as verified by restriction digestion and automatic sequencing. COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were plated on 2-cm2 wells at 250,000 cells/well. 20 h later, cells were transfected with 1 μg of plasmid DNA using LipofectAMINE 2000, following the manufacturer's recommendations. To prepare conditioned mediums, cells were serum starved 24 h post-transfection, and the medium was collected 48–72 later. MCF-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Crude plasma membranes were essentially prepared as described by Schroering and Carey (5Schroering A. Carey J. J. Biol. Chem. 1998; 273: 30643-30650Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) with minor modifications. Briefly, transfected cells were washed with phosphate-buffered saline (PBS) and lysed with buffer A (2 mm MgCl2, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 20 mm Hepes pH 7.4). Cell lysates were centrifuged at 6,000 × g, 4 °C for 10 min to prepare low speed pellet (Pi, containing the plasma membranes) and supernatants (S), which contain the soluble proteins and the remaining membranes. Low speed pellets (Pi) were washed in buffer B (buffer A + 1 mNaCl), incubated for 15 min at 4 °C, centrifuged, and washed with buffer A for desalting. In some experiments, pellets were washed with 50 mm Na2CO3, pH 12. Final pellets (P) and supernatants (S), prepared from equivalent amounts of cells, were mixed with β-mercaptoethanol containing SDS sample buffer, heated, and separated by SDS-PAGE. Proteins were electrotransferred onto nitrocellulose membranes, blocked with 10% fat-free skim milk in TBS and incubated with the anti-GFP antibody or the anti-HRGβ3 polyclonal antibody in blocking buffer (1:2,000) for 1 h. Membranes were washed with TBS-Tween (0.3%), incubated with the horseradish peroxidase-conjugated anti-rabbit IgG, and developed with the ECL Plus system. SMDF-induced tyrosine phosphorylation of ErbB receptors was carried out as described by Ho et al. (4Ho W.H. Armanini M.P. Nuijens A. Philips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Briefly, MCF-7 cells were grown until ≥80% confluence in 24-well plates. Thereafter, cells were serum starved for 2 h and incubated with serum-free, conditioned medium or recombinant SMDF for 15 min at room temperature as indicated. The medium was removed, and cells were harvested with 100 μl of β-mercaptoethanol containing SDS sample buffer. Whole cell extracts were heat denatured, separated by SDS-PAGE, and analyzed by immunoblotting with the monoclonal anti-phosphotyrosine antibody (1:1,000). Cells were seeded on poly-l-lysine-coated coverslips at 50–200 × 103 cells/well and transfected as indicated previously. The medium was removed, and cells were washed three times for 5 min using PBS with 10% fetal bovine serum (PBS/FBS) at room temperature. Thereafter, cells were incubated with anti-HRGβ3 (1:200) in PBS/FBS at room temperature for 20 min, washed with PBS/FBS three times for 8 min, and incubated with biotinylated anti-rabbit IgG (1:200) in PBS/FBS at room temperature for 20 min. Washes were repeated, and cells were incubated with streptavidin Alexa 546 (1:200) in PBS/FBS, washed in the same conditions, and prepared for in vivo fluorescence microscopy in a Zeiss Axiophot microscope using a 20× objective. For confocal microscopy studies, cells were transfected, washed with PBS, and observed in vivo with a confocal microscope Olympus Fluoview 300 using a 478 nm argon laser. SMDF-encoding cDNA was cloned in pGEX-4T-1 and transformed in the BL21 codon plus strain. Bacterial cells were grown at 28 °C until reaching 0.6–0.8 A (600 nm). Thereafter, cells were induced with isopropyl-1-thio-β-d-galactopyranoside at 0.3 mm for 4 h and pelleted. The pellet was resuspended in PBS and 5 mm dithiothreitol and sonicated. Triton-X100 was added to reach 1% and centrifuged at 10.000 × g for 10 min. GST-SMDF was purified from the supernatant with GSH-agarose beads. After extensive washing, GST-SMDF was eluted from the beads with 10 mm GSH in 5 mm dithiothreitol, 150 mm NaCl, 20 mm Tris-HCl, pH 8.8. Protein concentration was calculated with the method of Bradford (8Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217317) Google Scholar). Analysis of detergent-insoluble complexes in flotation gradients was performed following standard protocols (7Frenzel K.E. Falls D.L. J. Neurochem. 2001; 77: 1-12Crossref PubMed Scopus (37) Google Scholar, 12Mañes S. Mira E. Gómez-Moutón C. Lacalle R.A. Séller P. Labrador J.P. Martı́nez-A C. EMBO J. 1999; 18: 6211-6220Crossref PubMed Scopus (278) Google Scholar, 13Lafont F. Verkade P. Galli T. Wimmer C. Louvard D. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3734-3738Crossref PubMed Scopus (208) Google Scholar). Briefly, about 2.5 × 106 transiently transfected cells were cooled on ice, washed with PBS, and scraped off in buffer C (150 mm NaCl, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 0.5% Triton X-100, 20 mm Hepes pH 7.4), passed 10 times through a 29-gauge needle, extracted at 4 °C for 30 min, and brought to 35% Optiprep. One ml of the extract was sequentially overlaid with 8 ml of 30% Optiprep in 0.5× buffer C and 400 μl of buffer C in an SW41 tube. After centrifugation (178,000 × g at 4 °C for 4 h), 10 1-ml fractions were collected from the top to the bottom of the gradient; 200 μl of each fraction was precipitated with trichloroacetic acid, pH-neutralized, and analyzed by immunoblotting using the anti-HRGβ3 and anti-GFP antibodies. For differential scanning calorimetry large multilamellar vesicles made from synthetic dimyristoylphosphatidylcholine (DMPC) or DMPC plus cholesterol at 10% molar percentage were used. Dried lipids films obtained from chloroform solutions were suspended in 100 mm NaCl, 10 mmHepes pH 7.4 to give a final concentration of 1 mm in terms of lipid phosphorus. The resuspended lipids were kept for 90 min above their phase transition temperatures and vortexed. The resulting liposomes were stored overnight at 4 °C to assure a complete hydration of the sample prior to the differential scanning calorimetry measurements. Thermograms were recorded on a high resolution Microcal MC-2 differential scanning microcalorimeter, equipped with a DA-2 digital interface and data collection, as described (14Encinar J.A. Fernández A.M. Gavilanes F. Albar J.P. Ferragut J.A. Gonzalez-Ros J.M. Biophys. J. 1996; 71: 1313-1323Abstract Full Text PDF PubMed Scopus (13) Google Scholar). Lipid dispersions containing the peptides at different molar ratios, and the corresponding buffer in the reference cell were thermally equilibrated in the microcalorimeter, at ≈10 °C for 45 min, before heat was applied. Differences in the heat capacity between the sample and the reference cell were obtained by raising the temperature at a constant rate of 45 °C/h. Transition temperatures and enthalpies were calculated by fitting the observed transitions to a single van't Hoff component. To gain insights into SMDF membrane insertion, topology, and localization, we designed a YFP-SMDF fusion protein by cloning the YFP at the N terminus of the neuregulin. For this purpose, the SMDF cDNA was amplified from a human DRG cDNA library and cloned into the pEYFP-C1 vector. The subcelullar location and functional activity of both SMDF and YFP-SMDF were compared upon their transient heterologous expression in COS-7 cells. Immunocytochemical studies using the antibody anti-HGRβ3, raised against the C terminus of the neuregulin, show that both SMDF and YFP-SMDF were highly expressed in the cell surface, as depicted by the labeling of nonpermeabilized COS-7 cells (Fig. 1 A, a and e). A pattern of green fluorescence was present in YFP-SMDF- and YFP-transfected cells (Fig. 1 A, b and d). As expected, anti-HGR-β3 immunoreactivity was absent in YFP-expressing cells (Fig. 1 Ac). These results suggest that YFP-SMDF is highly expressed in COS-7 cells and that a fraction of this protein is located in the plasma membrane, with the ectodomain exposed on the cell surface. SMDF is released into the extracellular medium, presumably by proteolytic processing (4Ho W.H. Armanini M.P. Nuijens A. Philips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 5Schroering A. Carey J. J. Biol. Chem. 1998; 273: 30643-30650Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Membrane release of neuregulins is an important step for their functional activity because it may be required for the efficient interaction with their receptors in target cells (1Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar,2Buonano A. Fisbach G.D. Curr. Opin. Neurobiol. 2001; 11: 287-296Crossref PubMed Scopus (433) Google Scholar). Hence, we investigated whether the YFP-SMDF fusion protein was properly processed and released to the extracellular milieu. Immunoblot analysis of culture media from SMDF- and YFP-SMDF-transfected cells disclosed a diffuse, faint band of ≈50 KDa which was recognized by the anti-HRGβ3 antibody (Fig. 1 B, lanes 2 and 3). Notice that the molecular mass of this band is smaller than the major, 83-kDa SMDF immunoreactive form observed in cellular lysates (Fig. 1 B, lane 1). Because the antibody recognized the C terminus, these observations indicate that the 50 kDa band corresponds to the C-terminal ectodomain of the neuregulin which contains the EGF-like motif. Thus, the presence of the YFP protein at the N terminus did not affect the proteolytic processing of the neuregulin. We next evaluated the functional activity of both SMDF and YFP-SMDF. For this purpose, we used the epithelial cell line MCF-7, which expresses ErbB receptors (4Ho W.H. Armanini M.P. Nuijens A. Philips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 15Osheroff P.L. Tsai S.P. Chiang N.Y. King K.L., Li, R. Lewis G.D. Wong K. Henzel W. Mather J. Growth Factors. 1999; 16: 241-253Crossref PubMed Scopus (9) Google Scholar). Functional activity of the neuregulin was determined as the extent of ErbB tyrosine autophosphorylation induced by conditioned media obtained from COS-7 cells cultures expressing either SMDF or YFP-SMDF. As illustrated in Fig. 1 C, both types of conditioned medium stimulated the tyrosine phosphorylation of a 185-kDa protein in MCF-7 cells. This band corresponds to the ErbB receptors (4Ho W.H. Armanini M.P. Nuijens A. Philips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The extent of receptor phosphorylation decreased as a function of the dilution factor of the conditioned media from both SMDF- and YFP-SMDF-transfected cells (Fig. 1 C). This functional activity was specific because it was not detected in conditioned media from cells transfected with pEYFP-C1 or an inverted SMDF construct, but it was observed when purified recombinant GST-SMDF was used (Fig. 1 C). At variance with other neuregulins (16Li Q. Loeb J.A. J. Biol. Chem. 2001; 276: 38068-38075Abstract Full Text Full Text PDF PubMed Google Scholar), receptor-induced phosphorylation by both SMDF and YFP-SMDF ectodomains was reversible, as evidenced by the decline of ErbB phosphorylation 2 h after removal of conditioned media (Fig. 1 D). Taken together, these observations demonstrate that the YFP-SMDF fusion protein reproduces all properties of the wild type III β3 neuregulin. SMDF has been proposed to belong to the group II of membrane proteins, having the N terminus facing the cytosol and the C terminus extracellularly exposed (5Schroering A. Carey J. J. Biol. Chem. 1998; 273: 30643-30650Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The molecular determinants of this topology are still elusive. We addressed this question by evaluating the subcellular location of stepwise deletion mutants carried out on the YFP-SMDF fusion protein (Fig. 2). Because all truncations were carried out on the YFP-SMDF, we will refer to the truncated proteins as the segment deleted, indicating the first and last amino acids removed. First, we questioned the role of the potential TM domain located in the N terminus segment Ile76-Val100. For this task, we designed two truncated proteins, namely Δ65–296 and Δ108–296, which lack all of the C-terminal ectodomain from residue Glu65 and Ser108, respectively. As illustrated in Fig. 3, confocal microscopy images show that deletion mutant Δ108–296 (Fig. 3 b) exhibited a cell surface labeling pattern identical to that of YFP-SMDF (Fig. 3 a). Similar results were obtained with the deletion mutant Δ1–64, where the N-terminal domain was deleted up to residue Ala64. In contrast, the N-terminal Δ65–296 and the C-terminal Δ1–101 fragments displayed a distribution of fluorescence similar to that of YFP protein. Thus, removal of the amino acid stretch Ile76-Val100 disrupts the insertion of the protein in the plasma membrane. This finding was substantiated further by biochemical and immunological analysis of subcellular fractions derived from YFP-SMDF full-length and deletion mutants transfected in COS-7 cells. Cell cultures were harvested, lysed, and enriched plasma membrane fractions were pelleted by centrifugation. To prevent contamination with peripheral proteins, plasma membrane fractions were washed with 1.0 m NaCl and/or with pH 12. Under these conditions, YFP was found in the supernatant fraction (Fig 4, P1 and S1), whereas YPF-SMDF was encountered mainly in the pelleted membrane fraction (Fig. 4, P2 and S2), consistent with its location in the cell surface. The Δ108–296 truncated protein was also largely associated with low speed pellet fractions (Fig. 4, P3 and S3). In contrast, deletion mutant Δ65–296 was only detected in the supernatant fraction (Fig. 4, P4 and S4), similar to free YFP. Unexpectedly, Δ101–296, which lacks the TM segment, was found primarily in the plasma membrane-enriched fractions (Fig. 4, P5 and S5), suggesting the presence in this protein of a plasma membrane anchoring domain. Taken together, these findings demonstrate that the internal hydrophobic sequence is an uncleaved, membrane insertion signal sequence and suggest the existence of an additional, previously unrecognized membrane anchoring site in the ectodomain of SMDF. Amino acid sequence and hydropathy analysis of the protein domain downstream of the TM segment did not reveal the presence of additional hydrophobic stretches that may explain the ability of C-terminal fragments to associate with membrane fractions. Accordingly, to identify this protein domain we designed additional YFP-SMDF truncated proteins that explore the role of the C-terminal domain (Fig. 2). Deletion mutants were expressed in COS-7 cells, and their subcellular location was investigated with biochemical and immunological methods. As illustrated in Fig. 5, deletion up to Ala282 at the end of the EGF-like motif did not eliminate completely membrane interaction of the truncated proteins, as evidenced by the significant presence of the C-terminal fragments Δ5–142, Δ5–214, and Δ5–282 in the plasma membrane-enriched fractions (P1, S1, P2, S2, P3, and S3). This observation suggests that the C-terminal stretch (Ser283-Glu296) is able to anchor a significant portion of the YFP to cellular membranes. In support of this notion, the double truncated fusion protein Δ1–101/Δ274–296, where the TM and the C terminus have been deleted, was not found in the membrane fractions (Fig. 5, P4 and S4). As for Δ5–282, all other truncated proteins containing the segment Ser283-Glu296(Fig. 2) were detected mainly in the pellet fraction. It is interesting to note that immunocytochemical studies on intact cells using the anti-HRGβ3 antibody did not show cell surface expression of the C-terminal fragments (data not shown), indicating an intracellular location of the SMDF ectodomain. This distribution is consistent with confocal fluorescence images, showing a wide cellular distribution pattern (Fig. 3 e). Collectively, these observations imply that the C terminus of SMDF also functions as a membrane anchoring domain and indicate that the group II topology of the protein is determined by the TM located in the N terminus of the protein. The amino acid composition and sequence of the segment Ser283-Glu296 (SFYSTSTPFLSLPE) is rather polar, containing few hydrophobic residues. To evaluate whether this sequence was able to associate with membranes, we studied the interaction of the synthetic peptides WSFYSTSTPFLSLPE and SFYSTSTPWLSLPE with artificial lipid vesicles. Tryptophan residues were included in the sequence to provide intrinsic fluorescence properties to these amino acid sequences. Fluorescence intensity and anisotropy measurements show that both peptides interact marginally with lipid vesicles composed of phosphatidylcholine or phosphatidylcholine/cholesterol (data not shown). Similarly, the thermotropic properties of lipid vesicles made of DMPC and DMPC/cholesterol were not changed significantly by the presence of the synthetic peptides (Fig. 6). Thus, peptides patterned after th" @default.
• W2011126063 created "2016-06-24" @default.
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• W2011126063 date "2002-05-01" @default.
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• W2011126063 title "Molecular Determinants of the Sensory and Motor Neuron-derived Factor Insertion into Plasma Membrane" @default.
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• W2011126063 doi "https://doi.org/10.1074/jbc.m201587200" @default.
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