SGIP1α Is an Endocytic Protein That Directly Interacts with Phospholipids and Eps15
2007; Elsevier BV; Volume: 282; Issue: 36 Linguagem: Inglês
10.1074/jbc.m703815200
ISSN1083-351X
AutoresAkiyoshi Uezu, Ayaka Horiuchi, Kousuke Kanda, Naoya Kikuchi, Kazuaki Umeda, Kazuya Tsujita, Shiro Suetsugu, Norie Araki, Hideyuki Yamamoto, Tadaomi Takenawa, Hiroyuki Nakanishi,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoSGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1α. SGIP1α bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1α furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1α was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1α was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1α at clathrin-coated pits/vesicles. SGIP1α overexpression reduced transferrin and EGF endocytosis. SGIP1α knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1α plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15. SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1α. SGIP1α bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1α furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1α was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1α was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1α at clathrin-coated pits/vesicles. SGIP1α overexpression reduced transferrin and EGF endocytosis. SGIP1α knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1α plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15. Clathrin-mediated endocytosis governs not only the routine uptake of membranes and nutrient receptors but also the internalization of several ligand-stimulated receptors, channels, and transporters (1Conner S.D. Schmid S.L. Nature. 2003; 422,: 37-44Crossref PubMed Scopus (3017) Google Scholar). Clathrin triskelions assemble into polyhedral lattices on the cytoplasmic surface of the plasma membrane. Binding of clathrin to the plasma membrane is mediated by adaptor proteins that interact with clathrin and with specialized cytoplasmic motifs of transmembrane proteins and/or phospholipids (2Kirchhausen T. Nat. Rev. Mol. Cell Biol. 2000; 1,: 187-198Crossref PubMed Scopus (418) Google Scholar, 3Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1,: 161-172Crossref PubMed Scopus (421) Google Scholar, 4Owen D.J. Collins B.M. Evans P.R. Annu. Rev. Cell Dev. Biol. 2004; 20,: 153-191Crossref PubMed Scopus (352) Google Scholar, 5Sorkin A. Curr. Opin. Cell Biol. 2004; 16,: 392-399Crossref PubMed Scopus (167) Google Scholar, 6Traub L.M. Biochim. Biophys. Acta. 2005; 1744,: 415-437Crossref PubMed Scopus (167) Google Scholar). The major adaptor protein is the AP-2 complex that consists ofα-,β2-, σ2-, and μ2-adaptins. The AP-2 complex recruits other adaptor proteins, such as amphiphysin,β-arrestin, epsin, and Eps15. These adaptor proteins can also interact with each other and with other components, such as dynamin, of clathrin-mediated endocytosis machinery.Accompanied by the binding of clathrin and its adaptor proteins to the plasma membrane, membrane curvature is generated to form a coated pit (1Conner S.D. Schmid S.L. Nature. 2003; 422,: 37-44Crossref PubMed Scopus (3017) Google Scholar). The process of membrane curvature leads to the formation of a deeply invaginated membrane followed by the fission of a nascent coated vesicle. Adaptor proteins, such as epsin and amphiphysin, directly bind to and deform liposomes into tubules in vitro (3Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1,: 161-172Crossref PubMed Scopus (421) Google Scholar, 7Takei K. Slepnev V.I. Haucke V. De Camilli P. Nat. Cell Biol. 1999; 1,: 33-39Crossref PubMed Scopus (510) Google Scholar, 8Itoh T. Koshiba S. Kigawa T. Kikuchi A. Yokoyama S. Takenawa T. Science. 2001; 291,: 1047-1051Crossref PubMed Scopus (384) Google Scholar, 9Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419,: 361-366Crossref PubMed Scopus (788) Google Scholar, 10Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. McMahon H.T. Science. 2004; 303,: 495-499Crossref PubMed Scopus (1334) Google Scholar). These proteins, together with dynamin, play crucial roles in membrane curvature and fission for the formation of clathrin-coated vesicles (1Conner S.D. Schmid S.L. Nature. 2003; 422,: 37-44Crossref PubMed Scopus (3017) Google Scholar). They directly interact with membrane phosphoinositides through phospholipid-binding domains such as the ENTH 5The abbreviations used are: ENTHepsin N-terminal homologyFBP17formin-binding protein 17SGIP1Src homology 3-domain growth factor receptor-bound 2-like interacting protein 1aaamino acid(s)AbsantibodiesmAbmonoclonal AbpAbpolyclonal AbBARBin-Amphiphysin-RvsCBBCoomassie Brilliant BlueDTTdithiothreitolEFCextended FCHFCHFes-CIP4 homologyEGFPenhanced green fluorescent proteinGSTglutathione S-transferaseMBPmaltose-binding proteinMPmembrane phospholipid-bindingMTmicrotubulePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPI(4,5)P2PI(4,5)-bisphosphatePSphosphatidylserineRNAiRNA interferencesiRNAsmall interfering RNATfntransferrinEGFepidermal growth factorPBSphosphate-buffered salinePIPES1,4-piperazinediethanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.5The abbreviations used are: ENTHepsin N-terminal homologyFBP17formin-binding protein 17SGIP1Src homology 3-domain growth factor receptor-bound 2-like interacting protein 1aaamino acid(s)AbsantibodiesmAbmonoclonal AbpAbpolyclonal AbBARBin-Amphiphysin-RvsCBBCoomassie Brilliant BlueDTTdithiothreitolEFCextended FCHFCHFes-CIP4 homologyEGFPenhanced green fluorescent proteinGSTglutathione S-transferaseMBPmaltose-binding proteinMPmembrane phospholipid-bindingMTmicrotubulePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPI(4,5)P2PI(4,5)-bisphosphatePSphosphatidylserineRNAiRNA interferencesiRNAsmall interfering RNATfntransferrinEGFepidermal growth factorPBSphosphate-buffered salinePIPES1,4-piperazinediethanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.domain in epsin, the BAR domain in amphiphysin, the EFC/FCH and BAR domain in FBP17, and the pleckstrin homology domain in dynamin (11Itoh T. Erdmann K.S. Roux A. Habermann B. Werner H. De Camilli P. Dev. Cell. 2005; 9,: 791-804Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 12Tsujita K. Suetsugu S. Sasaki N. Furutani M. Oikawa T. Takenawa T. J. Cell Biol. 2006; 172,: 269-279Crossref PubMed Scopus (299) Google Scholar, 13Itoh T. De Camilli P. Biochim. Biophys. Acta. 2006; 1761,: 897-912Crossref PubMed Scopus (297) Google Scholar). These domains deform the plasma membrane into narrow tubules.We attempted to isolate a novel tubulin- and/or MT-binding protein and purified a protein with a molecular mass of about 100 kDa (p100). During the study, p100 was found to be highly homologous to a recently reported protein named Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1), the function of which is not fully understood (14Trevaskis J. Walder K. Foletta V. Kerr-Bayles L. McMillan J. Cooper A. Lee S. Bolton K. Prior M. Fahey R. Whitecross K. Morton G.J. Schwartz M.W. Collier G.R. Endocrinology. 2005; 146,: 3757-3764Crossref PubMed Scopus (31) Google Scholar). It is likely that p100 is a longer splicing variant of SGIP1; therefore, it was named SGIP1α. It remains elusive whether its tubulin binding is significant, but we have found that SGIP1α is an endocytic protein, which directly interacts with phosphoinositides and Eps15.EXPERIMENTAL PROCEDURESTubulin Blot Overlay—Tubulin blot overlay was performed as described (15Kremer L. Dominguez J.E. Avila J. Anal. Biochem. 1988; 175,: 91-95Crossref PubMed Scopus (18) Google Scholar, 16Balaban N. Goldman R. Cell Motil. Cytoskeleton. 1992; 21,: 138-146Crossref PubMed Scopus (14) Google Scholar) with some modifications. Samples to be tested were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with PBS containing 5% fat-free powder milk overnight at 4 °C and then equilibrated with PEM buffer (100 mm PIPES/NaOH, pH 6.8, 1 mm EGTA, 1 mm MgCl2, and 1 mm DTT) for 10 min at room temperature. The membrane was then incubated with 30 μg/ml tubulin in PEM buffer containing 1% bovine serum albumin for 1 h at 4° C. After washing in PBS, the membrane was subjected to immunoblot analysis using anti-tubulin α mAb (DM1A, Sigma-Aldrich).Purification of p100 and Mass Spectrometry—All the purification procedures were carried out at 0-4 °C. Twenty rat brains were homogenized in 200 ml of buffer A (20 mm Tris/HCl, pH 8.0, 2 mm EDTA, 5 mm EGTA, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, and 1 μg/ml pepstatin A) followed by ultracentrifugation. The pellet was rehomogenized in 200 ml of buffer A followed by ultracentrifugation. The pellet was again homogenized in 100 ml of buffer A containing 1.5 m NaCl. The homogenate was gently stirred for 30 min followed by ultracentrifugation. The supernatant was then dialyzed against buffer B (20 mm HEPES/NaOH, pH 7.5, 1 mm EDTA, and 1 mm DTT) overnight followed by ultracentrifugation. The pellet was resuspended in 65 ml of buffer C (buffer B containing 6 m urea) and gently stirred for 30 min. After the sample was centrifuged, the supernatant was applied to an SP-Sepharose Fast Flow column (2.6 × 10 cm, GE Healthcare Bio-Science Corp.) equilibrated with buffer C. Elution was performed with 60 ml of buffer C containing 0.5 m NaCl. Fractions of 5 ml each were collected. The active fractions (fractions 6-12) were collected and diluted with 210 ml of buffer D (20 mm Tris/HCl, pH 7.5, 1 mm EDTA, 1 mm DTT, and 4 m urea). The sample was applied to a HiPrep 16/10 Q Fast Flow column (1.6 × 10 cm, GE Healthcare Bio-Science Corp.) equilibrated with buffer D. Elution was performed with a 200-ml linear gradient of NaCl (0-0.5 m) in buffer D. Fractions of 4 ml each were collected. The active fractions (fractions 22-27) were collected. The sample was subjected to precipitation by the chloroform/methanol/water system (17Pohl T. Methods Enzymol. 1990; 182,: 68-83Crossref PubMed Scopus (54) Google Scholar) and dissolved in 15 ml of buffer E (10 mm potassium phosphate, pH 7.8, 1 mm DTT, 0.6% CHAPS, and 4 m urea). The sample was applied to a TSKgel (0.75 × 7.5 cm, HA-1000, Tosoh) equilibrated with buffer E. Elution was performed with a 30-ml linear gradient of potassium phosphate (10-500 mm) in buffer E. Fractions of 1 ml each were collected. The active fractions (fractions 11-18) were collected and subjected to precipitation by the chloroform/methanol/water system (17Pohl T. Methods Enzymol. 1990; 182,: 68-83Crossref PubMed Scopus (54) Google Scholar). The sample was dissolved in 5 ml of buffer F (20 mm bis-Tris/HCl, pH 5.5, 1 mm EDTA, 1 mm DTT, and 4 m urea) and applied to a Mono Q 5/50 GL column (GE Healthcare Bio-Science Corp.) equilibrated with buffer F. Elution was performed with a 20-ml linear gradient of NaCl (0-500 mm) in buffer F. Fractions of 0.5 ml each were collected. The active fractions (fractions 16-19) were subjected to SDS-PAGE followed by silver staining (18Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68,: 850-858Crossref PubMed Scopus (7768) Google Scholar). After the protein band corresponding to p100 was excised and subjected to in-gel trypsin digestion, the resultant peptides were desalted by Zip tips C18 (Millipore) and subjected to infusion nanoESI QQTOF mass spectrometry (QSTAR Pulsar i Applied Biosystems/MDS SCIEX). Partial aa sequences were obtained with peptide ion fragmentation due to high collision energy. The sequences obtained were subjected to a search for sequence similarity against the current NCBI data base using the Mascot Search Program (Matrix Science Ltd.).Construction of Expression Vectors—Oligonucleotide primers, 5′-CGG GAT CCA TGA TGG AAG GAC TGA AAA AAC GTA CA-3′ and 5′-ATC TCG AGT TAG TTA TCT GCC AAG TAC TTT CCT GC-3′, were designed, and mouse SGIP1α cDNA was obtained by PCR using mouse cDNA as a template. Rat Eps15 cDNA was obtained by PCR using rat brain cDNA as a template (DDBJ/EMBL/GenBank™ accession number AB262963). Expression vectors were constructed in pGex5X-3 (GE Healthcare Bio-Science Corp.), pMal C2 (New England Biolabs), pEGFP-C1 (Clontech), and pCMV-Myc (19Nakanishi H. Obaishi H. Satoh A. Wada M. Mandai K. Satoh K. Nishioka H. Matsuura Y. Mizoguchi A. Takai Y. J. Cell Biol. 1997; 139,: 951-961Crossref PubMed Scopus (162) Google Scholar). SGIP1α-1 (aa 1-280), SGIP1α-2 (aa 261-580), SGIP1α-3 (aa 561-854), SGIP1α-4 (aa 1-97), SGIP1α-5 (aa 98-280), SGIP1α-6 (aa 251-390), SGIP1α-7 (aa 428-854), and rat Eps15 (aa 593-834) were obtained by PCR. SGIP1α mutant (T2001C, C2004G, G2007A, A2010G, and C2013T) was generated using the site-directed mutagenesis kit (Stratagene) without changing the aa sequence. GST fusion and MBP fusion proteins were purified using glutathione-Sepharose beads (GE Healthcare Bio-Science Corp.) and amylose resin beads (New England Biolabs Inc.), respectively.Abs—A rabbit pAb against SGIP1α was raised against GST-SGIP1α-6 (aa 251-390). The antiserum was affinity-purified with the fusion protein covalently coupled to N-hydroxysuccinimidyl-activated Sepharose (GE Healthcare Bio-Science Corp.). The following Abs were purchased from commercial sources: mouse anti-Myc mAb (9E10) (American Type Culture Collection); mouse anti-tubulin α mAb (clone DM1A) (Sigma-Aldrich); rabbit anti-EGFP pAb and mouse anti-EGFP mAb (MBL Co.); mouse anti-α adaptin mAb, mouse anti-γ adaptin mAb, and mouse anti-Eps15 mAb (BD Biosciences); rabbit anti-Eps15 pAb (Covance); and secondary Abs conjugated with Alexa Fluor 488 and 594 (Invitrogen).Cell Culture, Transfection, and Immunofluorescence Microscopy—Cells were cultured at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfection was performed using Lipofectamine 2000 reagent (Invitrogen). Cells were fixed with 1% formaldehyde in PBS for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and immunostained with various Abs. DiIC16 (3Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1,: 161-172Crossref PubMed Scopus (421) Google Scholar) (Invitrogen) staining was performed as described (20Kamioka Y. Fukuhara S. Sawa H. Nagashima K. Masuda M. Matsuda M. Mochizuki N. J. Biol. Chem. 2004; 279,: 40091-40099Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Images were taken with a fluorescence microscope (BX51, Olympus) or a confocal microscopy system (BX50 and Fluoview FV300; Olympus) at room temperature. The fluorochromes used included Alexa Fluor 488 and 594, Texas-red conjugated EGF, and DiIC16 (3Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1,: 161-172Crossref PubMed Scopus (421) Google Scholar) (Invitrogen). A ×60 oil immersion objective, NA 1.40 (Olympus) was used. Images were assembled with PhotoShop (Adobe). In each plate, photographs were cropped, and each fluorochrome was adjusted for identical brightness and contrast to represent the observed images.The quantification of colocalization was performed using Meta-Morph imaging system software (21Murph M.M. Scaccia L.A. Volpicelli L.A. Radhakrishna H. J. Cell Sci. 2003; 116,: 1969-1980Crossref PubMed Scopus (59) Google Scholar). Briefly, background was subtracted from unprocessed images, and the percentage of SGIP1α pixels overlapping Eps15, α-adaptin, or EGF pixels was measured. For the colocalization in the plasma membrane region, images were acquired to display the entire cell surface adhering to culture dishes (22Puri C. Tosoni D. Comai R. Rabellino A. Segat D. Caneva F. Luzzi P. Di Fiore P.P. Tacchetti C. Mol. Biol. Cell. 2005; 16,: 2704-2718Crossref PubMed Scopus (122) Google Scholar). The colocalization percentage was determined in a restricted region of the images extending 15 pixels from the cell edge toward the cytoplasm. For the colocalization in the intracellular region, images were acquired at upper planes from the ventral surface. The colocalization percentage was determined in a region of the images excluding 15 pixels from the cell edge. Data were shown as the means + S.E. of three independent experiments.Liposome Binding and Liposome Tubulation Assays—Liposome binding assay was performed as described (12Tsujita K. Suetsugu S. Sasaki N. Furutani M. Oikawa T. Takenawa T. J. Cell Biol. 2006; 172,: 269-279Crossref PubMed Scopus (299) Google Scholar). PE/PC liposomes consisted of 70% PE, 20% PC, and 10% PI or various phosphoinositides. Where indicated, liposomes consisted of 80% PE, 20% PC, and various percentages of PS (with a corresponding reduction in PE). MBP-SGIP1α-4 (50 μg/ml) was incubated with 1 mg/ml liposomes for 15 min at room temperature followed by centrifugation. To calculate the Kd value, various doses of GST-SGIP1α-4 were incubated with 0.2 mg/ml liposomes (60% PE, 20% PC, and 20% PI(4,5)P2) for 30 min at 4 °C followed by centrifugation. Comparable amounts of the supernatant and pellet fractions were subjected to SDS-PAGE followed by CBB staining. The protein amount was quantified by scanning using NIH image (version 1.61). Liposome tubulation assay was carried out as described (12Tsujita K. Suetsugu S. Sasaki N. Furutani M. Oikawa T. Takenawa T. J. Cell Biol. 2006; 172,: 269-279Crossref PubMed Scopus (299) Google Scholar).In Vitro Binding Assays and Immunoprecipitation—To examine the in vitro binding of SGIP1α to Eps15, MBP-Eps15 or MBP alone (200 pmol each) was incubated with GST-SGIP1α-3 or GST alone (200 pmol each) immobilized on beads in PBS containing 0.1% Triton X-100. After washing, bound proteins were subjected to SDS-PAGE followed by CBB staining.Immunoprecipitation was performed as follows. Cells were lysed in lysis buffer (50 mm Tris/HCl, pH 7.5, 5 mm EGTA, 5 mm EDTA, 150 mm NaCl, 15 mm NaF, 1.5 mm Na3VO4, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 1 μg/ml pepstatin A) followed by centrifugation. The supernatant (1 mg of protein) was incubated for 3 h at 4 °C with anti-SGIP1α pAb or control rabbit IgG. Protein G-Sepharose beads were added to the sample, which was further incubated for 3 h at 4 °C. After the beads were thoroughly washed with the same buffer, bound proteins were subjected to SDS-PAGE followed by immunoblot analysis.RNAi—Stealth double-stranded RNAs were purchased from Invitrogen. The sequence of siRNA specific to SGIP1α was 5′-UUU ACC CAG AAU UCC UUG GUA UUG G-3′ (corresponding nucleotide 1997-2021 relative to the start codon). A double-stranded RNA targeting luciferase (5′-CGU ACG CGG AAU ACU UCG AAA UGU C-3′) was used as a control. N1E115 cells were transfected with 20 nm siRNA using Lipofectamine 2000 reagent (Invitrogen). After 24 h, a second transfection was performed, and the cells were cultured for 72 h and subjected to various experiments.Endocytosis Assays—Endocytosis in COS7 cells was assayed using fluorescent ligands as described (12Tsujita K. Suetsugu S. Sasaki N. Furutani M. Oikawa T. Takenawa T. J. Cell Biol. 2006; 172,: 269-279Crossref PubMed Scopus (299) Google Scholar, 20Kamioka Y. Fukuhara S. Sawa H. Nagashima K. Masuda M. Matsuda M. Mochizuki N. J. Biol. Chem. 2004; 279,: 40091-40099Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Endocytosis assays in N1E115 cells using fluorescent ligands were performed as described (12Tsujita K. Suetsugu S. Sasaki N. Furutani M. Oikawa T. Takenawa T. J. Cell Biol. 2006; 172,: 269-279Crossref PubMed Scopus (299) Google Scholar, 20Kamioka Y. Fukuhara S. Sawa H. Nagashima K. Masuda M. Matsuda M. Mochizuki N. J. Biol. Chem. 2004; 279,: 40091-40099Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) with slight modifications. Briefly, cells were starved with serum-free Dulbecco's modified Eagle's medium for 2 h and incubated with 25 μg/ml Alexa Fluor 594-conjugated Tfn (Invitrogen) or 20 ng/ml Texas red-conjugated, biotinylated EGF (Invitrogen) for 10 min at 37 °C. After acid stripping, cells were fixed with formaldehyde. The average intensity of internalized ligands per cell area was determined using MetaMorph imaging system software (23Strachan L.R. Condic M.L. J. Cell Biol. 2004; 167,: 545-554Crossref PubMed Scopus (48) Google Scholar). Endocytosis assays in N1E115 cells using radioactive ligands were performed as described (24Subtil A. Dautry-Varsat A. J. Cell Sci. 1997; 110,: 2441-2447Crossref PubMed Google Scholar, 25Nesterov A. Carter R.E. Sorkina T. Gill G.N. Sorkin A. EMBO J. 1999; 18,: 2489-2499Crossref PubMed Scopus (184) Google Scholar) with slight modifications. Briefly, cells were starved for 2 h and incubated with 10 nm 125I-labeled Tfn (PerkinElmer) or 1.5 ng/ml 125I-labeled EGF (PerkinElmer) for the indicated periods of time at 37 °C in Dulbecco's modified Eagle's medium containing 1% bovine serum albumin. Internalized and surface radioactivity was quantified by a gamma counter. Nonspecific binding was measured for each time point in the presence of 100-fold molar excess of the same unlabeled ligand.Other Procedures—Tubulin was prepared from fresh porcine brains as described (26Shelanski M.L. Gaskin F. Cantor C.R. Proc. Natl. Acad. Sci. U. S. A. 1973; 70,: 765-768Crossref PubMed Scopus (1967) Google Scholar, 27Jr. Williams R.C. Lee J.C. Methods Enzymol. 1982; 85,: 376-385Crossref PubMed Scopus (405) Google Scholar). The yeast two-hybrid library constructed from adult rat brain cDNA was screened using pBTM116HA-SGIP1α-7 (aa 428-854) as bait as described (28Shimizu K. Kawabe H. Minami S. Honda T. Takaishi K. Shirataki H. Takai Y. J. Biol. Chem. 1996; 271,: 27013-27017Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 29Takahashi K. Nakanishi H. Miyahara M. Mandai K. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145,: 539-549Crossref PubMed Scopus (438) Google Scholar). Protein concentrations were performed with bovine serum albumin as a reference protein (30Bradford M.M. Anal. Biochem. 1976; 72,: 248-254Crossref PubMed Scopus (213377) Google Scholar). SDS-PAGE was performed as described (31Laemmli U.K. Nature. 1970; 227,: 680-685Crossref PubMed Scopus (206024) Google Scholar).RESULTSIdentification of p100 as a Tubulin-binding Protein—To detect tubulin- and/or MT-binding proteins, we performed tubulin blot overlay. When various rat tissue homogenates were subjected to blot overlay, several bands with various molecular masses were detected (Fig. 1A). Of these protein bands, a thick band of about 100 kDa (p100) was detected only in the brain. By successive column chromatographies, p100 was copurified with another band of about 110 kDa (p110) (supplemental Fig. 1). These protein bands were separately excised out from the gels, digested with trypsin, and subjected to mass spectrometry. From the p100 protein band, four peptide sequences were obtained (supplemental Fig. 2A). A data base search revealed that the peptide sequences of p100 were identical to those of a mouse hypothetical protein (GenBank accession number BC017596). p110 was found to be CLIP-115, a brain-specific MT-binding protein (32De Zeeuw C.I. Hoogenraad C.C. Goedknegt E. Hertzberg E. Neubauer A. Grosveld F. Galjart N. Neuron. 1997; 19,: 1187-1199Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). We performed PCR to amplify cDNA of the hypothetical protein using mouse brain cDNA as a template. Our cloned cDNA encoded a protein that consisted of 854 aa and showed a calculated molecular weight of 91,719 (DDBJ/EMBL/GenBank accession number AB262964) (supplemental Fig. 2A). This protein contained a proline-rich domain in the middle region, whereas it did not possess any conserved domains in the N-terminal or C-terminal region (see Fig. 2A).FIGURE 2Membrane tubulation by SGIP1α overexpression.A, the N-terminal region (aa 1-97, MP domain) responsible for the formation of tubular structures. Upper panel, structures of full-length SGIP1α and various fragments of SGIP1α. Lower panel, COS7 cells were transfected with pEGFP-SGIP1α (full-length), pEGFP-SGIP1α-1 (aa 1-280), pEGFP-SGIP1α-2 (aa 261-580), pEGFP-SGIP1α-3 (aa 561-854), pEGFP-SGIP1α-4 (aa 1-97, MP domain), or pEGFP-SGIP1α-5(aa 98-280). Cells were examined with a fluorescence microscope. Scale bars, 10 μm. B, origination of tubular structures from the plasma membrane. COS7 cells transfected with pEGFP-SGIP1α-4 (MP domain) were fixed without permeabilization and then stained with DiIC16 (3Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1,: 161-172Crossref PubMed Scopus (421) Google Scholar). Samples were examined with a fluorescence microscope. Insets, enlarged images of dashed boxes. Scale bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)During the study, the mouse hypothetical protein was found to be a recently reported neuronal protein, named SGIP1 (14Trevaskis J. Walder K. Foletta V. Kerr-Bayles L. McMillan J. Cooper A. Lee S. Bolton K. Prior M. Fahey R. Whitecross K. Morton G.J. Schwartz M.W. Collier G.R. Endocrinology. 2005; 146,: 3757-3764Crossref PubMed Scopus (31) Google Scholar). When the aa sequences of our clone and SGIP1 were aligned, they showed 94% identity (supplemental Fig. 2A). Our clone had two inserted peptide sequences at its N-terminal and middle regions. It is likely that our clone is a longer splicing variant of SGIP1; therefore, the protein encoded by our clone was named SGIP1α. To confirm whether SGIP1α is detected by tubulin blot overlay, we expressed a Myc-tagged protein in COS7 cells. Myc-tagged SGIP1α was detected by blot overlay (Fig. 1B).Membrane Tubulation by SGIP1α Overexpression—To examine the binding of SGIP1α to tubulin and/or MTs in intact cells, we examined the subcellular distribution of SGIP1α. When transiently expressed at a high level in COS7 cells, EGFP-SGIP1α (full-length) showed short tubular structures (Fig. 2A). EGFP-SGIP1α-1 (aa 1-280) and -4 (aa 1-97) also showed tubular structures, whereas other fragments, including EGFP-SGIP1α-2 (aa 261-580), -3 (aa 561-854), and -5 (aa 98-280), did not. These results indicate that the N-terminal region (aa 1-97) of SGIP1α is responsible for the formation of tubular structures. These structures appeared to be different from those of MTs but were reminiscent of those recently reported to be formed by phospholipid-binding BAR and EFC domains (11Itoh T. Erdmann K.S. Roux A. Habermann B. Werner H. De Camilli P. Dev. Cell. 2005; 9,: 791-804Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 12Tsujita K. Suetsugu S. Sasaki N. Furutani M. Oikawa T. Takenawa T. J. Cell Biol. 2006; 172,: 269-279Crossref PubMed Scopus (299) Google Scholar, 20Kamioka Y. Fukuhara S. Sawa H. Nagashima K. Masuda M. Matsuda M. Mochizuki N. J. Biol. Chem. 2004; 279,: 40091-40099Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). To examine the possibility that the N-terminal region (aa 1-97) binds to the plasma membrane and deforms it into tubules, COS7 cells overexpressing EGFP-SGIP1α-4 (aa 1-97) were stained with DiIC16 (3Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1,: 161-172Crossref PubMed Scopus (421) Google Scholar). DiIC16 (3Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1,: 161-172Crossref PubMed Scopus (421) Google Scholar) is a lipophilic fluorescent probe used for plasma membrane staining (33Mukherjee S. Maxfield F.R. Traffic. 2000; 1,: 203-211Crossref PubMed Scopus (191) Google Scholar). The tubular structures of EGFP-SGIP1α-4 were completely overlapped with DiIC16 (3Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1,: 161-172Crossref PubMed Scopus (421) Google Scholar) staining (Fig. 2B). This result indicates that tubular structures originate from the plasma membrane. It remains elusive whether SGIP1α binds to tubulin and/or MTs in intact cells. Based on the observation that the N-terminal region (aa 1-97) of SGIP1α binds to membrane phospholipids as described below, we named this region the MP (membrane phospholipid-binding) domain, which shows no significant homology to any proteins in the current protein data base.Direct Binding of SGIP1α to Phospholipids—To examine whether the MP domain directly interacts with membrane phospholipids in vitro, we performed liposome cosedimentation assay. Although MBP-SGIP1α-4 (MP domain) did not bind to synthetic liposomes composed of PE and PC, it strongly bound to liposomes containing PS, PI 3-phosphate, PI 4-phosphate, PI 3,4-bisphosphate, PI 3,5-bisphosphate, or PI(4,5)P2 (Fig. 3A). The MP domain faintly bound to liposomes containing PI, PI 5-phosphate, or PI 3,4,5-trisphosphate. The percentage of PS in liposomes for maximal binding was about 10%. The MP domain bound to brain lipids (Folch fraction) rich in PS (about 50% of total lipids). This binding specificity of the MP domain is similar to that of the FBP17 EFC domain (11Itoh T. Erdmann K.S. Roux A. Habermann B. Werner H. De Camilli P. Dev. Cell. 2005; 9,: 791-804Abstract Full Tex
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