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• W2012095629 abstract "The four-transmembrane domain proteins synaptophysin and synaptogyrin represent the major constituents of synaptic vesicles. Our previous studies in PC12 cells demonstrated that synaptogyrin or its nonneuronal paralog cellugyrin targets efficiently to synaptic-like microvesicles (SLMVs) and dramatically increases the synaptophysin content of SLMVs (Belfort, G. M., and Kandror, K. V. (2003) J. Biol. Chem. 278, 47971–47978). Here, we explored the mechanism of these phenomena and found that ectopic expression of cellugyrin increases the number of SLMVs in PC12 cells. Mutagenesis studies revealed that cellugyrin's hydrophilic cytoplasmic domains are not involved in vesicle biogenesis, whereas small conserved hydrophobic hairpins in the first luminal loop and the carboxyl terminus of cellugyrin were found to be critical for the formation of SLMVs. In addition, the length but not the primary sequence of the second luminal loop was essential for SLMV biogenesis. We suggest that changing the length of this loop similar to disruption of the short hydrophobic hairpins alters the position of the vicinal transmembrane domains that may be crucial for protein function. The four-transmembrane domain proteins synaptophysin and synaptogyrin represent the major constituents of synaptic vesicles. Our previous studies in PC12 cells demonstrated that synaptogyrin or its nonneuronal paralog cellugyrin targets efficiently to synaptic-like microvesicles (SLMVs) and dramatically increases the synaptophysin content of SLMVs (Belfort, G. M., and Kandror, K. V. (2003) J. Biol. Chem. 278, 47971–47978). Here, we explored the mechanism of these phenomena and found that ectopic expression of cellugyrin increases the number of SLMVs in PC12 cells. Mutagenesis studies revealed that cellugyrin's hydrophilic cytoplasmic domains are not involved in vesicle biogenesis, whereas small conserved hydrophobic hairpins in the first luminal loop and the carboxyl terminus of cellugyrin were found to be critical for the formation of SLMVs. In addition, the length but not the primary sequence of the second luminal loop was essential for SLMV biogenesis. We suggest that changing the length of this loop similar to disruption of the short hydrophobic hairpins alters the position of the vicinal transmembrane domains that may be crucial for protein function. Understanding the mechanisms by which a cell sequesters specific proteins in unique subcellular compartments is fundamental to understanding how the cell maintains distinct organelles. For the past 25 years, the PC12 neuroendocrine cell line has provided a convenient system for studying the biogenesis of synaptic like microvesicles (SLMVs) 1The abbreviations used are: SLMV, synaptic-like microvesicle; PNS, postnuclear supernatant(s); PAS, postabsorptive supernatant(s); TE, Triton elution; SE, SDS elution; TM, transmembrane; ER, endoplasmic reticulum. (1Johnston P. Cameron P.L. Stukenbrok H. Jahn R. De Camilli P. Sudhof T.C. EMBO J. 1989; 8: 2863-2872Crossref PubMed Scopus (109) Google Scholar). SLMVs were chosen as a model organelle, because, like neuronal synaptic vesicles, their small size (∼50 nm) restricts the number of proteins present in each vesicle. Based on the estimates of 3 × 106 daltons of protein/synaptic vesicle and 50,000 daltons/average protein, ∼60 proteins could be contained in a vesicle of this size (2Jahn R. Sudhof T.C. J. Neurochem. 1993; 61: 12-21Crossref PubMed Scopus (85) Google Scholar). Two main methods have been used to study the biogenesis of SLMVs. Several groups have studied cell-free or perforated cell vesicle budding systems in order to identify cytosolic factors critical for vesicle budding. Others studying specific proteins that target to SLMVs have identified regions in their primary amino acid sequences that are required for targeting to that compartment. Two main pathways of SLMV biogenesis have emerged from in vitro studies. First, vesicles can bud from endosomes in a brefeldin A-sensitive mechanism that requires the AP3 adaptor protein and the ARF1 and Rab4 GTPases (3Faundez V. Horng J.T. Kelly R.B. J. Cell Biol. 1997; 138: 505-515Crossref PubMed Scopus (88) Google Scholar, 4Lichtenstein Y. Desnos C. Faundez V. Kelly R.B. Clift-O'Grady L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11223-11228Crossref PubMed Scopus (53) Google Scholar, 5Faundez V. Horng J.T. Kelly R.B. Cell. 1998; 93: 423-432Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 6de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (58) Google Scholar). By the second pathway, SLMVs can bud from the plasma membrane in a brefeldin A-insensitive mechanism that involves the AP2 adapter, clathrin, and dynamin (7Shi G. Faundez V. Roos J. Dell'Angelica E.C. Kelly R.B. J. Cell Biol. 1998; 143: 947-955Crossref PubMed Scopus (84) Google Scholar). Further work with perforated PC12 cells demonstrated that endophilin I, a dynamin-binding protein, facilitates SLMV budding, probably by modifying the membrane lipid composition (8Schmidt A. Wolde M. Thiele C. Fest W. Kratzin H. Podtelejnikov V. Witke W. Huttner W.B. Soling H.D. Nature. 1999; 401: 133-141Crossref PubMed Scopus (449) Google Scholar). Two categories of proteins are present in SLMVs in PC12 cells: neuronal synaptic vesicle proteins and other proteins that are not found in brain synaptic vesicles. Of those proteins belonging to the former category, synaptophysin, synaptobrevin/VAMPII, the vesicular acetylcholine transporter, and synaptotagmin I have all been at least partially analyzed for SLMV targeting sequences (9Leube R.E. J. Cell Sci. 1995; 108: 883-894Crossref PubMed Google Scholar, 10Linstedt D. Kelly R.B. J. Physiol. 1991; 85: 90-96Google Scholar, 11Grote E. Hao J.C. Bennett M.K. Kelly R.B. Cell. 1995; 81: 581-589Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 12Varoqui H. Erickson J.D. J. Biol. Chem. 1998; 273: 9094-9098Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 13Blagoveshchenskaya D. Hewitt E.W. Cutler D.F. Mol. Biol. Cell. 1999; 10: 3979-3990Crossref PubMed Scopus (60) Google Scholar). The group of nonsynaptic vesicle proteins localized in SLMVs can be further divided into the nonneuronal paralogs of synaptic vesicle proteins (cellubrevin, cellugyrin, and pantophysin) (14Chilcote T.J. Galli T. Mundigl O. Edelmann L. McPherson P.S. Takei K. De Camilli P. J. Cell Biol. 1995; 129: 219-231Crossref PubMed Scopus (118) Google Scholar, 15Belfort G.M. Kandror K.V. J. Biol. Chem. 2003; 278: 47971-47978Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 16Haass N.K. Kartenbeck M. Leube R.E. J. Cell Biol. 1996; 134: 731-746Crossref PubMed Scopus (68) Google Scholar) and those proteins that are unrelated to synaptic vesicle proteins (tyrosinase and P-selectin) (13Blagoveshchenskaya D. Hewitt E.W. Cutler D.F. Mol. Biol. Cell. 1999; 10: 3979-3990Crossref PubMed Scopus (60) Google Scholar, 17Norcott J.P. Solari R. Cutler D.F. J. Cell Biol. 1996; 134: 1229-1240Crossref PubMed Scopus (52) Google Scholar). These studies, which examined targeting motifs, failed to identify a universal sequence applicable to all SLMV proteins. The one commonality among all of the targeting motifs identified so far is that they are all located in cytoplasmic domains. The question then arises as to what mechanisms maintain the structural integrity of SLMVs, such that, despite the complexity of their trafficking pathways in the cells and apparent heterogeneity of targeting signals, these vesicles maintain uniform and specific protein composition? Undoubtedly, adaptor proteins provide a certain level of specificity upon vesicle budding from the donor membranes. However, given the fact that at least two adaptors, AP-2 and AP-3, with different binding specificities, are involved in the formation of vesicles with the same protein composition from different compartments, we speculate that additional factors should participate in the formation of SLMVs. Cellugyrin (also referred to as synaptogyrin 2) is the nonneuronal paralog of the synaptic vesicle protein, synaptogyrin 1. Both cellugyrin and synaptogyrin 1 target to SLMVs in PC12 cells, and both do so with a much higher efficiency than any other protein tested so far (15Belfort G.M. Kandror K.V. J. Biol. Chem. 2003; 278: 47971-47978Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Increasing the level of cellugyrin or synaptogyrin 1 in PC12 cells also causes a concomitant increase of endogenous synaptophysin in SLMVs. Here, we report that cellugyrin expression is sufficient to induce the biogenesis of SLMVs, as measured by two independent methods. Furthermore, despite the finding that cellugyrin and synaptogyrin are capable of targeting very efficiently to microvesicles in all cell types tested, the capacity to facilitate targeting of synaptophysin to microvesicles was not replicated in a nonneuronal cell line (COS7 cells). These results suggest that the capacity of cellugyrin to facilitate the targeting of synaptophysin is not an artifact of overexpression but rather represents an epiphenomenon of cellugyrin targeting to microvesicles that requires cell type-specific factors. In support of this latter hypothesis, molecular dissection of cellugyrin via recombinant DNA techniques revealed that the targeting of cellugyrin mutants to SLMVs can be uncoupled from cellugyrin's effects on synaptophysin targeting. These studies demonstrated that conserved hydrophobic sequences in the carboxyl-terminal domain and the first luminal loop, in addition to the lengths, but not the primary sequences, of the second luminal loop and the cytoplasmic loop are key determinants for efficient SLMV targeting. Furthermore, inefficiencies in the targeting of these mutants to SLMVs yielded an even more significant decrease in the facilitation of synaptophysin targeting. We hypothesize that the highly efficient targeting of cellugyrin and synaptogyrin 1 to SLMVs and their ability to facilitate the targeting of synaptophysin is dependent on their capacity to increase the number of SLMVs and that this function is mediated by the regions identified in our targeting studies. Materials—Plasmid pCMV5-p38, which expresses rat synaptophysin, pCMV-Cgyr, which expresses rat cellugyrin, and pCMV5-p29, which expresses rat synaptogyrin 1, were all kind gifts from Dr. T. C. Südhof (University of Texas Southwestern). Plasmid pBABE-mycGlut4, which contains a 110-amino acid 7× Myc tag, was the kind gift of Jun Shi (Boston University School of Medicine). Plasmids pcDNA3.1/myc-His(+) and pcDNA3.1His(+) were purchased from Invitrogen. Anti-cellugyrin monoclonal antibody was purchased from BD Biosciences. Anti-HisG monoclonal antibody, which recognizes the amino-terminal His tag, was purchased from Invitrogen. Anti-synaptophysin monoclonal antibody was purchased from Chemicon. Anti-VAMPII/synaptobrevin monoclonal antibody and anti-synaptogyrin polyclonal antibody was purchased from Synaptic Systems. Anti-Myc monoclonal and polyclonal antibodies were purchased from Cell Signaling. Anti-calnexin polyclonal antibody was purchased from Stressgen. Anti-tubulin monoclonal antibody was purchased from Sigma. Recombinant DNA Constructs—Plasmid pHis-cellugyrin, encoding rat cellugyrin with an N-terminal His tag, was reported previously (15Belfort G.M. Kandror K.V. J. Biol. Chem. 2003; 278: 47971-47978Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Table I contains information on how the plasmids used in these studies were constructed.Table IRecombinant DNA constructsPlasmidNo. of PCR stepsTemplateOligonucleotidesVector: EnzymespMyc/His-Cellugyrin1pCMV-Cgyr5′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′pcDNA3.1/myc-His(+): EcoRI/XhoI5′-ATGACGTACTGCCTCGAGGTACACTGGGGG-3′pHis-Synaptophysin1pCMV5-p385′-AATAGCCTGAAAGATATCATGGACGTGGTG-3′pcDNA3.1His(+): EcoRV/XhoI5′-ATGACGTACTGCCTCGAGTTACATCTGATT-3′pHis-Synaptogyrin1pCMV5-p295′-AATAGCCTGAAAGAATTCATGGAAGGGGGT-3′pcDNA3.1His(+): EcoRI/XhoI5′-CTTGGGTAACTCGAGGTAGCCCTGCGA-3′pMyc/His-CR1-1771pCMV-Cgyr5′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′pcDNA3.1/myc-His(+): EcoRI/XhoI5′-ATAACGCGTAAACTCGAGGTAGGCCAGGGAGGCCAG-3′pHis-SCR67-761pCMV-Cgyr5′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′pcDNA3.1His(+): EcoRI/XhoI5′-GTTAACATGATCACGCTCAGACTGGTCAGATGA-3′2pCMV-Cgyr5′-GATCATGTTAACTTATTTAATGCCTGCCGCTAC-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′3PCR from steps 1 and 25′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′pMyc/His-CR1-177-SCR67-761pHis-SCR67-765′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′pcDNA3.1/myc-His(+): EcoRI/XhoI5′-ATAACGCGTAAACTCGAGGTAGGCCAGGGAGGCCAG-3′pHis-67-69toA1pCMV-Cgyr5′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′pcDNA3.1His(+): EcoRI/XhoI5′-CACGGCGGCGGCCTGGTCAGATGA-3′2pCMV-Cgyr5′-GCCGCCGCCGTGTTCAACCGG-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′3PCR from steps 1 and 25′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′pHis-70-73toA1pCMV-Cgyr5′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′pcDNA3.1His(+): EcoRI/XhoI5′-GGCGGCGGCGGCACAGTGTAGCTG-3′2pCMV-Cgyr5′-GCCGCCGCCGCCAACGAAGATGCC-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′3PCR from steps 1 and 25′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′pHis-74-76toA1pCMV-Cgyr5′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′pcDNA3.1His(+): EcoRI/XhoI5′-GGCGGCGGCGGCCCGGTTGAACAC-3′2pCMV-Cgyr5′-GCCGCCGCCGCCTGCCGCTAC-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′3PCR from steps 1 and 25′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′pHis-SCR104-1121pCMV-Cgyr5′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′pcDNA3.1His(+): EcoRI/XhoI5′-TGCTGAGCGCTGGATATCAGAGAAAAACGC-3′2pCMV-Cgyr5′-CAGCGCTCAGCAAAAACCAACTATCTGGTCATT-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′3PCR from steps 1 and 25′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′pHis-SCR142-1511pCMV-Cgyr5′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′pcDNA3.1His(+): EcoRI/XhoI5′-GGCCGTAAGATCCACAACTTTTGCCCACTGGTT-3′2pCMV-Cgyr5′-GATCTTACGGCCGGCCCAGACGCAGACTCAGCC-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′3PCR from steps 1 and 25′-AATAGCCTGAAAGAATTCATGCCCTTGAGGGTCGGC-3′5′-CTTGGGTAACTCGAGTGATCAGTACACTGGGGGAGG-3′pHis- mycinSCR104-112No PCRAnneal oligogonucleotides and insert5′-TCAGCAGAACAAAAACTCATCTCAGAAGAGGATCTGCGC-3′pHis-SCR104-112: BlpI5′-TGAGCGCAGATCCTCTTCTGAGATGAGTTTTTGTTCTGC-3′pHis-mycinSCR142-151No PCRAnneal oligogonucleotides and insert5′-CCCAGAACAAAAACTCATCTCAGAAGAGGATCTGGCCGG-3′pHis-SCR142-151: FseI5′-CCAGATCCTCTTCTGAGATGAGTTTTTGTTCTGGGCCGG-3′pHis-7mycinSCR104-1121pBABE-mycGlut45′-AATAGCCTGAAACGCTCAGCAGGGTCCTGCAGA-3′pHis-SCR104-112: BlpI5′-ATGACGTACTGCTGCTGAGCGGGTCCCTTAAGC-3′ Open table in a new tab Cell Culture and Transfections—The pheochromocytoma cell line, PC12, was grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 2 mm l-glutamine, 50 μg/ml gentamicin, 100 units/ml penicillin, and 100 μg/ml streptomycin in 5% CO2 at 37 °C. COS7 cells, a monkey kidney cell line, were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 50 μg/ml gentamicin, 100 units/ml penicillin, and 100 μg/ml streptomycin in 5% CO2 at 37 °C. Transfections were performed with Lipofectamine2000 reagent (Invitrogen) according to the manufacturer's instructions. Optimal transfections were obtained with 5 μg of DNA/10-cm plate and a Lipofectamine2000 reagent to plasmid ratio of 3 μl/μg of DNA. The efficiency of transfection was over 90%. Stably transfected cell lines were selected by culturing transiently transfected cells in the presence of G418 for 30 days. Vesicle Isolation and Fractionation of PC12 Cells—Confluent 15- or 10-cm plates were washed once with PBS. Cells were removed from the plates with cell dissociation medium (Sigma) for PC12 cells or trypsin/EDTA (Invitrogen) for COS7. Cells in suspension were pelleted at 400 × g for 10 min. Pelleted cells were resuspended in Buffer A (150 mm NaCl, 10 mm HEPES, pH 7.4, 1 mm EGTA, 0.1 mm MgCl2) with protease inhibitors (1 μm aprotinin, 5 mm benzamidine, 2 μm leupeptin, 1 μm pepstatin, 1 mm phenylmethylsulfonyl fluoride). Resuspended cells were homogenized with 11 strokes through a ball bearing cell cracker (European Molecular Biology Laboratory) and centrifuged at 1000 × g for 5 min to generate a postnuclear supernatant (PNS). A high speed supernatant (S2) that contains only microvesicles and cytosolic proteins was generated by centrifugation of the PNS at 27,000 × g (15,000 rpm) for 35 min in a Ti42.2 rotor (Beckman) (18Clift-O'Grady L. Linstedt D. Lowe W. Grote E. Kelly R.B. J. Cell Biol. 1990; 110: 1693-1703Crossref PubMed Scopus (146) Google Scholar). Density gradient centrifugation was performed by layering ∼1 mg of S2, adjusted to 230 μl, onto a 4.6-ml 10–50% sucrose gradient in Buffer A (w/v). Density gradients were centrifuged at 280,000 × g (48,000 rpm) in an SW55 rotor (Beckman) for 16 h. Fractions were collected from the bottom via a peristaltic pump. Vesicle decoration/shift analysis was performed by incubating 1 mg of S2 with 2.5 μg of purified nonspecific mouse IgG or anti-synaptophysin monoclonal antibodies, together with 2 μg of Nanogold-conjugated goat anti-mouse Fab fragments (Nanoprobes). This mixture was rotated at 4 °C for 2 h. The total volume of decorated vesicles (always adjusted to 230 μl) was then loaded on a 4.6-ml linear 10–30% sucrose gradient in Buffer A (w/v). Gradients were centrifuged at 280,000 × g (48,000 rpm) in an SW55 rotor (Beckman) for 1 h and 15 min. Fractions were collected from the bottom via a peristaltic pump. Vesicle Isolation and Fractionation of COS7 Cells—Confluent 15- or 10-cm plates were washed once with PBS. Cells were removed from the plates with trypsin/EDTA (Invitrogen). Cells in suspension were pelleted at 400 × g for 10 min. Pelleted cells were resuspended in Buffer A with protease inhibitors. Resuspended cells were homogenized with 11 strokes through a ball bearing cell cracker (European Molecular Biology Laboratory) and centrifuged at 16,000 × g for 30 min. The pellet (16,000 × g pellet) represents total expression, and the supernatant (16,000 × g supernatant) represents the microvesicular fraction. The 16,000 × g pellet was extracted with a 1% solution of Triton X-100 in Buffer A. This isolation method was tested in tandem with the PNS/S2 isolation method (see above) in PC12 cells and found to be functionally equivalent. Radioactive Labeling and Organelle Immunoisolation—Whole cell lipids were labeled as previously reported (19Schreiber B.M. Veverbrants M. Fine R.E. Blusztajn J.K. Salmona M. Patel Sipe J.D. Biochem. J. 1999; 344: 7-13Crossref PubMed Google Scholar). Briefly, cells were incubated overnight with 8 μCi of [1-14C]acetic acid, sodium salt (40–60 mCi/mmol) (PerkinElmer Life Sciences). A PNS was prepared and then assayed for protein concentration. PNS from empty vector- and pHis-cellugyrin-transfected cells were adjusted to the same protein concentration. A high speed supernatant was prepared (S2). Goat anti-mouse magnetic beads (Dynal) were precoated for 2 h with 2 μg of nonspecific IgG or synaptophysin antibody and washed with Buffer A. Equal quantities of IgG or synaptophysin-coated beads were mixed overnight in S2 from empty vector- or synaptophysin-transfected cells. Unbound material, referred to here as postabsorptive supernatants (PAS) was removed. The beads were washed three times with Buffer A. Triton elution (TE), with 1% Triton X-100 in Buffer A, was used to elute the lipids from the beads. SDS elution (SE), performed with Laemmli sample buffer, was used to remove the bound antibody (anti-synaptophysin) and its ligand (synaptophysin) from the bead (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). Radioactivity of the culture media at time 0 and 24 h, PNS, S2, PAS, TE, and SE was quantified in a 1217 cintillation counter (LKB Wallace). In addition, PAS, TE, and SE were subjected to Western blot analysis. Immunofluorescence—PC12 cells transfected with pMyc/His-CR1–177-SCR67–76, which codes for rat cellugyrin with a truncated carboxyl terminus, a scrambled first luminal loop, and a carboxyl-terminal Myc/His tag, were lifted and grown on coverslips coated with poly-l-lysine overnight. Cells were then fixed with 4% paraformaldehyde in PBS for 30 min, washed with PBS, permeabilized with 0.2% Triton X-100 for 5 min, blocked with 4% donkey serum, and probed with either monoclonal anti-Myc and polyclonal anti-calnexin or polyclonal anti-Myc and monoclonal anti-synaptophysin antibodies followed by Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) and Alexa 488-conjugated donkey anti-mouse IgG (Molecular Probes, Inc., Eugene, OR). Incubation with primary and secondary antibodies lasted for 60 min at room temperature and was followed by six quick rinses with PBS. Next, nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride according to the manufacturer's instructions (Molecular Probes). The SlowFade-Light Antifade kit (Molecular Probes) was used for mounting coverslips onto slides. Staining was examined by fluorescence microscopy (Axiovert 200M; Zeiss). Gel Electrophoresis and Immunoblotting—Protein samples were separated by SDS-PAGE according to Laemmli and transferred to polyvinylidene difluoride membranes in 25 mm Tris, 192 mm glycine (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). Following transfer, the membrane was blocked with 10% nonfat dry milk in PBST for 1 h at 25 °C and probed with specific antibodies overnight. The following day membranes were washed three times with phosphate-buffered saline with 0.05% Tween and incubated with horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. After three more washes, the membranes were incubated in ECL reagent (PerkinElmer Life Sciences) for 1 min and then exposed to an Eastman Kodak Co. 440 image station. Data analysis was performed with Kodak 1D image analysis software. Hydropathy Plot Generation—The hydropathy plot of rat cellugyrin was generated by the Kyte-Doolittle method available on the Biology Workbench (available on the World Wide Web at workbench.sdsc. edu/) (21Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17297) Google Scholar). Statistical Analysis—Student's unpaired two-tailed t test was used to evaluate the statistical significance of the differences in targeting efficiencies. Targeting efficiency is defined as the amount of a specific protein in the S2 fraction normalized to the total amount of the same protein in the PNS. A paired t test was used to evaluate the statistical significance of the difference between synaptophysin-attributable radioactive counts in the Triton X-100 extract from empty vector and pHis-cellugyrin-transfected cells. Synaptophysin-attributable counts are defined as the counts precipitated with synaptophysin antibody minus the counts precipitated with nonspecific IgG. Cellugyrin Increases the Number of SLMVs in the Cell—In order to further characterize the biological role of cellugyrin, we explored the impact of exogenous cellugyrin expression on the physical characteristics of the SLMV. In a previous publication (15Belfort G.M. Kandror K.V. J. Biol. Chem. 2003; 278: 47971-47978Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), we showed that increasing the cellugyrin levels in the SLMV had no impact on vesicle size, since sedimentation in glycerol velocity gradients was unchanged. Now we asked whether the vesicles changed in buoyant density, hypothesizing that an increase in protein per vesicle should increase the buoyant density of the vesicles (Fig. 1). High speed supernatants (S2) from empty vector and pHis-cellugyrin-transfected cells were loaded on continuous 10–50% sucrose gradients and centrifuged for 16 h at 280,000 × g. The peak of cellugyrin and synaptophysin in both empty vector and His-cellugyrin-transfected cells is between fractions 11 and 15 (Fig. 1). Note that both endogenous and transfected cellugyrin signals are detectable in the lower cellugyrin panel. These experiments reveal that the vesicle pool from cellugyrin-transfected cells are the same buoyant density as those from empty vector-transfected cells. Given that the vesicles are the same size and buoyant density, we hypothesized that cellugyrin increases the number of vesicles per cell and that these new vesicles accommodate the increased accumulation of proteins. We tested this hypothesis using two independent approaches. First we used a method developed in our laboratory called antibody decoration/shift analysis. This method involves the addition of an excess of a single monoclonal antibody to a high speed supernatant (S2) containing the SLMVs. During the incubation period, the antibody binds to the cytoplasmic epitope of the target protein in a 1:1 stoichiometric ratio. Excess Nanogold-conjugated goat anti-mouse Fab fragments are included in the incubation to add more mass to each antibody. Next, the decorated vesicles are loaded on to a linear 10–30% sucrose gradient and centrifuged (see “Experimental Procedures”). Vesicles with a high copy number of the target protein migrate farther than vesicles with a lower copy number, because they are bound by more specific antibodies and in turn more Nanogold-labeled Fab fragments. In Fig. 2, we compared the shift of vesicles from empty vector- and cellugyrin-transfected cells using synaptophysin as the shifting antibody and nonspecific purified mouse IgG as the control. In the presence of nonspecific IgG, vesicles from both empty vector- and His-cellugyrin-transfected cells sediment with a peak between fractions 15 and 17. Anti-synaptophysin antibody shifts both peaks to between fractions 1 and 13. These data suggest that vesicles from cellugyrin-over-expressing cells and empty vector controls contain the same number of synaptophysin molecules. This implies that the increase in synaptophysin seen in the high speed supernatant (S2) is in fact explained by a greater number of vesicles. In order to verify the result obtained by antibody decoration/shift analysis, radioactive lipid labeling experiments were performed (Fig. 3, A and B). Cells transiently (Fig. 3A) or stably (Fig. 3B) transfected with either empty vector or His-cellugyrin were incubated in [1-14C]acetate overnight. A PNS was isolated, followed by adjustment of the protein concentrations of the different PNS samples so that they were equal. Then a high speed supernatant (S2) was obtained and split into two aliquots, one for immunoadsorption with anti-synaptophysin-coated goat antimouse magnetic beads and the other for mock immunoadsorption with nonspecific IgG-coated beads. The precipitated lipids along with vesicular proteins were eluted with 1% Triton X-100 (TE). Antibody and ligand were then eluted with SDS-containing sample buffer (SE). Liquid scintillation counting of the TE samples revealed ∼2.5-fold increase in the lipid content in these vesicles (p < 0.03) (Fig. 3, A and B). Given that the vesicles are the same size and density, this strongly suggests that expression of cellugyrin leads to an increase in the number of SLMVs. Fig. 3C demonstrates a Western blot analysis of fractions obtained during organelle immunoisolation. Cellugyrin's Effect on Synaptophysin Targeting Is Specific for PC12 Cells—Cellugyrin is a marker for ubiquitous microvesicles present in all cell types tested, including the COS7 monkey kidney cell line (15Belfort G.M. Kandror K.V. J. Biol. Chem. 2003; 278: 47971-47978Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). In order to explore whether or not gyrins can exert their effects in nonneuronal cells, we co-transfected His-cellugyrin (or His-synaptogyrin) with His-synaptophysin in COS7 cells (Fig. 4). Note that synaptophysin ectopically expressed in COS7 cells represents a doublet, which is consistent with earlier results of Linstedt and Kelly (22Linstedt A.D. Kelly R.B. Neuron. 1991; 7: 309-317Abstract Full Text PDF PubMed Scopus (100) Google Scholar). Total expression levels per microgram of transfected DNA of all three proteins were similar to one another in COS7 as well as in PC12 cells. In agreement with previously published results (15Belfort G.M. Kandror K.V. J. Biol. Chem. 2003; 278: 47971-47978Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), a significant fraction of ectopically expressed cellugyrin and synaptogyrin were found in the fraction of light vesicles recovered in the 16,000 × g supernatant of both COS7 and PC12 cells, suggesting that proteins of the gyrin family can facilitate formation of microvesicles in various different cells. Synaptophysin alone was not targeted efficiently to microvesicles in either cell type. In addition, cellugyrin and synaptogyrin did not facilitate the targeting of synaptophysin to the microvesicular compartment in COS7 cells, as they do in PC12 cells. These results argue against the possibility that the facilitation of synaptophysin targeting by either cellugyrin or synaptogyrin 1 represents an artifact of protein overexpression. Instead, these data suggest that synaptophysin targeting to the newly formed compartment requires a factor or factors that are only expressed in certain cell types. Mapping of Functional Domains of Cellugyrin—A comprehensive analysis of the rat cellugyrin protein was undertaken using recombinant DNA technology to identify domains responsible for the formation of SLMVs as well as cellugyrin's effect on synaptophysin targeting in PC12 cells (15Belfort G.M. Kandror K.V. J. Biol. Chem. 2003; 278: 47971-47978Abstract Full Text Full Text P" @default.
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• W2012095629 title "Cellugyrin Induces Biogenesis of Synaptic-like Microvesicles in PC12 Cells" @default.
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