Evidence for a Primary Endocytic Vesicle Involved in Synaptic Vesicle Biogenesis
2000; Elsevier BV; Volume: 275; Issue: 10 Linguagem: Inglês
10.1074/jbc.275.10.7004
ISSN1083-351X
AutoresChester J. Provoda, Michael T. Waring, Kathleen M. Buckley,
Tópico(s)Retinal Development and Disorders
ResumoThe regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from “classical” synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles. The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from “classical” synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles. GLUT3, GLUT4, plasma membrane glucose transporter isoform types 1, 3, and 4, respectively transferrin receptor dopamine transporter hemagglutinin endoplasmic reticulum endoglycosidase H peptide:N-glycosidase F monoclonal antibody phosphate-buffered saline phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis bovine serum albumin room temperature horseradish peroxidase polyacrylamide gel electrophoresis The biogenesis of synaptic vesicles is a complex orchestration of events culminating in a vesicle population responsible for the uptake, storage, and regulated secretion of neurotransmitters. Current models of synaptic vesicle biogenesis suggest that at least two pathways exist for the formation of new synaptic vesicles after exocytosis: directly from the plasma membrane and indirectly via an intermediate endosomal compartment (1.Cremona O. De Camilli P. Curr. Opin. Neurobiol. 1997; 7: 323-330Crossref PubMed Scopus (196) Google Scholar, 2.Hannah M.J. Schmidt A.A. Huttner W.B. Annu. Rev. Cell Dev. Biol. 1999; 15: 733-798Crossref PubMed Scopus (152) Google Scholar). In neurons, both a direct pathway, which is at the active zone, and a more distal indirect pathway have been demonstrated at synapses in the Drosophila shibire ts1 mutant (3.Koenig J.H. Ikeda K. J. Cell Biol. 1996; 135: 797-808Crossref PubMed Scopus (229) Google Scholar). Studies of membrane recycling in hippocampal synapses using the fluorescent dye FM1-43 also suggest that synaptic vesicles formed by endocytosis can fuse with the plasma membrane directly, without passing through an endosomal compartment (4.Murthy V.N. Stevens C.F. Nature. 1998; 392: 497-501Crossref PubMed Scopus (227) Google Scholar). In the PC12 neuroendocrine cell line, synaptic vesicles are also derived from two different pathways as follows: directly from the plasma membrane (5.Schmidt A. Hannah M.J. Huttner W.B. J. Cell Biol. 1997; 137: 445-458Crossref PubMed Scopus (103) Google Scholar, 6.Shi G. Faundez V. Roos J. Dell'Angelica E.C. Kelly R.B. J. Cell Biol. 1998; 143: 947-955Crossref PubMed Scopus (84) Google Scholar) as well as from an endosomal intermediate (7.Grote E. Kelly R.B. J. Cell Biol. 1996; 132: 537-547Crossref PubMed Scopus (76) Google Scholar, 8.Desnos C. Clift-O'Grady L. Kelly R.B. J. Cell Biol. 1995; 130: 1041-1049Crossref PubMed Scopus (61) Google Scholar, 9.Lichtenstein 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, 10.Blagoveshchenskaya A.D. Hewitt E.W. Cutler D.F. J. Cell Biol. 1999; 145: 1419-1433Crossref PubMed Scopus (40) Google Scholar). Together these data form an emerging model of multiple pathways to recycle both synaptic vesicle proteins and synaptic vesicle membrane. Although presumably all synaptic vesicle proteins undergo endocytic trafficking, relatively little is known about the intermediate steps in the pathway to mature synaptic vesicles. In order to understand fully the mechanisms underlying synaptic vesicle biogenesis, it is critical that the synaptic vesicle protein trafficking pathways are fully elucidated. In neurons, synaptic vesicle proteins are present in heterogeneous vesicle populations, but the relationship of these compartments to the process of synaptic vesicle formation is almost impossible to determine, given the difficulty of performing a kinetic analysis of synaptic vesicle protein trafficking in brain (11.Huttner W.B. Schiebler W. Greengard P. De Camilli P. J. Cell Biol. 1983; 96: 1374-1388Crossref PubMed Scopus (919) Google Scholar, 12.Clift-O'Grady L. Linstedt A.D. Lowe A.W. Grote E. Kelly R.B. J. Cell Biol. 1990; 110: 1693-1703Crossref PubMed Scopus (152) Google Scholar, 13.Thoidis G. Chen P. Pushkin A.V. Vallega G. Leeman S.E. Fine R.E. Kandror K.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 183-188Crossref PubMed Scopus (25) Google Scholar). In contrast, PC12 cells have proved to be a useful system for analyzing the trafficking of synaptic vesicle proteins and the biogenesis of synaptic vesicles (5.Schmidt A. Hannah M.J. Huttner W.B. J. Cell Biol. 1997; 137: 445-458Crossref PubMed Scopus (103) Google Scholar, 6.Shi G. Faundez V. Roos J. Dell'Angelica E.C. Kelly R.B. J. Cell Biol. 1998; 143: 947-955Crossref PubMed Scopus (84) Google Scholar, 8.Desnos C. Clift-O'Grady L. Kelly R.B. J. Cell Biol. 1995; 130: 1041-1049Crossref PubMed Scopus (61) Google Scholar, 9.Lichtenstein 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, 10.Blagoveshchenskaya A.D. Hewitt E.W. Cutler D.F. J. Cell Biol. 1999; 145: 1419-1433Crossref PubMed Scopus (40) Google Scholar, 12.Clift-O'Grady L. Linstedt A.D. Lowe A.W. Grote E. Kelly R.B. J. Cell Biol. 1990; 110: 1693-1703Crossref PubMed Scopus (152) Google Scholar, 14.Cameron P. Mundigl O. De Camilli P. J. Cell Sci. 1993; 17: 93-100Crossref Google Scholar, 15.Clift-O'Grady L. Desnos C. Lichtenstein Y. Faundez V. Horng J.T. Kelly R.B. Methods Companion Methods Enzymol. 1998; 16: 150-159Crossref Scopus (50) Google Scholar, 16.Grote E. Hao J.C. Bennett M.K. Kelly R.B. Cell. 1995; 81: 581-589Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 17.Norcott J.P. Solari R. Cutler D.F. J. Cell Biol. 1996; 134: 1229-1240Crossref PubMed Scopus (52) Google Scholar, 18.Schmidt A. Huttner W.B. Methods Companion Methods Enzymol. 1998; 16: 160-169Crossref Scopus (30) Google Scholar). We have characterized a vesicular compartment in PC12 cells containing the synaptic vesicle proteins synaptophysin, SV2, and synaptotagmin. These vesicles are distinct in size and protein composition from synaptic vesicles; they contain two different endogenous glucose transporters, GLUT11 and GLUT3, as well as the transferrin receptor (TfR). Moreover, we show that exogenously expressed proteins such as GLUT4 and the sodium-dependent dopamine transporter (DAT) are also present in these vesicles. Our data provide evidence that this compartment is not derived by vesiculation of biosynthetic organelles or the plasma membrane and that vesicles with similar properties are present in rat brain. We suggest that this vesicle is part of the endocytic recycling system in neurons and PC12 cells, potentially operating as a primary endocytic vesicle involved in trafficking synaptic vesicle proteins that have escaped the direct targeting pathway to synaptic vesicles. PC12 cells were grown in high glucose Dulbecco's modified Eagle's media (Life Technologies, Inc.) supplemented with 2 mm glutamine, 10 units/ml penicillin/streptomycin, 5% horse serum, and 5% defined/supplemented bovine calf serum (HyClone, Logan, UT) in a humidified 37 °C incubator at 10% CO2. For transfection, cells were grown in 15-cm dishes to ∼75% confluence (∼3 × 107cells). The transfection method was adapted from Grote et al. (16.Grote E. Hao J.C. Bennett M.K. Kelly R.B. Cell. 1995; 81: 581-589Abstract Full Text PDF PubMed Scopus (146) Google Scholar), and the electroporation conditions were optimized for our cells and expression vector. Briefly, cells from one 15-cm dish were harvested by trituration in Ca2+/Mg2+-free PBS and pelleted for 5 min at 1000 × g. Cells were resuspended in 750 μl per dish electroporation buffer (137 mm NaCl, 5 mm KCl, 0.7 mmNa2HPO4, 6 mm dextrose, 20 mm HEPES, pH 7.2, equilibrated to room temperature (RT)) in the presence of 50 μg of plasmid cDNA and transferred to 0.4-cm electroporation cuvettes (Bio-Rad). After 3–5 min the cells were pulsed at 300 V (rather than 250 V (16.Grote E. Hao J.C. Bennett M.K. Kelly R.B. Cell. 1995; 81: 581-589Abstract Full Text PDF PubMed Scopus (146) Google Scholar)) and 500 microfarads and then transferred immediately to 10 ml of growth media supplemented with 3 mm EGTA prewarmed to 37 °C. After incubating for 30 min at 37 °C with gentle mixing, cells were pelleted for 5 min and re-plated onto two 15-cm polylysine-coated dishes. Sodium butyrate was added to a final concentration of 5 mm 24–30 h after electroporation, and the cells were harvested for experiments 16–18 h after the addition of sodium butyrate. The rat GLUT1 cDNA in the retroviral expression vector pDOJ was obtained from Dr. Morris J. Birnbaum (19.Verhey K.J. Hausdorff S.F. Birnbaum M.J. J. Cell Biol. 1993; 123: 137-147Crossref PubMed Scopus (66) Google Scholar). An epitope specific for the human GLUT1 amino acid sequence was engineered by changing amino acid 239 from Arg to His (19.Verhey K.J. Hausdorff S.F. Birnbaum M.J. J. Cell Biol. 1993; 123: 137-147Crossref PubMed Scopus (66) Google Scholar). The GLUT1 cDNA was ligated into the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA). The mycepitope (AAAEQKLISEEDLLI) including a STOP codon was added to the 3′-end of the coding region by first engineering by polymerase chain reaction an in-frame NotI restriction site between the last coding nucleotide and the STOP codon. The DNA between thisNotI site and the XbaI site in the polylinker was excised and replaced with a synthetic double-stranded DNA encoding themyc epitope. The HA epitope (IDYPYDVPDYA) was inserted into this cDNA by polymerase chain reaction between amino acids 53 and 54 in the exofacial loop between transmembrane domains 1 and 2. This chimera has previously been shown to be correctly processed in COS-7 and Chinese hamster ovary cells (20.Czech M.P. Chawla A. Woon C.W. Buxton J. Armoni M. Tang W. Joly M. Corvera S. J. Cell Biol. 1993; 123: 127-135Crossref PubMed Scopus (71) Google Scholar). The sequences of all constructs were confirmed by sequencing with Sequenase (Stratagene, La Jolla, CA). The rat GLUT4 cDNA was also obtained from Dr. Morris J. Birnbaum (University of Pennsylvania) and subcloned into the expression vector pCMV1. 2A. E. West, unpublished data. The human dopamine transporter was expressed by transfection of pcDNA3.1-DAT (21.Melikian H. Buckley K.M. J. Neurosci. 1999; 19: 7699-7710Crossref PubMed Google Scholar). PC12 cells were homogenized and analyzed in glycerol velocity gradients as in Clift-O'Grady et al. (12.Clift-O'Grady L. Linstedt A.D. Lowe A.W. Grote E. Kelly R.B. J. Cell Biol. 1990; 110: 1693-1703Crossref PubMed Scopus (152) Google Scholar) and Schmidt et al. (5.Schmidt A. Hannah M.J. Huttner W.B. J. Cell Biol. 1997; 137: 445-458Crossref PubMed Scopus (103) Google Scholar), with modifications. Cells were harvested in Ca2+/Mg2+-free PBS, pelleted for 5 min, and resuspended at 4 °C in 250 μl of homogenization buffer consisting of either 10 mm HEPES or 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA (HB) with 2 mm PMSF, 2 μg/ml aprotinin/leupeptin/pepstatin. All subsequent steps were carried out at 4 °C unless otherwise noted. Cells were homogenized with 10 passes through a stainless steel ball-bearing homogenizer (Berni-Tech Engineering, Saratoga, CA) with a 12-μm clearance. The homogenate was spun for 10 min at 1,400 × g in a microcentrifuge, and the supernatant was transferred to a Beckman thick-walled polycarbonate tube. The pellet was resuspended in 250 μl of HB plus protease inhibitors and passed through the homogenizer 4 times. This homogenate was then spun as before (P1) and the supernatants combined (S1). The S1 was spun at 40,000 rpm (66,0000 × g av) for 15 min in a Beckman (Fullerton, CA) TLA 100.4 rotor (P2) (5.Schmidt A. Hannah M.J. Huttner W.B. J. Cell Biol. 1997; 137: 445-458Crossref PubMed Scopus (103) Google Scholar). The supernatant (S2) was then used for further analysis. As a control for artifacts of our homogenization method, cells were also homogenized with a Teflon/glass homogenizer with 15 strokes at 500 rpm on ice in HB plus protease inhibitors. Under these conditions our homogenization efficiency was >90%. Identical results were obtained regardless of the homogenization method. The S2 (∼500 μl; ∼2 mg protein) was loaded onto a 4.5-ml 5–25% (v/v) linear gradient of glycerol diluted in HB, with a 200-μl 50% sucrose pad at the bottom of the gradient. Gradients were poured and fractions collected using an Auto Densi-Flow II apparatus (Labconco, Kansas City, MO). The gradient was spun in a Beckman SW55 rotor for 45 min at 48,000 rpm (218,438 × g av) at maximum acceleration and deceleration. Sixteen fractions (0.3 ml each 1–15, 0.6 ml fraction 16) were collected dropwise from the top of the gradient. Gradient fractions to be analyzed by SDS-PAGE were precipitated with 5% (v/v) trichloroacetic acid using sodium deoxycholate (200 μg/ml) as a carrier. The samples were incubated on ice for 10 min, spun for 30 min at 13,000 rpm in a microcentrifuge at 4 °C, and the pellets were resuspended in 20 μl of 1× SDS sample buffer consisting of 8m urea, 5% SDS, 20% glycerol, 1 mm EDTA, 1m 2-mercaptoethanol, and 0.25 m Tris, pH 6.8. In some cases, as noted in the figure legends, membranes were pelleted from individual gradient fractions prior to SDS-PAGE and Western blotting. Each fraction was diluted with 10 volumes of HB and spun at 85,000 rpm for 90 min in a Beckman (Fullerton, CA) TLA 100.4 rotor. Intact organelles were isolated from pooled gradient fractions using M-500 subcellular Dynabeads (Dynal, Great Neck, NY). Beads were first coated with goat anti-mouse IgG (Pierce) according to manufacturer's protocol. These beads were incubated overnight at 4 °C in HB, 0.1% BSA with SY38 (Roche Molecular Biochemicals), a mouse monoclonal antibody to synaptophysin, at a ratio of 1 μg of SY38, 1 mg of coated beads and washed with HB, 0.1% BSA to remove unbound antibody. This amount of SY38/M-500 beads was incubated with 100 μg (protein) of vesicles overnight at 4 °C in HB, 0.1% BSA. Organelle immunoisolation using the anti-SV2 monoclonal antibody was performed similarly, except that 1 ml of anti-SV2 hybridoma supernatant was used to coat the goat anti-mouse magnetic beads. The beads and bound organelles were pelleted in a Magnetic Particle Concentrator (Dynal) and were washed twice with 1 ml of HB, 0.1% BSA, once with 1 ml of HB at 4 °C, 10 min each. The supernatants were precipitated with trichloroacetic acid. Beads were resuspended in 20 μl of 1× SDS-PAGE sample buffer for analysis. In some cases, organelle immunoisolation was performed on individual gradient fractions. Briefly, 0.6 mg of goat anti-mouse IgG-coated M-500 Dynabeads (Dynal, Great Neck, NY) were coated with 0.5 μg of mAb SY38. This amount of beads plus primary antibody was incubated with one gradient fraction overnight at 4 °C. Beads were then washed and the unbound organelles pelleted for 1.5 h at ∼200,000 ×g in a Beckman TLA100.4 rotor. Approximately 2 g of frozen rat cerebral cortex (Zivic-Miller, Portersville, PA) was homogenized in 40 ml of ice-cold buffer (0.32 m sucrose, 5 mmHEPES, 0.1 mm EGTA, pH 7.4) with 14 strokes in a glass-Teflon homogenizer. The homogenate was spun in an SS34 rotor at 3500 rpm for 10 min at 4 °C. The supernatant (S1) was then transferred into fresh tubes and spun in the same rotor at 8500 rpm for 10 min at 4 °C. The resulting pellet (P2) was resuspended in 5 ml of ice-cold homogenization buffer, layered on top of 20 ml of 0.8m sucrose in 5 mm HEPES, 0.1 mmEGTA, pH 7.4, and spun in an SS34 rotor at 8500 rpm for 25 min at 4 °C. The clear middle layer was removed, gradually mixed with an equal volume of 1× synaptosome buffer (128 mm NaCl, 2.4 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, 10 mm HEPES, 10 mm glucose, pH 7.4), and centrifuged at 10,000 rpm in an SS34 rotor for 10 min at 4 °C. The pellet was resuspended in 1 ml of ice-cold distilled H2O to lyse the synaptosomes, then transferred to a small homogenizer (Thomas number A with type 5 ridged pestle), and resuspended with 5 strokes at 200 rpm. 0.1 volume of 10× synaptosome buffer was then added, and a protein assay was performed (BCA protein assay, Pierce). 5 mg of homogenate was loaded on a 5–25% glycerol gradient and spun as described for PC12 glycerol gradients above. Samples were resuspended for analysis by SDS-PAGE in 20 μl of 1× sample buffer (5% SDS, 8m urea, 1 m 2-mercaptoethanol, 1 mmEDTA, 20% v/v glycerol, 0.2% bromphenol blue, 0.25 mTris, pH 6.8). Samples precipitated with trichloroacetic acid were resuspended in sample buffer, and the pH of the samples was adjusted to neutrality by the addition of 2 μl of 1 m Tris base, pH 10.5. Samples were incubated 1 h at RT (this incubation was necessary to avoid aggregation of the GLUT1 transporter) and subjected to electrophoresis in 10% polyacrylamide gels. Proteins were transferred to nitrocellulose overnight at ∼100 mA 4 °C, and the nitrocellulose was blocked in TBST (150 mm NaCl, 0.05% v/v Tween 20, 50 mm Tris, pH 7.5) containing either 5% w/v nonfat milk solids or 1% casein (Sigma). Proteins were detected on Western blots with enhanced chemiluminescence by exposure to Kodak Biomax film. Quantitation of protein bands was performed by scanning the exposed film using a Umax Powerlook II digital scanner (Umax Data Systems Inc., Hsinchu, Taiwan, Republic of China). Scanned images were imported into Adobe Photoshop version 4.0 (San Jose, CA) at a pixel resolution of 600 dpi using Input Level settings of “225,” “10,” and “1.0”. Images were inverted and imported into ImageQuant version 1.2 software (Molecular Dynamics, Sunnyvale, CA) for quantitative analysis. Bands to be quantitated were selected as identically sized rectangular objects, using the smallest sized rectangle that would accommodate all bands of a particular protein, and background-corrected using Object Averaging. Intact vesicles were purified as described above. Proteins bound to the beads were denatured with two successive incubations in 10 μl of 0.5% SDS, 1% 2-mercaptoethanol at RT for 30 min each. Sample volumes were adjusted to 30 μl with 1% Nonidet P-40, enzyme digestion buffer, and 250 units of endoglycosidase H (Endo H; New England Biolabs, Beverly, MA) or 250 units of peptide:N-glycosidase F (PNGase F; New England Biolabs) and incubated 16 h at RT. PC12 cells were transfected as described above, except that after transfection the cells were plated onto 15-cm dishes coated with 5 μg/cm2rat tail collagen (Collaborative Research, Bedford, MA). Forty-eight hours after transfection the cells were equilibrated to 4 °C for 1 h, after which the cells were washed 2–3 times with borate buffer (50 mm sodium borate, pH 9.0, 150 mmNaCl) at 4 °C. All subsequent steps were done at 4 °C. Cells were then incubated twice, for 15 min, with 5 ml 0.5 mg/ml sulfo-NHS-SS-biotin (Pierce) in borate buffer on a rocking platform with slow agitation. Cells were then washed once with PBS, followed by quenching of remaining biotin with serum-free Dulbecco's modified Eagle's media supplemented with 100 mm glycine. After removal of the quenching medium, cells were harvested in PBS plus 10 mm EDTA and 2 mm PMSF with a cell scraper and processed as described for glycerol gradient analysis or organelle immunoisolation. PC12 cells were transfected and plated onto collagen-coated dishes as described above. Medium was replaced 16–18 h after the addition of sodium butyrate with fresh, prewarmed medium and incubated for 1 h at 37 °C, after which the cells were incubated with fresh medium containing 5 μg/ml 12CA5 anti-HA monoclonal antibody for 1 h at 4 or 37 °C. Cells used for the 4 °C labeling control experiment were equilibrated to 4 °C for 20 min prior to the addition of antibody. After incubation with antibody the cells were washed twice with 4 °C PBS, after which the 37 °C cells were allowed an additional 10 min to equilibrate to 4 °C. All subsequent steps were carried out at 4 °C. The 37 °C cells were then incubated with 30 μg/ml Pronase E (Sigma) in PBS, 10 mm EDTA for 5 min to strip antibody remaining at the cell surface, after which all Pronase buffer was removed (22.Warren R.A. Green F.A. Stenberg P.E. Enns C.A. J. Biol. Chem. 1998; 273: 17056-17063Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Pronase treatment was immediately stopped by the addition of complete media containing 2 mm PMSF plus 5 μg/ml aprotinin/leupeptin/pepstatin/benzamidine. Cells were resuspended, pelleted, and washed 2 more times with PBS, 10 mm EDTA plus protease inhibitors. Final cell pellets were resuspended in HB plus protease inhibitors for homogenization and glycerol velocity gradient analysis (see above). The anti-synaptotagmin monoclonal antibody Cl 41.1 was the generous gift of Dr. Reinhard Jahn (Max Planck Institute, Göttingen, Germany). 12CA5 hybridoma cells were purchased from ATCC (Manassas, VA). For cell-surface labeling mAb 12CA5 was purified from hybridoma supernatant using the Pierce Immunopure (G) IgG purification kit. 3F10 anti-HA high affinity rat monoclonal antibody, SY38 anti-synaptophysin, anti-rat IgG biotin, anti-mouse IgG biotin, and anti-rabbit IgG biotin were all purchased from Roche Molecular Biochemicals. Rabbit anti-GLUT3, rabbit anti-GLUT1, and rabbit anti-GLUT4 polyclonal antibodies were purchased from Chemicon (Temecula, CA). Anti-Thy-1 mouse monoclonal antibody was purchased from PharMingen (San Diego, CA). Anti-α-mannosidase II mouse monoclonal antibody was purchased from Babco (Richmond, CA). Anti-transferrin receptor monoclonal antibody was purchased fromZymed Laboratories Inc. (South San Francisco, CA). Anti-calnexin rabbit polyclonal antibody was obtained from StressGen(Victoria, British Columbia, Canada). Streptavidin-horseradish peroxidase was purchased from Amersham Pharmacia Biotech. Previous experiments have examined the distribution of exogenously expressed GLUT1 and GLUT4 in PC12 cells. GLUT1 is predominantly expressed in the plasma membrane (23.Hudson A.W. Fingar D.C. Seidner G.A. Griffiths G. Burke B. Birnbaum M.J. J. Cell Biol. 1993; 122: 579-588Crossref PubMed Scopus (53) Google Scholar), whereas GLUT4 is found in both large dense core vesicles (23.Hudson A.W. Fingar D.C. Seidner G.A. Griffiths G. Burke B. Birnbaum M.J. J. Cell Biol. 1993; 122: 579-588Crossref PubMed Scopus (53) Google Scholar) and a vesicle population that sediments more rapidly than synaptic vesicles in glycerol velocity gradients (24.Herman G.A. Bonzelius F. Cieutat A.M. Kelly R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12750-12754Crossref PubMed Scopus (85) Google Scholar). Initially, we were interested in using GLUT1 as a control protein for experiments on synaptic vesicle protein targeting. When we transfected HA-tagged GLUT1 into PC12 cells and examined its distribution relative to synaptic vesicle proteins in glycerol velocity gradients, we found that although the majority of the protein was found in heavier membranes, including the plasma membrane and endosomes as described previously (see P1 and P2 in Fig. 9) (23.Hudson A.W. Fingar D.C. Seidner G.A. Griffiths G. Burke B. Birnbaum M.J. J. Cell Biol. 1993; 122: 579-588Crossref PubMed Scopus (53) Google Scholar), a peak of GLUT1HA immunoreactivity was reproducibly localized to a region of the gradient that overlapped with but was clearly distinct from the peak of immunoreactivity for the synaptic vesicle protein synaptophysin (Fig. 1 A). We occasionally observed a shoulder (Fig. 1 B, arrow), and infrequently a distinct second peak (Fig.2), of synaptic vesicle protein immunoreactivity, including synaptophysin, SV2, and synaptotagmin, at the same position as the GLUT1 peak. This distribution was also seen when we expressed a chimeric protein in which the amino-terminal cytoplasmic domain of GLUT1 was replaced with the amino-terminal cytoplasmic domain of SV2A (data not shown). To determine if this colocalization in the second vesicle peak in the gradient represented comigration of two different vesicle populations or colocalization of GLUT1 and synaptophysin in the same organelles, we immunoisolated vesicles containing synaptic vesicle proteins from this region of the glycerol gradient with an antibody to a cytoplasmic epitope of synaptophysin. Vesicles immunoprecipitated with synaptophysin antibodies were then analyzed by SDS-PAGE and Western blotting with antibodies to the HA epitope tag in GLUT1. Fig.3 A shows that antibodies to synaptophysin specifically and quantitatively immunoprecipitate the GLUT1HA present in these fractions, providing strong evidence for the presence of both proteins in the same vesicle population. Furthermore, no detectable GLUT1HA is immunoprecipitated by synaptophysin antibodies from fractions containing the major peak of synaptophysin immunoreactivity, suggesting that GLUT1 is excluded from the classical synaptic vesicles in PC12 cells. Fig. 3 B shows that antibodies to SV2, another synaptic vesicle-specific protein, are also capable of precipitating GLUT1HA, confirming that the presence of other synaptic vesicle proteins in this vesicle population are consistent with the bimodal distribution occasionally seen in Western blots (Fig.2).Figure 1The distribution of transfected GLUT1 on glycerol velocity gradients of PC12 cell homogenates. PC12 cells transiently transfected with HA epitope-tagged GLUT1 (GLUT1HA) were homogenized 48 h after transfection, and the subcellular distributions of GLUT1HA (▵) and synaptophysin (Sphysin) (●) were determined following separation of organelles in the homogenate in a 5–25% glycerol velocity gradient. In this and subsequent experiments, fractions were collected from the top of the gradient and loaded onto the gel from left to right. Proteins in each gradient fraction were resuspended in SDS sample buffer, resolved by 10% SDS-PAGE, transferred to nitrocellulose, and probed for the presence of GLUT1HA with 3F10 rat anti-HA monoclonal antibody and for synaptophysin with SY38 mouse monoclonal antibody. Proteins were detected using biotinylated secondary antibodies and streptavidin-HRP with enhanced chemiluminescence. A, a representative gradient of 4 different experiments: top,GLUT1HA; bottom, synaptophysin (Sphysin). The molecular weights (×10−3) of prestained protein standards are shown on the left. B, quantitation of proteins analyzed in A: GLUT1HA (▵) and synaptophysin (●). Protein bands were digitally scanned and quantitated by densitometry and the amount of protein expressed in arbitrary units (AU). GLUT1HA is present in membranes that copurify with a shoulder of synaptophysin immunoreactivity (fractions 8–10, arrow), distinct from the main peak of synaptophysin (fraction 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Bimodal distributions of SV2, synaptophysin, and synaptotagmin. SV2, synaptophysin and synaptotagmin occasionally exhibit marked bimodal distributions consistent with their presence in two distinct vesicle populations. Homogenates of PC12 cells were analyzed by separation in glycerol velocity gradients followed by Western blotting as described in Fig. 1, except that the membranes in each gradient fraction were pelleted for 2 h at 200,000 ×g prior to solubilization in SDS
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