Title: Cellular Localization, Oligomerization, and Membrane Association of the Hereditary Spastic Paraplegia 3A (SPG3A) Protein Atlastin
Abstract: Hereditary spastic paraplegias comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord. The early onset hereditary spastic paraplegia SPG3A is caused by mutations in the atlastin/human guanylate-binding protein-3 gene (renamed here atlastin-1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein superfamily of large GTPases. The atlastin-1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus. In cultured cortical neurons, atlastin-1 co-localized most prominently with markers of the Golgi apparatus, and immunogold electron microscopy revealed a predominant localization of atlastin-1 to the cis-Golgi. Yeast two-hybrid analyses and co-immunoprecipitation studies demonstrated that atlastin-1 can self-associate, and gel-exclusion chromatography and chemical cross-linking studies indicated that atlastin-1 exists as an oligomer in vivo, most likely a tetramer. Membrane fractionation and protease protection assays revealed that atlastin-1 is an integral membrane protein with two predicted transmembrane domains; both the N-terminal GTP-binding and C-terminal domains are exposed to the cytoplasm. Together, these findings indicate that the SPG3A protein atlastin-1 is a multimeric integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking. Hereditary spastic paraplegias comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord. The early onset hereditary spastic paraplegia SPG3A is caused by mutations in the atlastin/human guanylate-binding protein-3 gene (renamed here atlastin-1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein superfamily of large GTPases. The atlastin-1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus. In cultured cortical neurons, atlastin-1 co-localized most prominently with markers of the Golgi apparatus, and immunogold electron microscopy revealed a predominant localization of atlastin-1 to the cis-Golgi. Yeast two-hybrid analyses and co-immunoprecipitation studies demonstrated that atlastin-1 can self-associate, and gel-exclusion chromatography and chemical cross-linking studies indicated that atlastin-1 exists as an oligomer in vivo, most likely a tetramer. Membrane fractionation and protease protection assays revealed that atlastin-1 is an integral membrane protein with two predicted transmembrane domains; both the N-terminal GTP-binding and C-terminal domains are exposed to the cytoplasm. Together, these findings indicate that the SPG3A protein atlastin-1 is a multimeric integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking. Hereditary spastic paraplegias (HSPs) 1The abbreviations used are: HSPshereditary spastic paraplegiasGBPguanylate-binding proteinHAhemagglutininPBSphosphate-buffered salineNGSnormal goat serumFPLCfast protein liquid chromatographyCBPcalmodulin-binding peptideNIK/HGKNck-interacting kinase/hematopoietic progenitor kinase/germinal center kinase-like kinaseMAPKmitogen-activated protein kinaseSAPK/JNKstress-activated protein kinase/c-Jun N-terminal kinaseGTPγSguanosine 5′-O-(3-thiotriphosphate)DDP1deafness-dystonia-protein-1. are a group of neurological disorders characterized principally by progressive spasticity and weakness of the lower limbs (1.Tallaksen C.M.E. Dürr A. Brice A. Curr. Opin. Neurol. 2001; 14: 457-463Crossref PubMed Scopus (68) Google Scholar, 2.Crosby A.H. Proukakis C. Am. J. Hum. Genet. 2002; 71: 1009-1016Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 3.Fink J.K. Ann. Neurol. 2002; 51: 669-672Crossref PubMed Scopus (31) Google Scholar, 4.Fink J.K. Neurol. Clin. 2002; 20: 711-726Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5.Reid E. J. Med. Genet. 2003; 40: 81-86Crossref PubMed Scopus (137) Google Scholar). They typically exhibit axonal degeneration in the distal portions of long ascending dorsal column fibers and descending corticospinal tracts of the spinal cord, which constitute the longest motor and sensory axons in the central nervous system (6.Schwartz G.A. Liu C.-N. Arch. Neurol. Psychiatry. 1956; 75: 144-162Crossref Scopus (100) Google Scholar). HSPs have historically been classified as "pure" or "uncomplicated" if spastic paraplegia occurs in isolation and "complicated" if other neurological abnormalities are present (7.Harding A.E. Lancet. 1983; 1: 1151-1155Abstract PubMed Scopus (786) Google Scholar). More recently, the identification of new genetic loci for HSPs has permitted a molecular classification of HSPs (2.Crosby A.H. Proukakis C. Am. J. Hum. Genet. 2002; 71: 1009-1016Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 3.Fink J.K. Ann. Neurol. 2002; 51: 669-672Crossref PubMed Scopus (31) Google Scholar, 4.Fink J.K. Neurol. Clin. 2002; 20: 711-726Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5.Reid E. J. Med. Genet. 2003; 40: 81-86Crossref PubMed Scopus (137) Google Scholar). Of the 20 known loci (SPG1–20), 11 are autosomal dominant, six are autosomal recessive, and three are X-linked. Although most HSP patients experience progressive worsening of their symptoms, for some, the disorder does not appear to be progressive (3.Fink J.K. Ann. Neurol. 2002; 51: 669-672Crossref PubMed Scopus (31) Google Scholar, 4.Fink J.K. Neurol. Clin. 2002; 20: 711-726Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Thus, although some HSPs are clearly neurodegenerative disorders (e.g. SPG4), others such as SPG1 (due to mutations in the L1 cell adhesion molecule) and SPG3A (due to mutations in the atlastin GTPase) may be neurodevelopmental disorders (3.Fink J.K. Ann. Neurol. 2002; 51: 669-672Crossref PubMed Scopus (31) Google Scholar, 4.Fink J.K. Neurol. Clin. 2002; 20: 711-726Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). hereditary spastic paraplegias guanylate-binding protein hemagglutinin phosphate-buffered saline normal goat serum fast protein liquid chromatography calmodulin-binding peptide Nck-interacting kinase/hematopoietic progenitor kinase/germinal center kinase-like kinase mitogen-activated protein kinase stress-activated protein kinase/c-Jun N-terminal kinase guanosine 5′-O-(3-thiotriphosphate) deafness-dystonia-protein-1. Eight disease genes for HSPs have now been identified; and based on the proteins involved, several mechanisms for pathogenesis have been advanced. These include aberrant cell signaling or migration, abnormalities of mitochondrial chaperones, abnormalities of myelination, and defects in intracellular trafficking and transport (2.Crosby A.H. Proukakis C. Am. J. Hum. Genet. 2002; 71: 1009-1016Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 3.Fink J.K. Ann. Neurol. 2002; 51: 669-672Crossref PubMed Scopus (31) Google Scholar, 4.Fink J.K. Neurol. Clin. 2002; 20: 711-726Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5.Reid E. J. Med. Genet. 2003; 40: 81-86Crossref PubMed Scopus (137) Google Scholar). Proteins mutated in HSPs that have been implicated in cellular protein or vesicle trafficking include KIF5A (SPG10), spastin (SPG4), spartin (SPG20; Troyer syndrome), and atlastin (SPG3A) (reviewed in Refs. 2.Crosby A.H. Proukakis C. Am. J. Hum. Genet. 2002; 71: 1009-1016Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 3.Fink J.K. Ann. Neurol. 2002; 51: 669-672Crossref PubMed Scopus (31) Google Scholar, 4.Fink J.K. Neurol. Clin. 2002; 20: 711-726Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5.Reid E. J. Med. Genet. 2003; 40: 81-86Crossref PubMed Scopus (137) Google Scholar). KIF5A is a neuronal kinesin heavy chain motor protein involved in the transport of macromolecules and membranous organelles along the axon (8.Reid E. Kloos M. Ashley-Koch A. Hughes L. Bevan S. Svenson I.K. Graham F.L. Gaskell P.C. Dearlove A. Pericak-Vance M.A. Rubinsztein D.C. Marchuk D.A. Am. J. Hum. Genet. 2002; 71: 1189-1194Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). Spastin, a member of the AAA (for ATPases associated with a variety of cellular activities) protein family, associates with microtubules, and spastin overexpression causes microtubule disassembly (9.Errico A. Ballabio A. Rugarli E.I. Hum. Mol. Genet. 2002; 11: 153-163Crossref PubMed Scopus (284) Google Scholar). The spartin protein is similar to the VPS4, SNX15 (sorting nexin-15), and SKD1 proteins, which are involved in endosome morphology and protein trafficking of endosomal compartments (10.Patel H. Cross H. Proukakis C. Hershberger R. Bork P. Ciccarelli F.D. Patton M.A. McKusick V.A. Crosby A.H. Nat. Genet. 2002; 31: 347-348Crossref PubMed Scopus (212) Google Scholar). These latter proteins share with both spastin and spartin a region called the MIT (contained within microtubule-interacting and trafficking molecules) or ESP (present in End13/VPS4, SNX15, and PalB) domain (2.Crosby A.H. Proukakis C. Am. J. Hum. Genet. 2002; 71: 1009-1016Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 11.Ciccarelli F.D. Proukakis C. Patel H. Cross H. Azam S. Patton M.A. Bork P. Crosby A.H. Genomics. 2003; 81: 437-441Crossref PubMed Scopus (122) Google Scholar). Based on its similarity to members of the dynamin/Mx/guanylate-binding protein (GBP) superfamily of large GTPases (12.Prakash B. Praefcke G.J.K. Renault L. Wittinghofer A. Herrmann C. Nature. 2000; 403: 567-571Crossref PubMed Scopus (256) Google Scholar), the SPG3A protein atlastin (renamed here atlastin-1) has been implicated in intracellular trafficking, yet little is known regarding its cellular localization or function (13.Zhao X. Alvarado D. Rainier S. Lemons R. Hedera P. Weber C.H. Tukel T. Apak M. Heiman-Patterson T. Ming L. Bui M. Fink J.K. Nat. Genet. 2001; 29: 326-331Crossref PubMed Scopus (298) Google Scholar). Among the HSPs, SPG3A is particularly notable for the very early onset of pure spastic paraplegia. Five missense mutations and one single base insertion with premature termination of the predicted 558-amino acid coding region of atlastin-1 have been reported (13.Zhao X. Alvarado D. Rainier S. Lemons R. Hedera P. Weber C.H. Tukel T. Apak M. Heiman-Patterson T. Ming L. Bui M. Fink J.K. Nat. Genet. 2001; 29: 326-331Crossref PubMed Scopus (298) Google Scholar, 14.Muglia M. Magariello A. Nicoletti G. Patitucci A. Gabriele A.L. Conforti F.L. Mazzei R. Caracciolo M. Ardito B. Lastilla M. Tedeschi G. Quattrone A. Ann. Neurol. 2002; 51: 794-795Crossref PubMed Scopus (37) Google Scholar, 15.Tessa A. Casali C. Damiano M. Bruno C. Fortini D. Patrono C. Cricchi F. Valoppi M. Nappi G. Amabile G.A. Bertini E. Santorelli F.M. Neurology. 2002; 59: 2002-2005Crossref PubMed Scopus (42) Google Scholar, 16.Dalpozzo F. Rossetto M.G. Boaretto F. Sartori E. Mostacciuolo M.L. Daga A. Bassi M.T. Martinuzzi A. Neurology. 2003; 61: 580-581Crossref PubMed Scopus (30) Google Scholar). It has been speculated that these mutations may alter the structure, interactions, or GTPase activity of atlastin-1 (13.Zhao X. Alvarado D. Rainier S. Lemons R. Hedera P. Weber C.H. Tukel T. Apak M. Heiman-Patterson T. Ming L. Bui M. Fink J.K. Nat. Genet. 2001; 29: 326-331Crossref PubMed Scopus (298) Google Scholar, 14.Muglia M. Magariello A. Nicoletti G. Patitucci A. Gabriele A.L. Conforti F.L. Mazzei R. Caracciolo M. Ardito B. Lastilla M. Tedeschi G. Quattrone A. Ann. Neurol. 2002; 51: 794-795Crossref PubMed Scopus (37) Google Scholar, 15.Tessa A. Casali C. Damiano M. Bruno C. Fortini D. Patrono C. Cricchi F. Valoppi M. Nappi G. Amabile G.A. Bertini E. Santorelli F.M. Neurology. 2002; 59: 2002-2005Crossref PubMed Scopus (42) Google Scholar, 16.Dalpozzo F. Rossetto M.G. Boaretto F. Sartori E. Mostacciuolo M.L. Daga A. Bassi M.T. Martinuzzi A. Neurology. 2003; 61: 580-581Crossref PubMed Scopus (30) Google Scholar). Of the members of the dynamin/Mx/GBP superfamily, atlastin-1 is most similar to GBPs. Like GBPs, atlastin-1 possesses an RD loop instead of the classical (N/T)KXD sequence within the third motif of the guanylate-binding consensus triad (13.Zhao X. Alvarado D. Rainier S. Lemons R. Hedera P. Weber C.H. Tukel T. Apak M. Heiman-Patterson T. Ming L. Bui M. Fink J.K. Nat. Genet. 2001; 29: 326-331Crossref PubMed Scopus (298) Google Scholar). However, atlastin-1 lacks a C-terminal isoprenylation motif and the C-terminal α12/13 helix motif, two characteristic structural features of many of the GBPs (12.Prakash B. Praefcke G.J.K. Renault L. Wittinghofer A. Herrmann C. Nature. 2000; 403: 567-571Crossref PubMed Scopus (256) Google Scholar). Here, we demonstrate that atlastin-1 is an oligomeric GTPase that, unlike GBPs, is composed of subunits that are integral membrane proteins, with both N and C termini exposed to the cytoplasmic compartment. Atlastin-1 localizes prominently to the Golgi apparatus and is enriched in cerebral cortical pyramidal cells, a subpopulation of which exhibit a "long axonopathy" in patients with SPG3A. Sequence Analysis—Atlastin-1 homologs were identified with BLAST (17.Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). Chromosomal assignments were made using the NCBI Map-Viewer. 2Available at www.ncbi.nlm.nih.gov. Transmembrane helical domains were predicted using the SOSUI system (18.Hirokawa T. Boon-Chieng S. Mitaku S. Bioinformatics. 1998; 14: 378-379Crossref PubMed Scopus (1577) Google Scholar). Protein alignments were performed with ClustalW (19.Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56003) Google Scholar). Protein sequence similarities were calculated using BESTFIT analysis (Wisconsin Package Version 10.3, Accelrys, San Diego, CA). Eukaryotic DNA Expression Constructs and Cell Transfection—The full coding sequence of the atlastin-1 GTPase (GenBank™/EBI accession number NM_015915) was amplified by PCR using Pfu Turbo (Stratagene, La Jolla, CA) from a Marathon human brain (cerebral cortex) cDNA library (Clontech) and confirmed by DNA sequencing. The full-length atlastin-1 cDNA was cloned into the XmaI site of the eukaryotic expression vector pGW1 with Myc or hemagglutinin (HA) epitope tags at the N terminus as described previously (20.Blackstone C. Roberts R.G. Seeburg D.P. Sheng M. Biochem. Biophys. Res. Commun. 2003; 305: 345-352Crossref PubMed Scopus (13) Google Scholar). The full-length atlastin-1 cDNA was also cloned into the XmaI site of pRK5 (Genentech, South San Francisco, CA) for expression of the untagged protein. Torsin A (accession number AF007871) was cloned as a HindIII-BglII fragment into pGW1, preceding an in-frame C-terminal Myc tag. Site-directed mutagenesis was performed using the QuikChange method (Stratagene). African green monkey COS-7 cells (American Type Culture Collection CRL-1651) were maintained, transfected, and harvested as described previously (20.Blackstone C. Roberts R.G. Seeburg D.P. Sheng M. Biochem. Biophys. Res. Commun. 2003; 305: 345-352Crossref PubMed Scopus (13) Google Scholar). Antibodies—Affinity-purified antibodies against residues 1–18 (No. 5409; MAKNRRDRNSWGGFSEKTC-amide) and 544–558 (No. 4735; acetyl-CTPKSESTEQSEKKKM-OH) of atlastin-1 were prepared commercially (BioSource International, Hopkinton, MA), with terminal cysteines added to facilitate coupling. Antibodies were also prepared against residues 947–960 (No. 5174; acetyl-CEKLDAFIEALHQEK-OH) of human OPA1/Mgm1 (GenBank™/EBI accession number NM_ 015560) (21.Alexander C. Votruba M. Pesch U.E. Thiselton D.L. Mayer S. Moore A. Rodriguez M. Kellner U. Leo-Kottler B. Auberger G. Bhattacharya S.S. Wissinger B. Nat. Genet. 2000; 26: 211-215Crossref PubMed Scopus (1076) Google Scholar, 22.Delettre C. Lenaers G. Griffoin J.M. Gigarel N. Lorenzo C. Belenguer P. Pelloquin L. Grosgeorge J. Turc-Carel C. Perret E. Astarie-Dequeker C. Lasquellec L. Arnaud B. Ducommun B. Kaplan J. Hamel C.P. Nat. Genet. 2000; 26: 207-210Crossref PubMed Scopus (1169) Google Scholar). Mouse monoclonal anti-Myc (9E10), rabbit polyclonal anti-HA probe (Y-11), and goat anti-calregulin (T-19) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-deafness-dystonia protein-1 (DDP1)/TIMM8a antibodies have been described previously (20.Blackstone C. Roberts R.G. Seeburg D.P. Sheng M. Biochem. Biophys. Res. Commun. 2003; 305: 345-352Crossref PubMed Scopus (13) Google Scholar). Mouse monoclonal anti-calnexin (IgG1), anti-GM130 (IgG1), and anti-p115 (IgG1) antibodies were from Pharmingen. Mouse monoclonal anti-KDEL antibodies (clone 10C3, IgG2a) were from Stressgen Biotech Corp. (Victoria, British Columbia, Canada). Mouse monoclonal anti-microtubule-associated protein-2 antibody (clone HM-2, mouse ascites) was obtained from Sigma. Tissue Preparation and Subcellular Fractionation—Human tissue homogenates were obtained from Clontech. Brain subcellular fractions were prepared from Sprague-Dawley rats (150–175 g; Charles River Laboratories, Wilmington, MA). Dissected brains were homogenized in 0.32 m sucrose and 10 mm HEPES (pH 7.4) and centrifuged at 1330 × g for 3 min, generating a pellet (P1) and a supernatant (S1). The S1 supernatant was centrifuged at 21,200 × g for 10 min, producing a pellet (P2) and a supernatant (S2). The S2 supernatant was then centrifuged at 200,000 × g for 1 h, generating a P3 pellet and an S3 supernatant. Protein concentrations were determined by the BCA assay (Pierce) with bovine serum albumin as the standard. Gel Electrophoresis and Immunoblotting—Proteins were resolved by SDS-PAGE on 10 or 14% acrylamide gels and electrophoretically transferred to nitrocellulose (Hybond ECL, Amersham Biosciences). After blocking with 5% nonfat milk, 0.1% Tween 20, and Tris-buffered saline (pH 7.4) overnight, antibodies (0.1–1.0 μg/ml) were added for 1 h at 25 °C. After several washes with 0.1% Tween 20 and Tris-buffered saline, horseradish peroxidase-conjugated secondary antibodies (1:3000 dilution; Amersham Biosciences) were added for 30 min. Finally, after several washes with the blocking buffer, followed by Tris-buffered saline, immunoreactive proteins were revealed using Renaissance enhanced chemiluminescence reagent (PerkinElmer Life Sciences). Immunohistochemistry—Three adult male Sprague-Dawley rats (150–200 g) were perfused transcardially under deep pentobarbital anesthesia with 0.9% NaCl in 0.1 m phosphate buffer (pH 7.4), followed by 4% paraformaldehyde and finally 10% sucrose in 0.1 m phosphate buffer. Brains were post-fixed for 1 h and incubated overnight in phosphate-buffered 30% sucrose. Brains were then placed on a freezing microtome and cut into 50-μm-thick sections. These sections were collected in phosphate-buffered saline (PBS; pH 7.4) and stored at –20 °C in a cryoprotective solution consisting of 30% sucrose and 30% ethylene glycol in 0.05 m phosphate buffer (pH 7.2) until used. The immunohistochemical reaction was carried out using the ABC Elite kit (Vector Labs, Inc., Burlingame, CA) according to the manufacturer's instructions. Sections were preincubated in 1.0% normal goat serum (NGS), 10% bovine serum albumin, and PBS for 30 min at 25 °C and then incubated for 48 h at 4 °C with anti-atlastin-1 antibodies (No. 5409; 0.7 μg/ml) in 0.1% NGS, 1% bovine serum albumin, and PBS. In control experiments, the anti-atlastin-1 antibodies (No. 5409) were preincubated with the immunogenic peptide (0.25 μm) prior to use. The sections were then rinsed with PBS and incubated with biotinylated anti-rabbit IgG (Vector Labs, Inc.) in PBS with 1.5% NGS for 1 h at 25 °C. After rinsing with PBS, sections were incubated with the ABC complex (Vector Labs, Inc.) for 45 min and rinsed again. Peroxidase staining was revealed using 3,3′-diaminobenzidine (Fast DAB kit, Sigma). Sections were mounted on Superfrost Plus slides (Fisher), air-dried, dehydrated, and coverslipped with Cytoseal 60 (Stephens Scientific, Riverdale, NJ). Neuronal Culture and Immunocytochemistry—Primary cultures of rat cortical neurons were prepared from embryonic day 18 rat embryos and maintained as described previously (23.Zheng Y.-l. Li B.-S. Veeranna Pant H.C. J. Biol. Chem. 2003; 278: 24026-24032Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). After 6 days in culture, neurons were fixed with 4% formaldehyde for 30 min at 25 °C; washed several times with PBS; and then permeabilized and blocked for 30 min in 5% NGS, 0.2% saponin, and PBS. Cells were incubated overnight at 4 °C with antibodies against atlastin-1 (No. 5409; 10 μg/ml) and microtubule-associated protein-2 (1:200 dilution), p115 (0.5 μg/ml), GM130 (0.5 μg/ml), or KDEL (1.9 μg/ml) in 3% NGS, 0.05% saponin, and PBS. After washing three times with PBS, cells were incubated with Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies (1:500 dilution; Molecular Probes, Inc., Eugene, OR) in 3% NGS, 0.05% saponin, and PBS for 30 min, followed by three washes with PBS. Coverslips were then mounted using Gel/Mount (Biomeda, Foster City, CA). Fluorescent images were acquired with a Zeiss Axiovert 100M laser scanning confocal microscope and processed with Adobe Photoshop software. Immunogold Electron Microscopy—Rat cortical neuron cultures were prepared as described above. After 6 days in culture, neurons were fixed with 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4) for 30 min and then washed with 0.1 m phosphate buffer. The cells were permeabilized and blocked in 5% NGS, 0.1% saponin, and PBS for 1 h and incubated with anti-atlastin-1 antibodies (No. 5409) or without primary antibodies as a control in blocking buffer for 1 h. After washing with 1% NGS in PBS with 2% nonfat milk in PBS, cells were incubated with 1.4-nm Nanogold gold-conjugated anti-rabbit secondary antibodies (1:250 dilution; Nanoprobes, Yaphank, NY) in 2% nonfat milk in PBS for 1 h. After washing with 2% nonfat milk in PBS, the cells were fixed with 2% glutaraldehyde in PBS for 30 min. Finally, the cells were thoroughly washed with PBS and distilled water, silver-enhanced (HQ silver kit, Nanoprobes), and washed again with water and 0.1 m phosphate buffer. The cells were treated with 0.2% OsO4 in 0.1 m phosphate buffer for 30 min, mordanted en bloc with 0.25% uranyl acetate in acetate buffer (pH 5.0) overnight, washed and dehydrated with serial concentrations of ethanol, and finally infiltrated and embedded in epoxy resins. Thin sections of ∼70 nm were counterstained with uranyl acetate and lead citrate and examined under a Jeol 1200 EXII transmission electron microscope. Digital images were collected with an XR-100 CCD camera (Advanced Microscopy Techniques, Danvers, MA). Membrane Association Assays—COS-7 cells overexpressing atlastin-1 were washed twice with 10 mm Tris-HCl (pH 7.5) and then harvested in 10 mm Tris-HCl (pH 7.5), 10 mm NaCl, and 1.5 mm MgCl2. After multiple passes through a 25-gauge needle, the homogenate was centrifuged at 1330 × g. The post-nuclear supernatant was sonicated and then recentrifuged at 200,000 × g for 60 min, yielding a pellet (lysed membrane fraction) and a soluble fraction. The membrane fraction was treated with 1 m NaCl and 25 mm phosphate buffer (pH 7.4), with 100 mm glycine buffer (pH 2.8), with 100 mm carbonate buffer (pH 11.0), with 0.1% Triton X-100 and PBS, or with 1.0% sodium deoxycholate and PBS as indicated and centrifuged at 200,000 × g for 60 min to generate a final pellet and supernatant. In other experiments, soluble and membrane fractions were prepared from COS-7 cells overexpressing Myc-tagged wild-type atlastin-1 or various Myc-tagged atlastin-1 deletion constructs. Equal proportions of the soluble and membrane fractions were then immunoblotted with anti-Myc antibodies. Membranes from COS-7 cells overexpressing untagged atlastin-1 and P3 membrane fractions prepared from rat brain were subjected to phase partitioning with Triton X-114 as described by Bordier (24.Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar), and equal proportions of the aqueous and detergent phases were immunoblotted with anti-atlastin-1 antibodies (No. 5409). Protease Digestion and Deglycosylation Assays—COS-7 cells overexpressing untagged atlastin-1 were washed twice with 10 mm Tris-HCl (pH 7.5) and then collected in 10 mm Tris-HCl (pH 7.5), 10 mm NaCl, 1.5 mm MgCl2, and 10% sucrose. Cells were passed through a 25-gauge needle, and the homogenate was centrifuged at 3000 × g for 3 min. The pellet was discarded, and the supernatant was centrifuged at 130,000 × g for 60 min. This latter pellet was resuspended in the same buffer with or without proteinase K (EC 3.4.21.64; Sigma) at either 50 μm (30 min, 25 °C) or 200 μm (15 min, 37 °C). Reactions were terminated with 2 mm phenylmethylsulfonyl fluoride, followed immediately by lysis in SDS-PAGE sample buffer. Samples were then immunoblotted with antibodies against the C terminus (No. 4735) or N terminus (No. 5409) of atlastin-1 or with anti-calregulin antibodies. Protein deglycosylation with peptide N-glycosidase F (EC 3.5.1.52; New England Biolabs Inc., Beverly, MA) was performed as described previously (25.Blackstone C.D. Moss S.J. Martin L.J. Levey A.I. Price D.L. Huganir R.L. J. Neurochem. 1992; 58: 1118-1126Crossref PubMed Scopus (217) Google Scholar). Yeast Two-hybrid Tests—Yeast two-hybrid tests were performed using the L40 yeast strain harboring the reporter genes HIS3 and β-galactosidase under the control of upstream LexA-binding sites as described previously (20.Blackstone C. Roberts R.G. Seeburg D.P. Sheng M. Biochem. Biophys. Res. Commun. 2003; 305: 345-352Crossref PubMed Scopus (13) Google Scholar). Atlastin-1 deletion constructs were produced by PCR amplification using Pfu Turbo and cloned in-frame into pGAD10 prey and pBHA bait vectors (Clontech). All constructs were confirmed by DNA sequencing. Strength of interaction was assayed by β-galactosidase and HIS3 induction as described previously (20.Blackstone C. Roberts R.G. Seeburg D.P. Sheng M. Biochem. Biophys. Res. Commun. 2003; 305: 345-352Crossref PubMed Scopus (13) Google Scholar). Immunoprecipitation and Chemical Cross-linking—COS-7 cells co-transfected with HA- and Myc-atlastin-1 or transfected with Myc-atlastin-1 alone were washed twice with PBS and then harvested in 0.5% Triton X-100 and PBS and clarified by centrifugation at 130,000 × g for 30 min. Extracts (100 μg of protein) were incubated for 1–2 h at 4 °C with 5 μg of rabbit polyclonal anti-HA probe antibodies (Y-11) pre-coupled to protein A-Sepharose CL-4B (Amersham Biosciences). Beads were washed three times with 0.5% Triton X-100 and PBS. Bound proteins were resolved by SDS-PAGE and immunoblotted with mouse monoclonal anti-Myc antibodies. Chemical cross-linking with dithiobis(succinimidyl propionate) (Pierce) was performed using high speed pellets from the post-nuclear supernatant derived from Myc-atlastin-1-overexpressing COS-7 cells. Pellets were resuspended in PBS, and dithiobis(succinimidyl propionate) was added for 30 min on ice. Cross-linked products were resolved by SDS-PAGE under nonreducing conditions and immunoblotted with anti-Myc antibodies. Gel-exclusion FPLC—Myc-tagged wild-type or deletion mutants of atlastin-1 overexpressed in COS-7 cells were lysed in 0.1% Triton X-100 and PBS and clarified by centrifugation at 130,000 × g for 30 min. The soluble extract was applied to a Superdex 200 HR10/30 FPLC column (Amersham Biosciences) at a flow rate of 0.25 ml/min in 0.1% Triton X-100 and PBS. Fractions (0.25 ml) were collected, and proteins were resolved by SDS-PAGE and then immunoblotted using anti-Myc antibodies. Protein standards (Sigma and Amersham Biosciences) in 0.1% Triton X-100 and PBS were applied to the column to generate a standard curve, from which the native molecular masses for wild-type and deletion mutants of atlastin-1 were calculated. GTPase Activity Assays—The atlastin-1 cDNA was subcloned into pCAL-n-EK for the production of calmodulin-binding peptide (CBP) fusion proteins (Stratagene). Expression of CBP-atlastin-1 in Escherichia coli BL21(DE3) was induced by 100 μm isopropyl-β-d-thiogalactopyranoside for 4.5 h at 25 °C. After pelleting, cells were resuspended in 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 5 mm MgCl2, 2 mm CaCl2, 10% glycerol, 10 mm β-mercaptoethanol, 1.0% Triton X-100, and 0.5 mm phenylmethylsulfonyl fluoride and ruptured by two passages through a French pressure cell at 10,000 p.s.i. The extract was clarified by centrifugation at 50,000 × g for 30 min and then applied to calmodulin affinity resin (Stratagene). After washing with 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 5 mm MgCl2, 2 mm CaCl2, 10 mm β-mercaptoethanol, and 0.1% Triton X-100, bound fusion proteins were eluted with 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 10 mm β-mercaptoethanol, 2 mm EGTA, and 0.1% Triton X-100. Affinity-purified CBP-atlastin-1 fusion protein was dialyzed against assay buffer (20 mm HEPES (pH 7.2), 2 mm MgCl2, and 1 mm dithiothreitol). The reaction mixture for the GTPase assay included dialyzed CBP-atlastin-1 with 0.05% bovine serum albumin and 0.825 μm [α-32P]GTP (3000 Ci/mmol; ICN Biomedicals, Irvine, CA) in assay buffer. Samples of the reaction mixture at various time points (0–60 min) were spotted onto polyethyleneimine cellulose on polyester TLC plates (Sigma). Guanine nucleotides were separated by ascending chromatography in 1 m LiCl and 1.2 m formic acid. The [32P]GDP and [32P]GTP spots w