Characterization of Amyotrophic Lateral Sclerosis-linked P56S Mutation of Vesicle-associated Membrane Protein-associated Protein B (VAPB/ALS8)

The P56S mutation in VAPB (vesicle-associated membrane protein-associated protein B) causes autosomal dominant motoneuronal diseases. Although it was reported that the P56S mutation induces localization shift of VAPB from endoplasmic reticulum (ER) to non-ER compartments, it remains unclear what the physiological function of VAPB is and how the P56S mutation in VAPB causes motoneuronal diseases. Here we demonstrate that overexpression of wild type VAPB (wt-VAPB) promotes unfolded protein response (UPR), which is an ER reaction to suppress accumulation of misfolded proteins, and that small interfering RNA for VAPB attenuates UPR to chemically induced ER stresses, indicating that VAPB is physiologically involved in UPR. The P56S mutation nullifies the function of VAPB to mediate UPR by inhibiting folding of VAPB that results in insolubility and aggregate formation of VAPB in non-ER fractions. Furthermore, we have found that expression of P56S-VAPB inhibits UPR, mediated by endogenous wt-VAPB, by inducing aggregate formation and mislocalization into non-ER fractions of wt-VAPB. Consequently, the P56S mutation in a single allele of the VAPB gene may diminish the activity of VAPB to mediate UPR to less than half the normal level. We thus speculate that the malfunction of VAPB to mediate UPR, caused by the P56S mutation, may contribute to the development of motoneuronal degeneration linked to VAPB/ALS8. The P56S mutation in VAPB (vesicle-associated membrane protein-associated protein B) causes autosomal dominant motoneuronal diseases. Although it was reported that the P56S mutation induces localization shift of VAPB from endoplasmic reticulum (ER) to non-ER compartments, it remains unclear what the physiological function of VAPB is and how the P56S mutation in VAPB causes motoneuronal diseases. Here we demonstrate that overexpression of wild type VAPB (wt-VAPB) promotes unfolded protein response (UPR), which is an ER reaction to suppress accumulation of misfolded proteins, and that small interfering RNA for VAPB attenuates UPR to chemically induced ER stresses, indicating that VAPB is physiologically involved in UPR. The P56S mutation nullifies the function of VAPB to mediate UPR by inhibiting folding of VAPB that results in insolubility and aggregate formation of VAPB in non-ER fractions. Furthermore, we have found that expression of P56S-VAPB inhibits UPR, mediated by endogenous wt-VAPB, by inducing aggregate formation and mislocalization into non-ER fractions of wt-VAPB. Consequently, the P56S mutation in a single allele of the VAPB gene may diminish the activity of VAPB to mediate UPR to less than half the normal level. We thus speculate that the malfunction of VAPB to mediate UPR, caused by the P56S mutation, may contribute to the development of motoneuronal degeneration linked to VAPB/ALS8. Amyotrophic lateral sclerosis (ALS) 3The abbreviations used are: ALS, amyotrophic lateral sclerosis; SMA, spinal muscular atrophy; VAMP, vesicle-associated membrane protein; VAP, VAMP-associated protein; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; UPR, unfolded protein response; TMD, transmembrane domain; GST, glutathione S-transferase; ER, endoplasmic reticulum; wt-VAPB, wild type VAPB; HRP, horseradish peroxidase; DTT, dithiothreitol; MES, 2-(N-morpholino)ethanesulfonic acid; siVAPB, small interfering RNA for VAPB; MBS, MES-buffered saline. 3The abbreviations used are: ALS, amyotrophic lateral sclerosis; SMA, spinal muscular atrophy; VAMP, vesicle-associated membrane protein; VAP, VAMP-associated protein; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; UPR, unfolded protein response; TMD, transmembrane domain; GST, glutathione S-transferase; ER, endoplasmic reticulum; wt-VAPB, wild type VAPB; HRP, horseradish peroxidase; DTT, dithiothreitol; MES, 2-(N-morpholino)ethanesulfonic acid; siVAPB, small interfering RNA for VAPB; MBS, MES-buffered saline. is the most prevalent fatal motor neuron disease, characterized by progressive loss of upper and lower motor neurons (1Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Crossref PubMed Scopus (1167) Google Scholar, 2Bruijn L.I. Miller T.M. Cleveland D.W. Annu. Rev. Neurosci. 2004; 27: 723-749Crossref PubMed Scopus (1182) Google Scholar). Although typical cases occur sporadically, some patients have a genetic background.To date, four ALS-causative genes have been identified, and precise characterization of their physiological roles and abnormalities by ALS-causing mutations is bringing us clues as to how ALS and other motor neuron diseases occur. Overexpression of mutants of Cu/Zn-superoxide dismutase (SOD1), whose gene is the most characterized familial ALS-related one known as ALS1, causes neuronal cell death in vitro (3Ghadge G.D. Lee J.P. Bindokas V.P. Jordan J. Ma L. Miller R.J. Roos R.P. J. Neurosci. 1997; 17: 8756-8766Crossref PubMed Google Scholar, 4Kanekura K. Hashimoto Y. Niikura T. Aiso S. Matsuoka M. Nishimoto I. J. Biol. Chem. 2004; 279: 19247-19256Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and an ALS-like phenotype in vivo (5Gurney M.E. Pu H. Chiu A.Y. Dal Canto M.C. Polchow C.Y. Alexander D.D. Caliendo J. Hentati A. Kwon Y.W. Deng H.X. Chen W. Zhai P. Sufit R.L. Siddique T. Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3417) Google Scholar, 6Chiba T. Hashimoto Y. Tajima H. Yamada M. Kato R. Niikura T. Terashita K. Schulman H. Aiso S. Kita Y. Matsuoka M. Nishimoto I. J. Neurosci. Res. 2004; 78: 542-552Crossref PubMed Scopus (40) Google Scholar). A recently identified autosomal recessive ALS-causative gene, ALS2, encodes alsin protein (7Hadano S. Hand C.K. Osuga H. Yanagisawa Y. Otomo A. Devon R.S. Miyamoto N. Showguchi-Miyata J. Okada Y. Singaraja R. Figlewicz D.A. Kwiatkowski T. Hosler B.A. Sagie T. Skaug J. Nasir J. Brown Jr., R.H. Scherer S.W. Rouleau G.A. Hayden M.R. Ikeda J.E. Nat. Genet. 2001; 29: 166-173Crossref PubMed Scopus (585) Google Scholar, 8Yang Y. Hentati A. Deng H.X. Dabbagh O. Sasaki T. Hirano M. Hung W.Y. Ouahchi K. Yan J. Azim A.C. Cole N. Gascon G. Yagmour A. Ben-Hamida M. Pericak-Vance M. Hentati F. Siddique T. Nat. Genet. 2001; 29: 160-165Crossref PubMed Scopus (658) Google Scholar) that has several functional domains common to Rho guanine nucleotide-exchanging factors (RhoGEF) (9Kanekura K. Hashimoto Y. Kita Y. Sasabe J. Aiso S. Nishimoto I. Matsuoka M. J. Biol. Chem. 2005; 280: 4532-4543Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and Rab5GEF (10Otomo A. Hadano S. Okada T. Mizumura H. Kunita R. Nishijima H. Showguchi-Miyata J. Yanagisawa Y. Kohiki E. Suga E. Yasuda M. Osuga H. Nishimoto T. Narumiya S. Ikeda J.E. Hum. Mol. Genet. 2003; 12: 1671-1687Crossref PubMed Scopus (212) Google Scholar). We have recently demonstrated that alsin exerts neuroprotective function via its RhoGEF domain against neurotoxicity by SOD1 mutants in vitro (4Kanekura K. Hashimoto Y. Niikura T. Aiso S. Matsuoka M. Nishimoto I. J. Biol. Chem. 2004; 279: 19247-19256Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Ablation of the ALS2 gene caused mild motor disorder (11Hadano S. Benn S.C. Kakuta S. Otomo A. Sudo K. Kunita R. Suzuki-Utsunomiya K. Mizumura H. Shefner J.M. Cox G.A. Iwakura Y. Brown Jr., R.H. Ikeda J.E. Hum. Mol. Genet. 2006; 15: 233-250Crossref PubMed Scopus (113) Google Scholar). ALS4, a recently identified autosomal dominant ALS-associated gene, is thought to encode a DNA/RNA helicase whose function remains unknown (12Chen Y.Z. Bennett C.L. Huynh H.M. Blair I.P. Puls I. Irobi J. Dierick I. Abel A. Kennerson M.L. Rabin B.A. Nicholson G.A. Auer-Grumbach M. Wagner K. De Jonghe P. Griffin J.W. Fischbeck K.H. Timmerman V. Cornblath D.R. Chance P.F. Am. J. Hum. Genet. 2004; 74: 1128-1135Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar).ALS8, encoding mutated VAPB (vesicle-associated membrane protein-associated protein B), was most recently identified from a large Brazilian family with autosomal dominant motor neuron diseases. The P56S point mutation in VAPB caused a typical ALS phenotype with rapid progression or late onset spinal muscular atrophy (SMA) (13Nishimura A.L. Mitne-Neto M. Silva H.C. Richieri-Costa A. Middleton S. Cascio D. Kok F. Oliveira J.R. Gillingwater T. Webb J. Skehel P. Zatz M. Am. J. Hum. Genet. 2004; 75: 822-831Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar). This mutation has affected eight families totaling 1500 individuals, of whom 200 suffer from ALS/SMA (14Nishimura A.L. Al-Chalabi A. Zatz M. Hum. Genet. 2005; 118: 499-500Crossref PubMed Scopus (83) Google Scholar).The human VAP family proteins were initially identified as homologues of vesicle-associated membrane protein (VAMP)-associated protein (VAP) with a size of 33 kDa in Aplysia californica (aVAP33) (15Nishimura Y. Hayashi M. Inada H. Tanaka T. Biochem. Biophys. Res. Commun. 1999; 254: 21-26Crossref PubMed Scopus (144) Google Scholar) that is involved in exocytosis of neurotransmitters (16Skehel P.A. Martin K.C. Kandel E.R. Bartsch D. Science. 1995; 269: 1580-1583Crossref PubMed Scopus (145) Google Scholar). They include VAPA (also known as VAP33), VAPB, and VAPC. VAPB and VAPC are alternatively spliced variants. VAPA and VAPB, which interact with each other, associate with VAMP/synaptobrevin (15Nishimura Y. Hayashi M. Inada H. Tanaka T. Biochem. Biophys. Res. Commun. 1999; 254: 21-26Crossref PubMed Scopus (144) Google Scholar). It was subsequently demonstrated that the yeast VAP homologue, called SCS2 (suppressor of choline sensitivity 2), compensates for the defect of IRE1 and HAC1 (17Kagiwada S. Hosaka K. Murata M. Nikawa J. Takatsuki A. J. Bacteriol. 1998; 180: 1700-1708Crossref PubMed Google Scholar). IRE1 and HAC1 encode yeast homologues of mammalian IRE1 (inositol-requiring enzyme-1) and mammalian transcriptional factor XBP1 (X-box-binding protein-1). IRE1 and XBP1 play a pivotal role in inositol metabolism and unfolded protein response (UPR) (18Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Crossref PubMed Scopus (2093) Google Scholar), an ER process to suppress accumulation of unfolded protein in ER (19Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1919) Google Scholar). Several other reports have also suggested that VAPB may play important roles in the structural regulation of ER, protein transport, phospholipid metabolism, and also viral infection (17Kagiwada S. Hosaka K. Murata M. Nikawa J. Takatsuki A. J. Bacteriol. 1998; 180: 1700-1708Crossref PubMed Google Scholar, 20Amarilio R. Ramachandran S. Sabanay H. Lev S. J. Biol. Chem. 2005; 280: 5934-5944Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 21Hamamoto I. Nishimura Y. Okamoto T. Aizaki H. Liu M. Mori Y. Abe T. Suzuki T. Lai M.M. Miyamura T. Moriishi K. Matsuura Y. J. Virol. 2005; 79: 13473-13482Crossref PubMed Scopus (169) Google Scholar).Based on the foregoing studies, it is speculated that VAPB is involved in ER function, especially UPR induction mediated by the IRE1/XBP1 pathway, and in inositol metabolism (22Wyles J.P. McMaster C.R. Ridgway N.D. J. Biol. Chem. 2002; 277: 29908-29918Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 23Kagiwada S. Zen R. J. Biochem. (Tokyo). 2003; 133: 515-522Crossref PubMed Scopus (35) Google Scholar), although there is no direct evidence supporting this idea. Moreover, it remains unclear how the ALS-causing P56S-VAPB mutant participates in the pathomechanism of ALS. So far, the sole reported finding relating to the latter issue is that P56S-VAPB localizes in non-ER and non-Golgi compartments, whereas wt-VAPB does so in ER and the Golgi apparatus (13Nishimura A.L. Mitne-Neto M. Silva H.C. Richieri-Costa A. Middleton S. Cascio D. Kok F. Oliveira J.R. Gillingwater T. Webb J. Skehel P. Zatz M. Am. J. Hum. Genet. 2004; 75: 822-831Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar).In this study, we demonstrate that VAPB plays an important role in UPR. We further show that the P56S mutation causes almost complete loss of function of VAPB to mediate UPR by inducing its misfolding and localization shift to the non-ER compartments. In addition, P56S-VAPB suppresses UPR, mediated by wt-VAPB, by interfering with the folding of wt-VAPB. Inhibition of wt-VAPB-mediated UPR by coexpressed P56S-VAPB may occur possibly because wt-VAPB is firmly dimerized with P56S-VAPB and may be trapped into inactive homodimerized protein complexes. Considering the data together, we speculate that the function of VAPB to mediate UPR may be diminished to less than half of the normal level by the P56S mutation in a single allele of the VAPB gene. We further speculate that the malfunction of VAPB to mediate UPR, induced by the P56S mutation, may eventually contribute to the development of motoneuronal degeneration linked to ALS8 by permitting the accumulation of misfolded proteins in the ER.EXPERIMENTAL PROCEDURESAntibodies and Compounds—Rabbit anti-VAPB polyclonal antibody was purchased from Abcam (Cambridge, UK). Immunoblot analysis with this antibody only detects overexpression levels of VAPB, but not endogenous levels of VAPB, in NSC34 cells. Rabbit polyclonal anti-VAPB-P antibody was raised against an N-terminal human VAPB peptide (N-terminal 14 residues) by Tanpaku Seisei Kogyo. Anti-FLAG-M2 beads consisting of anti-FLAG M2 monoclonal antibody, horseradish peroxidase (HRP)-conjugated anti-FLAG M2 monoclonal antibody, and rabbit anti-actin polyclonal antibody were purchased from Sigma. Mouse monoclonal anti-Xpress antibody and HRP-conjugated anti-HisG antibody, which recognizes the N-terminal His6-Gly tag, were purchased from Invitrogen. HRP-conjugated goat anti-mouse secondary antibody and HRP-conjugated goat anti-rabbit secondary antibody were purchased from Bio-Rad (Hercules, CA). Rabbit polyclonal anti-calreticulin antibody and anti-calnexin antibody were purchased from Stressgen (Victoria, Canada). Texas Red-conjugated goat anti-rabbit polyclonal antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The following mouse monoclonal antibodies were purchased from companies: anti-Myc antibody, Biomol (Plymouth Meeting, PA); HRP-conjugated anti-HA antibody, Roche Applied Science; anti-GST-antibody, Upstate (Charlottesville, VA); anti-tubulin antibody, Oncogene (Cambridge, MA). Thapsigargin, bafilomycin A, and brefeldin A were purchased from Sigma. MG132 was purchased from Calbiochem.Constructions—Human cDNAs encoding VAPB (GenBank™ accession number NM_004738), VAPA (GenBank™ accession number BT019618), VAMP1 (GenBank™ accession number NM_014231), and VAMP2 (GenBank™ accession number NM_014232) were amplified from a human postcentral gyrus cDNA library (Biochain, Hayward, CA) by PCR with a sense primer (5′-CGGGATCCACCAATGGCGAAGGTGGAGCAGGTC-3′) and an antisense primer (5′-GGAATTCCTACAAGGCAATCTTCCCAATAATTAC-3′) for human VAPB, a sense primer (5′-CGGGATCCACCATGGCGAAGCACGAGCAG-3′) and an antisense primer (5′-GGAATTCCTACAAGATCAATTTCCCTAGAAAGAATC-3′) for VAPA, a sense primer (5′-CGGGATCCACCATGTCTGCTCCAGCTCAGC-3′) and an antisense primer (5′-GGAATTCAGTAAAAAAGTAGATTACAATAAACTACCACGATG-3′) for VAMP1, and a sense primer (5′-CGGGATCCACCATGTCTGCTACCGCTG-3′) and an antisense primer (5′-GGAATTCAGTGCTGAAGTAAACTATGATGATGATG-3′) for VAMP2, respectively.P56S-VAPB, P56S-VAPA, P56A-VAPB, P56K-VAPB, P56D-VAPB, P56del-VAPB, and K87D/M89D-VAPB were obtained by site-directed mutagenesis with a sense primer (5′-GGTACTGTGTGAGGTCCAACAGCGGAATCATCG-3′) and an antisense primer (5′-CGATGATTCCGCTGTTGGACCTCACACAGTACC-3′) for P56S-VAPB and P56S-VAPA, a sense primer (5′-GGTACTGTGTGAGGGCCAACAGCGGAATCATCG-3′) and an antisense primer (5′-CGATGATTCCGCTGTTGGCCCTCACACAGTACC-3′) for P56A-VAPB, a sense primer (5′-GGTACTGTGTGAGGAAGAACAGCGGAATCATCG-3′) and an antisense primer (5′-CGATGATTCCGCTGTTCTTCCTCACACAGTACC-3′) for P56K-VAPB, a sense primer (5′-GGTACTGTGTGAGGGACAAACAGCGGAATCATCG-3′) and an antisense primer (5′-CGATGATTCCGCTGTTGTCCCTCACACAGTACC-3′) for P56D-VAPB, a sense primer (5′-GGTACTGTGTGAGGAACAGCGGAATCATCG) and an antisense primer (5′-CGATGATTCCGCTGTTCCTCACACAGTACC-3′) for P56del-VAPB, and a sense primer (5′-CCCAATGAGAAAAGTAAAACACGACTTTGACGTTCAGTCTATGTTTGCTCC-3′) and an antisense primer (5′-GGAGCAAACATAGACTGAACGTCAAAGTCGTGTTTACTTTTCTCATTGGG-3′) for K87D/M89D-VAPB, respectively. Plasmid-based small interfering RNA for silencing of endogenous VAPB was constructed as follows. Two oligonucleotides, a sense fragment (5′-CGGGATCCCGTAGACTGAACCATAAACTTGTTTGATATCCGACAAGTTTATGGTTCAGTCTATTTTTTCCAAGGTACCCC-3′) and an antisense fragment (5′-GGGGTACCTTGGAAAAAATAGACTGAACCATAAACTTGTCGGATATCAAACAAGTTTATGGTTCAGTCTACGGGATCCCG), were annealed in vitro and subcloned into the BamHI-KpnI site of pRNA-U6.1/Shuttle vector (Genscript). pCAX-F-XBP1-Venus and pCAX-F-XBP1-ΔDBD-Venus constructs were kindly provided by Dr. Masayuki Miura (Tokyo University). pXJ-HA-ubiquitin plasmid was a kind gift from Dr. Victor Yu (National University of Singapore).Cell Culture and Transfection—Motoneuronal NSC34 cell, a hybrid cell line established from a mouse neuroblastoma cell line and mouse embryo spinal cord cells, was a kind gift from Dr. Neil Cashman (Toronto University). NSC34 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% of fetal bovine serum (Hyclone, Logan, UT). NSC34 cells were seeded onto a 6-cm culture dish at 2.1 × 105 cells/dish 24 h before transfection. Transfection was performed under the manufacturer's protocol. In brief, DNA (3 μg), Lipofectamine (6 μl), and PLUS reagent (12 μl) were premixed for each transfection. The premixed complexes were then added onto the cells cultured in serum-free Dulbecco's modified Eagle's medium, and 3 h after transfection, the culture media were changed to 10% fetal bovine serum plus Dulbecco's modified Eagle's medium.Immunoblot Analysis—Samples were mixed with equal amounts of 2× sample buffer containing 4% SDS and boiled for 5 min at 95 °C. The samples were applied to SDS-polyacrylamide gels and blotted onto polyvinylidene fluoride membranes. Immunoreactive bands were detected with ECL Western blotting detection reagents (Amersham Biosciences). Intensities of immunodetected signals were densitometrically estimated with NIH Image.Immunocytochemistry—COS7 cells, plated onto cell culture dishes, were transfected with N-terminally EGFP-tagged VAPB by lipofection with Lipofectamine and PLUS reagent. Fortyeight h after transfection, the cells were fixed with 4% paraformaldehyde plus phosphate-buffered saline. ER was probed by anti-calreticulin antibody (Stressgen) and visualized by Texas Red-conjugated goat anti-rabbit antibody (Jackson ImmunoReseach Laboratories). The cells were observed with confocal microscopy LSM510 (Carl Zeiss).Pull-down Assay—NSC34 cells, transiently expressing GST-fused proteins and His6-Xpress-tagged proteins, were harvested 48 h after transfection and lysed with a pull-down buffer (150 mm NaCl, 20 mm HEPES (pH 7.5), 1 mm EDTA, 1 mm dithiothreitol (DTT), 0.5% Triton X-100, protease inhibitors) by pipetting and a freeze-thaw cycle. After centrifugation, the cell lysates were precleared with Sepharose beads for 4 h and pulled down with glutathione-Sepharose beads (Amersham Biosciences) for 4 h. After being washed four times with the pull-down buffer, the precipitates were immunoblotted with anti-HisG antibody (for the detection of His6-Xpress-tagged proteins) and anti-GST antibody.Ubiquitination Assay—NSC34 cells, cotransfected with the vector encoding HA-ubiquitin in association with the pEF4/His vector, pEF4/His-wt-VABP, or pEF4/His-P56S-VAPB, were harvested 48 h after transfection for lysis with the radioimmune precipitation buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). The cell lysates were then precleared with Sepharose beads for 4 h and immunoprecipitated with anti-Xpress antibody. After being washed four times with the radioimmune precipitation buffer, the precipitates were immunoblotted with HRP-conjugated anti-HA antibody (to detect ubiquitinated proteins) and anti-HisG antibody (to detect VAPB).Fractionation into Soluble and Insoluble Fractions—NSC34 cells overexpressing VAPB were harvested 48 h after transfection for lysis with a cell lysis buffer (10 mm Tris-HCl (pH 7.4), 1% Triton X-100, 1 mm EDTA, protease inhibitors) by pipetting and a freeze-thaw cycle. The soluble fraction was defined as the supernatant of the cell lysates after centrifugation at 12,000 × g for 5 min. After complete removal of the supernatant, the pellets were resuspended in 500 μl of the cell lysis buffer and sonicated for 10 s to homogenize the mixture. The solutions were then centrifuged for 5 min at 12,000 × g, and the supernatant was completely removed. The resulting pellets, defined as the insoluble fractions, were solubilized by pipetting in the 4% SDS-containing sample buffer for subsequent immunoblot analysis.Fractionation by Sucrose Density Gradient Centrifugation—Untransfected NSC34 cells or NSC34 cells transfected with VAPB-encoding vectors were harvested for suspension in the 1% Triton X-100-MBS lysis buffer (1% Triton-X100, 25 mm MES (pH 6.7), 150 mm NaCl) and rotated for 20 min at 4 °C, followed by 10 passages through 26-gauge needles. The suspended total cell lysates, mixed with an equal volume of 80% sucrose plus MBS (final 40% sucrose), were initially loaded to 4.5-ml ultracentrifuge tubes (Beckman). A discontinuous sucrose gradient was then formed by sequentially layering 30% sucrose-MBS and 5% sucrose plus MBS. After the tubes were subjected to ultracentrifugation at 260,000 × g for 18 h in Beckman SW-Ti60 rotor at 4 °C, the gradient was divided into the same volume (350 μl) of fractions from the top to the bottom of the tube. The pellet was sonicated to suspend in 350 μl of the 1% Triton X-100-MBS lysis buffer. An equal volume (20 μl) of each fraction was then analyzed by Western blot.RESULTSThe P56S Mutation Induces the Insolubility of VAPB—We cloned a human wt-VAPB cDNA, from which the P56S-VAPB mutant cDNA was generated by site-directed mutagenesis (Fig. 1A). The 56th proline, located at the middle of the major sperm protein domain, is highly conserved among VAP family proteins derived from various species. We constructed their expression vectors to express various epitope-tagged proteins (Fig. 1B).It has been generally accepted that many neurodegenerative disease-linked mutations result in misfolding and aggregation of disease-related proteins (24Bruijn L.I. Houseweart M.K. Kato S. Anderson K.L. Anderson S.D. Ohama E. Reaume A.G. Scott R.W. Cleveland D.W. Science. 1998; 281: 1851-1854Crossref PubMed Scopus (981) Google Scholar, 25Bates G. Lancet. 2003; 361: 1642-1644Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 26Shults C.W. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1661-1668Crossref PubMed Scopus (347) Google Scholar). Misfolded proteins are prone to be easily polyubiquitinated (27Obata Y. Niikura T. Kanekura K. Hashimoto Y. Kawasumi M. Kita Y. Aiso S. Matsuoka M. Nishimoto I. J. Neurosci. Res. 2005; 81: 720-729Crossref PubMed Scopus (11) Google Scholar, 28Urushitani M. Kurisu J. Tsukita K. Takahashi R. J. Neurochem. 2002; 83: 1030-1042Crossref PubMed Scopus (221) Google Scholar). To assess whether the P56S mutation induces misfolding and aggregation of VAPB, we first examined the status of polyubiquitination of P56S-VAPB. As shown in Fig. 2A, P56S-VAPB was more ubiquitinated than wt-VAPB, supporting the idea that P56S-VAPB was also misfolded. Note that the expression level of P56S-VAPB is much lower than that of wt-VAPB despite transfection with same amount of plasmids.FIGURE 2P56S mutation enhances insolubility and polyubiquitination of VAPB. A, the P56S mutation enhanced polyubiquitination of VAPB. NSC34 cells expressing His6-Xpress-tagged wt-VAPB or P56S-VAPB in association with HA-ubiquitin (Ub) were lysed with the radioimmune precipitation buffer, and the total cell lysates were immunoprecipitated with anti-Xpress antibody. Inputs and immunoprecipitates (IP) were then immunoblotted with anti-HA antibody or anti-HisG antibody. B, the P56S mutation increased the Triton X-100-insoluble fraction of VAPB protein. NSC34 cells were transfected with the vector encoding His6-Xpress-tagged wt-VAPB or P56S-VAPB. The Triton X-100-soluble (sol.) and insoluble (insol.) fractions were immunoblotted with anti-VAPB-P antibody. The black and white arrowheads indicate the exogenous (exo.) and endogenous (endo.) VAPB. C and D, COS7 cells were transfected with the vectors encoding N-terminally-EGFP-fused wt-VAPB (wt-VAPB) (C) or N-terminally EGFP-fused P56S-VAPB (P56S-VAPB) (D). At 48 h after transfection, the cells were fixed and immunostained with anti-calreticulin (ER marker), followed by visualization with Texas Red-conjugated secondary antibody. E-G, untransfected NSC34 cells (E) or cells transfected with pEF4/His-wt-VAPB (F) or cells transfected with pEF4/His-P56S-VAPB (G), lysed in 1% Triton X-100-MBS, were subjected to sucrose density gradient centrifugation analysis. Each fraction (350 μl) was numbered in a top-to-bottom-of-tube order. Endogenous VAPB, His6-Xpress-VAPB, calnexin (ER membrane marker), or calreticulin (ER marker) were probed with anti-VAPB-P antibody, anti-HisG antibody, anti-calnexin antibody, or anti-calreticulin antibody, respectively. Percentages indicate sucrose concentrations. short, short chemoimmunofluorescent exposure; long, long chemoimmunofluorescent exposure; P, pellet.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Based on these findings, we tested whether the P56S mutation increased insolubility of VAPB in the Triton X-100-containing cell lysis buffer (1% Triton X-100, 10 mm Tris-HCl (pH 7.5), 1 mm EDTA, protease inhibitors). As shown in Fig. 2B, the amount of the Triton X-100-soluble P56S-VAPB was much smaller than that of Triton X-100-soluble wt-VAPB, whereas the amount of the Triton X-100-insoluble P56S-VAPB was much greater than the Triton X-100-insoluble wt-VAPB. As expected, a smeared ladder, presumably composed of super-shifted P56S-VAPB proteins, was detected in the insoluble fraction of P56S-VAPB. These higher molecular weight bands were assumed to be SDS-containing buffer-insoluble P56S-VAPB with some modifications or P56S-VAPB aggregates. Thus, it was concluded that the P56S mutation reduced the solubility of VAPB proteins by inducing misfolding of VAPB protein. Considering that VAPB is homodimerized or heterodimerized with other synaptic proteins via the C-terminal transmembrane domain (TMD) (15Nishimura Y. Hayashi M. Inada H. Tanaka T. Biochem. Biophys. Res. Commun. 1999; 254: 21-26Crossref PubMed Scopus (144) Google Scholar, 21Hamamoto I. Nishimura Y. Okamoto T. Aizaki H. Liu M. Mori Y. Abe T. Suzuki T. Lai M.M. Miyamura T. Moriishi K. Matsuura Y. J. Virol. 2005; 79: 13473-13482Crossref PubMed Scopus (169) Google Scholar) and that the P56S mutation does not interfere with this oligomerization (data not shown, but see Fig. 6), VAPB-interacting proteins may be trapped into insoluble aggregates of P56S-VAPB and contribute to the VAPB-containing smeared ladder.FIGURE 6P56S-VAPB keeps its interaction with synaptic vesicular proteins. NSC34 cells were harvested for pull-down analyses at 48 h after transfection. All pull-down assays were performed with glutathione-Sepharose beads. The precipitates were then immunoblotted with anti-HisG antibody (to detect His6-Xpress-tagged proteins) (top) or anti-GST antibody (to detect GST-tagged protein) (bottom). A, wt-VAPB (wt) or P56S-VAPB (P56S) forms a homodimer. NSC34 cells were cotransfected with the GST-encoding backbone vector (vec), the vector encoding GST-wt-VAPB or GST-P56S-VAPB, in association with the vector encoding His6-Xpress-wt-VAPB or His6-Xpress-P56S-VAPB. B, wt-VAPB or P56S-VAPB forms a heterodimer with wt-VAPA. NSC34 cells were cotransfected with GST-vec or the vector encoding GST-wt-VAPB or GST-P56S-VAPB in association with the vector encoding His6-Xpress-wt-VAPA or His6-Xpress-P56S-VAPA. C, wt-VAPB or P56S-VAPB forms a heterodimer with VAMP1 or VAMP2. NSC34 cells were cotransfected with the GST-vec or the vector encoding GST-VAMP1 or GST-VAMP2 in association with the vector encoding His6-Xpress-wt-VAPB or His6-Xpress-P56S-VAPB.View Large Image Figure ViewerDownload Hi-res image Download (PPT)P56S-VAPB Shows Different Subcellular Localization—wt-VAPB has been known to mainly localize in ER (13Nishimura A.L. Mitne-Neto M. Silva H.C. Richieri-Costa A. Middleton S. Cascio D. Kok F. Oliveira J.R. Gillingwater T. Webb J. Skehel P. Zatz M. Am. J. Hum. Genet. 2004; 75: 822-831Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar, 20Amarilio R. Ramachandran S. Sabanay H. Lev S. J. Biol. Chem. 2005; 280: 5934-5944Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 21Hamamoto I. Nishimura Y. Okamoto T. Aizaki H. Liu M. Mori Y. Abe T. Suzuki T. Lai M.M. Miyamura T. Moriishi K. Matsuura Y. J. Virol. 2005; 79: 13473-13482Crossref PubMed Scopus (169) Google Scholar). It has been also demonstrated that the P56S-VAPB mutant does not co-localize with the Golgi apparatus or ER (13Nishimura A.L. Mitne-Neto M. Silva H.C. Richieri-Costa A. Middleton S. Cascio D. Kok F. Oliveira J.R. Gillingwater T. Webb J. Skehel P. Zatz M. Am. J. Hum. Genet. 2004; 75: 822-831Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar). To visualize these pr