Differential Regulation of Endoplasmic Reticulum Structurethrough VAP-Nir Protein Interaction*

Received for publication, August 19, 2004, and in revised form, November 15, 2004Published, JBC Papers in Press, November 15, 2004, DOI 10.1074/jbc.M409566200

Roy Amarilio‡, Sreekumar Ramachandran‡, Helena Sabanay§, and Sima Lev‡¶

From the ‡Neurobiology Department and the §EM unit, Weizmann Institute of Science, Rehovot 76100, Israel

The endoplasmic reticulum (ER) exhibits a charac-teristic tubular structure that is dynamically re-arranged in response to specific physiological de-mands. However, the mechanisms by which the ERmaintains its characteristic structure are largely un-known. Here we show that the integral ER-membraneprotein VAP-B causes a striking rearrangement of theER through interaction with the Nir2 and Nir3 pro-teins. We provide evidence that Nir (Nir1, Nir2, andNir3)-VAP-B interactions are mediated through theconserved FFAT (two phenylalanines (FF) in acidictract) motif present in Nir proteins. However, eachinteraction affects the structural integrity of the ERdifferently. Whereas the Nir2-VAP-B interaction in-duces the formation of stacked ER membrane arrays,the Nir3-VAP-B interaction leads to a gross remodelingof the ER and the bundling of thick microtubules alongthe altered ER membranes. In contrast, the Nir1-VAP-B interaction has no apparent effect on ER struc-ture. We also show that the Nir2-VAP-B interactionattenuates protein export from the ER. These resultsdemonstrate new mechanisms for the regulation of ERstructure, all of which are mediated through interac-tion with an identical integral ER-membrane protein.

The endoplasmic reticulum (ER)1 is an extensive network ofmembranes comprised of an array of interconnecting tubulesand cisternae that emerges from the nuclear envelope (NE) andextends peripherally throughout the cell cytoplasm (1). It con-tains several structurally distinct domains, including the NE,the rough and smooth ER (rER and sER), and the regions thatcontact other organelles, such as the Golgi apparatus, the late

endosomes, the lysosomes, mitochondria, peroxisomes, and theplasma membrane (2). The ER functions in diverse metabolicprocesses including lipid synthesis, carbohydrate metabolism,and the detoxification of drugs. It is responsible for the synthe-sis, translocation, glycosylation, folding, assembly, and proc-essing of secretory and membrane proteins, and it functions inintracellular calcium storage and sequestering (3, 4).

While the function of the ER in membrane trafficking, lipidbiosynthesis, and calcium signaling have been extensivelystudied, the mechanism by which the ER maintains its char-acteristic structure in vivo remains largely unknown. Studiesfrom yeast and mammalian cells have shown that the sizeand/or structure of the ER is extremely sensitive to certaincellular stress conditions, such as the unfolded protein re-sponse (UPR) or to the overexpression of a subset of ER-resident membrane proteins (5, 6). Overexpression of 3-hy-droxymethyl-3-methylglutaryl coenzyme A (HMG-CoA)reductase, microsomal aldehyde dehydrogenase (msALDH),cytochrome P-450, and malfolded cytochrome P-450, causesthe proliferation of ER membranes and stacking of the ERcisternae into organized structures known as crystalloid ER,sinusoidal ER, or karmellae (7–12). The formation of thesestructures is easily visible and can be used as a quantitativemethod for studying ER membrane biogenesis (5). Neverthe-less, the underlying mechanism of their formation is notcompletely understood. It has been proposed that proteinsthat regulate lipid metabolism, such as HMG-CoA reductase,induce the formation of these structures because of the ex-tensive requirement of membrane biogenesis. However, sub-sequent studies suggested that the oligomerization stateof the ER integral membrane proteins is crucial for theirproduction (7, 13, 14).

The organization of the ER is also sensitive to drugs thatinduce microtubule depolymerization, such as colchicine or no-codazole. These drugs cause retraction of the ER tubules fromthe cell periphery and consequently, the formation of ER mem-brane aggregates around the nucleus. It has long been knownthat the ER uses the microtubules as a framework for extend-ing and maintaining its reticular organization in animal cells(1, 15). The interaction of the ER with microtubules is mediatedby motor proteins, which interact directly with microtubulesand concomitantly with the ER membranes via protein-proteininteractions. Their interaction with the ER and their motoractivity are required for the sliding of ER tubules along sta-tionary microtubules and consequently, motility of the ER net-work. In contrast, the position of the ER within the cells and itsmotility by microtubule movement or tip-attachment mecha-nisms (3) require static association of the ER membranes withmicrotubules, and this is thought to be mediated by proteinssuch as p63 or cytoplasmic linker proteins (CLIPs). p63 is atype II integral ER membrane protein that binds directly tomicrotubules via its cytoplasmic domain and thereby links the

* This work was supported by the Israel Science Foundation (GrantNo. 1073/03), the Israel Cancer Research Foundation, and by the Harryand Jeanette Weinberg Fund for the Molecular Genetics of Cancer. Thecosts of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

¶ Sima Lev is an incumbent of the Helena Rubinstein Career Develop-ment Chair. To whom correspondence should be addressed: Dept. ofNeurobiology, Weizmann Institute of Science, Rehovot 76100, Israel. Tel.:972-8-934-2126; Fax: 972-8-934-4131; E-mail: sima.lev@weizmann.ac.il.

1 The abbreviations used are: ER, endoplasmic reticulum; CLIPs,cytoplasmic linker proteins; HMG-CoA, 3-hydroxymethyl-3-methyl-glutaryl coenzyme A; NE, nuclear envelope; NSF, NEM-sensitiveprotein; SNAP, soluble NSF protein attachment protein; SNARE,SNAP receptor; OSBP, oxysterol-binding protein; PDI, protein-disul-fide isomerase; PI, phosphatidylinositol; rdgB, retinal degenerationB; TMD, transmembrane domain; Scs2p, Saccharomyces cerevisiaesuppressor of choline sensitivity; VAMP, vesicle-associated mem-brane protein; VAP, VAMP-associated protein; VSV-G, vesicular sto-matitis virus G; HA, hemagglutinin; PBS, phosphate-buffered saline;DTT, dithiothreitol; FFAT, FF in acidic tract; GST, glutathione S-transferase; HEK, human embryonic kidney cells; YFP, yellow fluo-rescent protein; EM, electron microscopy.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 7, Issue of February 18, pp. 5934–5944, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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ER membranes to the microtubule network (16, 17). CLIPs aresoluble non-motor microtubule-binding proteins that link mi-crotubules to intracellular organelles by binding a putativemembrane receptor. Among the CLIPs, CLIP-170 links endo-somes to microtubules and might be involved in the regulationof ER extension (18).

VAP-B is also a type II integral membrane protein of �31kDa that has been previously localized to the ER and thepre-Golgi intermediates (19, 20). It belongs to a highly con-served family of proteins, which are implicated in the regula-tion of neurotransmitter release, ER-Golgi and intra-Golgitransport, Glu4 (glucose transporter 4) trafficking, stabiliza-tion of presynaptic microtubules, and the expression of phos-pholipid biosynthetic genes (19, 21–25). These diverse func-tions have been demonstrated in different species and celltypes, and are mediated by different members of this family.Nevertheless, the overall structures of VAP proteins are simi-lar and consist of a large N-terminal region facing the cyto-plasm and a hydrophobic C terminus that functions as a trans-membrane domain (TMD) (20). The cytoplasmic regioncontains a conserved N-terminal domain of 100 amino acids,which shares high sequence similarity with the nematode ma-jor sperm protein (MSP). This 100 amino acid domain containsa highly conserved sequence of 16 amino acids. The central partof the cytoplasmic region contains a coiled-coil domain of �40amino acids, which is a common motif in many t-SNARE (sol-uble N-ethylmaleimide-sensitive factor attachment protein(SNAP) receptor) proteins (26).

The VAP proteins interact with several intracellular proteinsand have the ability to interact with each other (27–29). Orig-inally, the Aplysia ApVAP33 was isolated as a VAMP/synapto-brevin-interacting protein using the yeast two-hybrid screen(21). Subsequent studies demonstrated the interaction of themammalian VAP-A with additional SNAREs, including syn-taxin 1A, rbet1, rsec22, �SNAP, and NSF (29). VAP-A alsointeracts with the tight junction protein occludin (22), withmicrotubules (20, 24), and with OSBP (oxysterol-binding pro-tein) (28). In this study, we isolated VAP-B as an interactingprotein with Nir2, using a pull-down experiment and massspectrometry analysis. Similar to VAP-B, Nir2 also belongs toa highly conserved family of proteins, the Nir/rdgB, which areimplicated in the regulation of membrane trafficking, phospho-lipid metabolism, and signaling (30, 31). Here we show that thethree Nir proteins: Nir1, Nir2, and Nir3, interact with VAP-Bvia their FFAT motif, and that Nir-VAP interactions differen-tially affect the organization of the ER.

EXPERIMENTAL PROCEDURES

Recombinant DNA and Antibodies—The cDNA of human VAP-B wasisolated by PCR using a first-strand cDNA, which was synthesized fromtotal RNA of HeLa cells (SuperScript II first strand synthesis system,Invitrogen), as a template and the following sense and antisense oligo-nucleutide primers; 5�-CCGGGATCCGCGAAGGTGGAGCA-3� and 5�-CGGGATATCCTACAAGGCAATCTTCCCAAT-3�, respectively. Theamplified PCR product was subcloned into the pCAN-Myc1 and pGE-X4T1 expression vectors. The FFAT mutant of Nir2 was generated byreplacing amino acids 349–353 (EFFDA) with ALLAG using three se-quential PCR steps with the following sense and antisense primers;5�-GAGATCCTGGCCAACCGG-3� and 5�-GTGCCCAGCGAGGAGAGC-TTCCTCGGAGCTGTTCTC-3� for the first PCR reaction; 5�-GAAGCT-CTCCTCGCTGGGCACGAAGGCTTCTCGGAC-3� and 5�-GATGGCCT-GGAACTTCGGGGC-3� for the second PCR reaction. The two PCR pro-ducts were mixed together and amplified by a third set of sense andantisense oligonucleotide primers; 5�-GAGATCCTGGCCAACCGG-3�and 5�-GATGGCCTGGAACTTCGGGGC-3�, respectively. The PCR pro-duct was digested with PstI and BglII and used to replace thecorresponding fragment in Nir2 cDNA. The cDNAs of the three Nirswere subcloned into pRK5 mammalian expression vector downstreamof the CMV promoter. Hemagglutinin (HA)-tag was fused to the C-terminal of Nir1, Nir2, and Nir3 coding sequences, essentially as de-

scribed previously (30). Restriction enzyme analysis and DNAsequencing verified the DNA constructs. YFP-VSV-G construct waskindly provided by Koret Hirschberg (Tel-Aviv University, Israel).Antibody against Nir2 was raised in rabbits as described previously(32). Polyclonal antibody against VAP-B was raised in rabbits immu-nized with a recombinant GST-VAP-B fusion protein. The antiserumwas first run through a GST-bound agarose column, to remove theanti-GST antibodies. The flow-through was then affinity-purified on aGST-VAP-B-bound agarose column. Monoclonal antibody against�-tubulin was purchased from Sigma. Monoclonal and polyclonal anti-bodies against HA and Myc were purchased from Santa CruzBiotechnology, Inc. Antibody against PDI was purchased from ABR(Affinity BioReagents). Alexa-488 donkey anti-mouse and anti-rabbitIgG were purchased from Molecular Probes. Cy3-conjugated goat anti-rabbit or goat anti-mouse IgG were from Jackson ImmunoResearchLaboratories (West Grove, PA).

Cell Cultures, Transfections, and Indirect Immunofluorescence—HEK293 and HeLa cells were grown in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% fetal bovine serum, penicillin(100 units/ml), and streptomycin (100 mg/ml). The cells were trans-fected by the calcium phosphate method as described previously (33).HeLa cells grown on glass coverslips were transfected as indicated,fixed in 4% paraformaldehyde in PBS for 20 min at room temperature,and immunostained essentially as described previously (33).

Cell Labeling, Extraction, Immunoprecipitations, and Pull-down Ex-periments—HeLa cells were washed with methionine-free DMEM andincubated in the same medium containing 10% dialyzed fetal bovineserum and 400 �Ci/ml [35S]methionine/cysteine (EXPRE35S35S, Du-pont/PerkinElmer Life Sciences) for 6 h. The cells were harvested andlysed in lysis buffer containing: 20 mM Tris-HCl, pH 7.5, 1% NonidetP-40, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,10 �g/ml leupeptin, and 10 �g/ml aprotinin. The lysate was centrifugedat 15,000 � g for 15 min at 4 °C, and the supernatant was used in eitherpull-down or immunoprecipitation experiments. Immunoprecipitationswere performed essentially as described previously (34). GST fusionproteins were expressed in bacteria and purified by standard proce-dures (Amersham Biosciences). For protein identification by mass spec-trometry, GST, or GST-Nir2-(205–424) were cross-linked to the agarosebeads using dimethyl pimelimidate (Sigma). Following cross-linkingtermination and washing, the beads were incubated with HeLa celllysate (from 107 cells) for 3 h at 4 °C, extensively washed, boiled inSDS-sample buffer, and separated by SDS-PAGE. The gel was stainedwith Gel Code and destained by multiple washing steps with double-distilled water. The protein bands were excised from the gel and sub-jected to protein identification by peptide mapping using MALDI-TOFmass spectrometry after in-gel trypsinization (Technion, Israel).

Chemical Cross-linking—HEK293 cells were transiently transfectedwith an expression vector encoding VAP-B-Myc. 24 h later, the cellswere washed with PBS and incubated at 4 °C for 60 min with 2 mM DSP(dithiobis(succinimidyl propionate); Pierce), a reversible chemical cross-linker. The cross-linking reaction was terminated by incubating thecells with 100 mM glycine for 15 min at 4 °C. For reversing the cross-linking, the cells were incubated with 100 mM DTT for 30 min at 37 °C.The cells were then washed in PBS and lysed in buffer containing 50mM Tris-HCl, pH 7.5, 2 mM EDTA, 100 mM NaCl, 1% Triton X-100, 5�g/ml leupeptin, 5 �g/ml aprotinin, and 1 mM phenylmethylsulfonylfluoride. Insoluble materials were removed by centrifugation at16,000 � g for 20 min at 4 °C. SDS sample buffer with or without 100mM DTT was added, the samples were boiled for 5 min and separated bySDS-PAGE.

Sucrose Density Gradient Sedimentation—HEK293 cells expressingthe indicated proteins were washed with PBS and lysed in buffercontaining 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 0.5% Triton X-100, 1mM phenylmethylsulfonyl fluoride, 5 �g/ml leupeptin, and 5 �g/mlaprotinin. Insoluble materials were pelleted by centrifugation at16,000 � g for 1 h at 4 °C. Supernatants (0.5 ml) were layered onto 10ml of continuous 5–30% (w/v) sucrose density gradients in a buffercontaining 50 mM Tris-HCl, pH 7.5 and either 0.1% Triton X-100 or 1%SDS, as indicated. The gradients were centrifuged at 100,000 � g for18 h at 4 °C, fractionated from the top to the bottom into 20 fractions of0.5 ml, and analyzed by Western blotting. Protein markers includingthyroglobulin (669 kDa), catalase (232 kDa), and bovine serum albumin(67 kDa) were separated and fractionated under the same conditionsand detected by SDS-PAGE and Coomassie Blue staining.

VSV-G Transport Assay—HeLa cells grown on glass coverslips weretransfected with an expression vector encoding the temperature-sensi-tive mutant of vesicular somatitis virus glycoprotein (VSV-G) fused toYFP (YFP-VSV-G), either alone or together with expression vectors

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encoding Nir2-HA, VAP-B-Myc, or Nir2-HA and VAP-B-Myc. Following5 h, the cells were shifted to 40 °C to accumulate YFP-VSV-G in the ER.The cells were then incubated at 32 °C for different time periods in thepresence of cycloheximide (100 �g/ml) to permit the transport of VSV-Gfrom the ER along the secretory pathway. The cells were fixed at theindicated time points, immunostained with antibody against Myc orHA, and analyzed by confocal microscopy.

Transmission Electron Microscopy—Hela cells grown in 35-mm Fal-con dishes, were transfected as indicated, and fixed for 60 min inKarnovsky’s fixative (3% paraformaldehyde, 2% glutaraldehyde, 5 mM

CaCl2 in 0.1 M cacodylate buffer, pH 7.4 containing 0.1 M sucrose). Thecells were then washed and scraped. The cell pellet was embedded inagar noble (1.7%) and postfixed with 1% OsO4, 0.5% potassium dichro-mate, and 0.5% potassium haxacyanoferrate in 0.1 M cacodylate buffer.The cells were stained en bloc with 2% aqueous uranyl acetate, followedby ethanol dehydration. Sections were cut using a diamond knife (Di-atome, Biel, Switzerland), and stained by 2% uranyl acetate in 50%ethanol and lead citrate. The samples were examined with transmis-sion electron microscope FEI CM12 Eindhoven, Holland, at an acceler-ating voltage of 120 kV and recorded with a SIS Biocam CCD, 1024 �1024 pixel camera.

RESULTS

Isolation of VAP-B as a Nir2-interacting Protein—To gain abetter insight into Nir2 cellular functions, we looked forNir2-interacting proteins using coimmunoprecipitation as-says. Control and Nir2-HA-transfected HeLa cells were met-abolically labeled with [35S]methionine, lysed, and immuno-precipitated with anti-HA antibody. Several proteins weredetected in the Nir2 immunocomplex in a specific manner,including a prominent one of �31 kDa (Fig. 1A). This proteinwas also found in the immunoprecipitates of several Nir2-

truncated mutants, including a mutant that lacks the first340 amino acids. However, it was not present in the immuno-complex of a truncated mutant lacking the first 440 aminoacids of Nir2 (Fig. 1B), suggesting that the interaction of Nir2with the 31 kDa protein is mediated through amino acids341–439. These results were further confirmed by a pull-down experiment using a GST fusion protein consisting ofamino acids 205–424 of Nir2 as an affinity column and[35S]methionine-labeled HeLa cell lysate. The 31 kDa proteinwas recovered on the Nir2 affinity column in a specific man-ner (Fig. 1C). Therefore, this column was used in the subse-quent large scale pull-down experiment as described under“Experimental Procedures,” and the 31 kDa protein was iden-tified by mass spectrometry. This analysis identified twopeptides corresponding to amino acids 20–31 and 201–216 ofthe human VAP-B protein. Since VAP-B protein consists 243amino acids and apparently migrates as a �31 kDa proteinon SDS-PAGE (19), we assumed that it is the 31 kDa proteinthat interacts with Nir2. To explore this possibility, the hu-man VAP-B cDNA was isolated and subcloned into a mam-malian expression vector containing an N-terminal Myc tag.The Myc-tagged VAP-B protein was expressed in the ex-pected size, and its interaction with Nir2 was determined bypull-down experiments using the GST-Nir2 fusion protein(amino acids 205–424) immobilized on glutathione beads asan affinity column. As shown in Fig. 1D, VAP-B interactswith the GST-Nir2 fusion protein but fails to interact withGST, suggesting that the 31 kDa protein is indeed VAP-B.

FIG. 1. Isolation and identification ofa 31 kDa protein that interacts withNir2. A, HeLa cells expressing wild-typeNir2-HA and control non-transfected cellswere metabolically labeled with [35S]me-thionine, lysed, and subjected to immuno-precipitation with anti-HA antibody. Thesamples were separated by 10% SDS-PAGE, and then subjected to autoradiogra-phy. The 31 kDa protein that coimmuno-precipitated with Nir2-HA is marked by anarrow. B, HeLa cells that express eitherthe wild-type Nir2-HA, the indicated Nir2-HA-truncated mutants, or control non-transfected cells were metabolically la-beled, lysed, and immunoprecipitated asdescribed above. The 31 kDa protein wasnot detected in the control or the immuno-complex of a mutant lacking the first 440amino acids of Nir2. C, GST or GST-Nir2-(205–424) fusion protein immobilized onglutathione-agarose beads were incubatedwith [35S]methionine-labeled HeLa cell ly-sate. The samples were extensivelywashed, resolved by SDS-PAGE, and sub-jected to autoradiography. D, HEK293 cellswere transiently transfected with an ex-pression vector encoding VAP-B-Myc. Thecell lysate was incubated with GST or GST-Nir2-(205– 424) bound to glutathione-agarose beads. Following washing, thesamples were resolved by SDS-PAGE,transferred to nitrocellulose membrane,and immunoblotted with anti-Mycantibody.

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Nirs Interact with VAP-B via Their Conserved FFAT Motif—Recently, the yeast homolog of VAP-B, Scs2p was shown tointeract with Opi1p, through a conserved EFFDAXE motifdesignated FFAT (35). This motif is also found in the Nir2protein at amino acids 349–355 and is highly conserved amongthe other Nir/rdgB family members. The presence of this motifwithin the region that mediates the interaction with the 31kDa protein (amino acids 341 to 439; Fig. 1B) further supportsour results and strongly suggests that the 31 kDa protein isindeed the VAP-B protein. To determine whether the interac-tion of Nir2 with VAP-B is mediated through its FFAT motif,the conserved EFFDA sequence was replaced by ALLAG. Thismutagenesis completely abolished the interaction of Nir2 withVAP-B (Fig. 2A), indicating that the interaction of Nir2 withVAP-B is mediated by its FFAT motif. To demonstrate theinteraction between endogenous VAP-B and Nir2 proteins, weraised a polyclonal antibody against VAP-B as described under“Experimental Procedures.” This antibody recognizes both thetransfected and the endogenous VAP-B protein by immunopre-

cipitation and immunoblotting (Fig. 2B). Moreover, Westernanalysis of endogenous VAP-B immunoprecipitated from HeLacells, revealed the presence of endogenous Nir2 in the VAP-Bimmunocomplex. Similarly, VAP-B was detected in the immu-nocomplex of Nir2. These results suggest that Nir2 and VAP-Binteract with each other in vivo (Fig. 2C).

Next, we assessed whether the other Nir proteins; Nir1 andNir3, interact with VAP-B, as they also contain a FFAT motif(Fig. 2D). The results shown in Fig. 2E, clearly demonstratethe interaction of Nir1 and Nir3 with VAP-B, suggesting thatthe three Nir proteins interact with VAP-B via their FFATmotif.

The Interaction of VAP-B with Nir2 Affects the ER Struc-ture—The VAP-B protein was previously localized to the ERand to pre-Golgi intermediates by biochemical and immunocy-tochemical methods (19). We have previously shown that Nir2mainly localizes in the Golgi apparatus, but it is also found inthe ER in interphase cells (36). To determine the localization ofVAP-B in HeLa cells, we used the anti-VAP-B antibody in

FIG. 2. The three Nir proteins interact with VAP-B. A, Nir2 interacts with VAP-B via its FFAT motif. HEK293 cells were either transfectedwith expression vectors encoding Nir2-HA or VAP-B-Myc or cotransfected with VAP-B-Myc and wild-type Nir2-HA, or its FFAT mutant (FM). Thecells were lysed and subjected to immunoprecipitations followed by immunoblotting with the indicated antibodies. The expression level of theproteins was determined by Western blotting of cell lysate (10% of total) using the indicated antibodies. B, specificity of the anti-VAP-B antibody.Antibody against VAP-B was raised in rabbits as described under “Experimental Procedures,” and used in immunoprecipitation or immunoblottingof lysate prepared from HEK293 cells expressing either the VAP-B-Myc or the Plk1-Myc protein, as indicated. Endogenous VAP-B wasimmunoprecipitated from HeLa cells (right panel) using either preimmune (PI) or VAP-B immune serum (I). The samples were resolved bySDS-PAGE, and immunoblotted with the indicated antibodies. C, coimmunoprecipitation of endogenous VAP-B and Nir2 proteins. HeLa cell lysatewas subjected to immunoprecipitation following immunoblotting by the indicated antibodies. Preimmune (PI) and immune (I) serum. D, structureof Nir and VAP-B proteins. The three Nir proteins contain a conserved C-terminal region of �300 amino acids, a DDHD domain, six hydrophobicstretches that are marked by vertical lines, and an acidic region that consists of the FFAT motif; EFFDAXE. Nir2 and Nir3 contain an N-terminalPI transfer domain. VAP-B consists of a large cytoplasmic N-terminal region, a TMD, and a short luminal C-terminal tail of 4 amino acids. Thecytoplasmic region consists of an N-terminal domain of 100 amino acids that shares high sequence homology with MSP (white) and within whichis a very conserved domain (VCD) of 16 amino acids, and a coiled-coil domain (CCD). E, three Nir proteins; Nir1, Nir2, and Nir3 interact withVAP-B. HEK293 cells were either transfected with an expression vector encoding VAP-B-Myc or cotransfected with VAP-B-Myc and Nir1-HA,Nir2-HA, or Nir3-HA. The three Nir proteins were immunoprecipitated by anti-HA antibody, and their association with VAP-B was determinedby immunoblotting with anti-Myc antibody, while VAP-B was immunoprecipitated with anti-Myc antibody and its association with Nirs wasdetermined by immunoblotting with anti-HA antibody. The expression level of the proteins was determined by Western blotting of cell lysate (10%of total) using the specified antibodies.

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indirect immunofluorescence and confocal microscopy analy-ses. The VAP-B protein exhibits a typical ER reticular local-ization. Similar localization was observed with the transfectedVAP-B-Myc protein, and double immunostaining with antibodyagainst protein-disulfide isomerase (PDI), an ER marker, re-vealed their strong colocalization (Fig. 3A), suggesting thatendogenous and transfected VAP-B proteins are localized inthe ER.

To gain a better understanding on Nir-VAP-B interactions,we coexpressed them together in HeLa cells and examinedtheir subcellular localization using indirect immunofluores-cence and confocal microscopy analyses. Overexpression ofwild-type Nir2 with VAP-B caused to production of large het-erogeneous granular structures that were dispersed around thenucleus throughout the cytosol, in which Nir2 and VAP-B werestrongly colocalized. These structures were not detected in cellsthat express Nir2 alone (data not shown), or coexpressed theFFAT mutant of Nir2 and VAP-B (Fig. 3B). These resultssuggest that the interaction of Nir2 with VAP-B, and not sim-ply their overexpression, induces the formation of these un-

usual structures. To determine the origin of these structures,we used several organelle-specific markers and analyzed theirlocalization by confocal microscopy. As shown in Fig. 3B, theER-resident protein PDI was strongly localized to these struc-tures, whereas neither the Golgi markers, nor the endosomal orlysosomal markers were detected in these structures (data notshown). These results suggest that these structures wereformed from the ER, and that Nir2-VAP-B interaction re-arranges the normal structure of the ER. Furthermore, coex-pression of Nir2 and VAP-B had no detectable effect on the cis,medial, or trans Golgi morphology, on COPI vesicles, on earlyor late endosomes, or on lysosomes, as determined by localiza-tion of their respective markers; p58, NAGT-I, sialyltransfer-ase, �-COP, EEA1, rab7, and LAMP1 (data not shown). Thus,coexpression of Nir2 and VAP-B specifically modifies the ERstructure.

To better characterize the effect of the Nir2-VAP-B interac-tion on ER structure, we used transmission electron micros-copy (EM) analysis. The EM images shown in Fig. 4, demon-strate the remarkable reorganization of the ER, which is

FIG. 3. Coexpression of VAP-B andNir2 proteins induces the productionof ER-associated granular struc-tures. A, endogenous and transfectedVAP-B proteins are localized to the ER.Shown are confocal images of HeLa cellsdouble immunostained with anti-VAP-Band anti-PDI antibodies (upper panel), orHeLa cells transfected with VAP-B-Mycdouble-immunostained with anti-Mycand anti-PDI antibodies. Colocalizationappears in yellow in the merged image.Bar, 10 �m. B, HeLa cells were cotrans-fected with expression vectors encodingVAP-B-Myc and either wild-type Nir2-HAor its FFAT mutant, Nir2(FM), as indi-cated. Thirty hours later, the cells werefixed and double-immunostained with an-ti-Myc monoclonal antibody and anti-HApolyclonal antibody. Bar, 10 �m.

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characterized by various structures consisting multiple mem-brane arrays that were organized in diverse forms. Some ofthem emerged from the NE, while others appeared in periph-eral locations, consistent with our confocal microscopy analy-sis. Sometimes they were visualized as circular packed cister-nae, or linear stacks of cisternae located far from the cellnucleus. Membrane wheels, or closed or partially opened loopswere also observed (data not shown). Similar structures havebeen previously obtained by overexpression of several integralER membrane proteins, including HMG-CoA (9), msALDH (7),and cytochrome b5 (37), among others.

VAP-B Undergoes Oligomerization in Mammalian Cells—The mechanism underlying the formation of stacked ER mem-brane arrays is not completely understood. However, severallines of evidence suggest that oligomerization of the membraneprotein is critical to this process. Accordingly, interaction be-tween the cytoplasmic domains of proteins on apposed ERmembranes causes stacking of the ER cisternae (7, 13, 14, 38).Other studies have suggested that oligomerization of the ERmembrane protein is insufficient for the induction of alter-ations in membrane assembly; rather, proper folding of theTMD is the critical induction factor (10, 37). We thereforeassessed the oligomerization state of VAP-B in mammaliancells. VAP-B-Myc was expressed in HEK293 cells, and its oli-gomerization state was analyzed following treatment withDSP, a membrane-permeable reducible cross-linker. The re-sults (Fig. 5A) indicated that VAP-B undergoes oligomerizationin mammalian cells. A clear band of �63 kDa, probably repre-

senting a dimer, was detected, whereas a band with the ex-pected migration for a tetramer, at �120 kDa, was less abun-dant. Furthermore, deletion of the first 63 amino acids ofVAP-B had no effect on its oligomerization, as demonstrated bythe same experimental approach (data not shown). These re-sults suggest that VAP-B undergoes self-oligomerization inmammalian cells, and that the TMD is probably critical for itsoligomerization. We further confirmed this result by sucrosedensity gradient analysis (Fig. 5B). In this set of experiments,we expressed either VAP-B or Nir2 alone, or coexpressedVAP-B with wild-type Nir2 or its FFAT mutant in HEK293cells. The cells were lysed as described under “ExperimentalProcedures” and layered onto a continuous sucrose densitygradient in the presence or absence of 1% SDS. Followingsedimentation, fractions were collected and analyzed by West-ern blotting. Protein markers, including thyroglobulin (669kDa), catalase (232 kDa), and bovine serum albumin (67 kDa),were separated on a parallel sucrose gradient, fractionatedunder the same conditions, and detected by SDS-PAGE andCoomassie Blue staining. A logarithmic plot of the molecularmass of the marker proteins as a function of gradient fractionindicated that VAP-B sediments as expected for a dimer, con-sistent with the data shown in Fig. 5A. The peak intensity ofthe VAP-B protein under native conditions was found in frac-tion 5, which has an estimated molecular mass of �63 kDa,while its peak under denaturing conditions (1% SDS) wasshifted to fraction 3. Nir2 was detected in fractions 6 through12, with a peak in fraction 10 corresponding to �153 kDa.Coexpression of VAP-B and the FFAT mutant of Nir2 had noeffect on the sedimentation profile of either VAP-B or Nir2,consistent with these proteins’ inability to interact with oneanother. However, coexpression of wild-type Nir2 and VAP-Binduced a dramatic shift in VAP-B sedimentation. The peaks ofVAP-B and Nir2 intensities appeared in fraction 12, corre-sponding to �220 kDa, and continued through to fraction 16(�440 kDa). Collectively, these results suggest that by itself,VAP-B is a dimer, and that its interaction with Nir2 createscomplexes of various sizes that may represent different oli-gomerization forms. Accordingly, we suggest (Fig. 5C) thatVAP-B undergoes dimerization in mammalian cells, probablymediated by the GXXXG motif present in its TMD (20, 27). Thebinding of Nir2 to VAP-B induces a conformational change inthe VAP-B cytoplasmic domain and facilitates its trans-oli-gomerization. This trans-oligomerization is mediated by ahead-to-head interaction of VAP-B cytoplasmic domains onapposing ER membranes, which zips up the apposing mem-brane, yielding stacked ER cisternae. Alternatively, a head-to-head interaction of Nir2 molecules could bridge VAP-Bproteins on apposing ER membranes and induce their stack-ing. The sedimentation profile of Nir2 alone (Fig. 5B), whichdemonstrates the formation of larger Nir2 structures, maysupport this possibility. Both of these possibilities rely onhead-to-head interactions, involving either VAP-B or Nir2molecules. However, it could be that the binding of Nir2 toVAP-B modifies the folding of VAP-B TMD, which would thentransmit a signal for the production of stacked membranearrays (Fig. 5C, option 3).

Coexpression of VAP-B and Nir2 Attenuates Protein Exportfrom the ER—Next, we assessed whether these structures haveany effect on protein export from the ER to the Golgi apparatusand subsequently to the plasma membrane. For this purpose,HeLa cells were cotransfected with either VAP-B and a tem-perature sensitive mutant of vesicular stomatitis virus-glyco-protein (ts045 VSV-G) fused to YFP, or with Nir2, VAP-B, andYFP-VSV-G. At 40 °C the VSV-G is synthesized and retained inthe ER because of its misfolding (39). However, upon shifting

FIG. 4. Transmission electron microscopy analysis of ER struc-ture in Nir2/VAP-B-expressing cells. Control or Nir2/VAP-B-ex-pressing HeLa cells were fixed as described under “Experimental Pro-cedures” and analyzed by electron microscopy. Selected electronmicrographs are shown. Low power view of control (panel A) and trans-fected HeLa cells (panels B, D, G), along with the high magnificationimages of the diverse ER structures consisting multiple membranearrays (panels C, E, F, H, I). A circular closed packed membrane arrayis shown in panel E, and the high magnification image demonstratingthe uniform width (�8 nm) of the cytoplasmic layer separating thelamellae is shown in F. Linear stacks of cisterna located near to or farfrom the nucleus or emerging from the NE are shown in panels I, H, andC, respectively. High magnification images of the asterisk-labeled struc-tures in panels B and D are shown in C and E, respectively, whereasthose that are labeled in panel G are shown in panels H and I. M,mitochondria, N; nucleus. Bars: 0.5 �m (A); 1 �m (B); 0.5 �m (C); 2 �m(D); 0.5 �m (E); 0.1 �m (F); 1 �m (G); 0.7 �m (H); 0.5 �m (I).

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the temperature to 32 °C, the VSV-G protein folds and is trans-ported to the Golgi apparatus and then to the cell surfacewithin 20–30 min and 60–90 min, respectively. The resultsshown in Fig. 6, clearly demonstrate the accumulation ofVSV-G in the granular structures at 40 °C (time 0) in cellscoexpressing Nir2 and VAP-B, consistent with the localizationof PDI in these structures and their ER origin. However, 30min after shifting the temperature to 32 °C, VSV-G was mainlylocalized in the Golgi apparatus in the control non-transfected,Nir2-transfected (data not shown), or the VAP-B-transfectedcells, and at 90 min was mainly at the plasma membrane. Incontrast, in cells coexpressing Nir2 and VAP-B, VSV-G wasretained in the granular structures even 1 h following shiftingthe temperature to 32 °C, suggesting that these structuresinhibit the export of VSV-G from the ER. The accumulation ofVSV-G in these structures was not caused by a continuoussynthesis of VSV-G protein, because the experiment was per-formed in the presence of cycloheximide.

Ectopic Expression of Nir3 and VAP-B Induces MicrotubuleBundling along the ER Membranes—Since Nir1 and Nir3 alsointeract with VAP-B, we assumed that their coexpression withVAP-B would affect the ER structure in a similar manner tothat of Nir2. We therefore coexpressed them with VAP-B andexamined their localization by immunofluorescence and confo-cal microscopy analyses. Coexpression of Nir1 with VAP-B hadno apparent effect on either Nir1 or VAP-B localizations (Fig.7A). In contrast, coexpression of Nir3 and VAP-B strikinglychanged the typical localization of VAP-B. It was visualized asthick bundles surrounding the nucleus that were extendedperipherally throughout the cytosol, to which Nir3 was colocal-ized (Fig. 7B). To determine whether coexpression of Nir3 andVAP-B-Myc has any effect on the ER structure, HeLa cells that

coexpress them were double immunostained with anti-PDI andanti-Myc antibodies and their localization was analyzed byconfocal microscopy. The results shown in Fig. 7C demonstratethat PDI staining was strikingly different in cells that coex-press VAP-B and Nir3 as compared with the control non-trans-fected HeLa cells (Fig. 3A). In the cotransfected cells, PDIimmunostaining appeared in tubular-like structures that werecolocalized with VAP-B. Thus, in contrast to Nir1 and Nir2,coexpression of Nir3 with VAP-B modified the ER structureinto a tubular pattern. A similar pattern has been previouslyobtained when the integral ER-membrane protein p63 wasoverexpressed in COS cells (16). Its overexpression caused torearrangement of the ER and concomitantly bundling of micro-tubules along the altered ER membranes. This similarity led usto examine the morphology of the microtubules in cells thatcoexpress Nir3 and VAP-B compared with their morphology incells that either express Nir3 or VAP-B alone. As shown in Fig.7D, coexpression of Nir3 and VAP-B dramatically modified theorganization of the microtubules; they were unusually thickand not organized, the microtubule organization center was notvisible, and more importantly, they aligned with VAP-B or Nir3immunostaining. These results suggest that the VAP-B-Nir3interaction links the ER membranes to the microtubule net-work, and thereby modifies both the ER and microtubule orga-nization. It is noteworthy that coexpression of Nir3 and VAP-Bcaused to partial dispersal of the Golgi (data not shown), con-sistent with the role of microtubules in positioning and main-tenance of the Golgi apparatus (1).

The mouse and the Drosophila VAP-A proteins have beenpreviously shown to directly interact with microtubules, yet,through an unidentified motif (20, 24). Since the DrosophilaVAP-A (DVAP-33A) was proposed to stabilize microtubules, we

FIG. 5. Oligomerization of VAP-B protein. A, cross-linking of VAP-B. HEK293 cells expressing VAP-B-Myc were incubated in the presenceor absence of 2 mM DSP for 1 h at 4 °C, neutralized, solubilized, and subjected to nonreducing (�DTT) or reducing (�DTT) SDS-PAGE. Theoligomerization state of VAP-B was determined by Western blotting using anti-Myc antibody. Arrows indicate monomers, dimers, and tetramers.B, sucrose density gradient sedimentation analysis of VAP-B complexes. HEK293 cells expressing the indicated proteins (right) were solubilizedas described under “Experimental Procedures,” and subjected to a 5–30% (w/v) sucrose gradient centrifugation. Where indicated, 1% SDS wasincluded in the gradient. Fractions were collected from the top to the bottom, separated by SDS-PAGE, and analyzed by Western blotting usingeither antibodies against Myc (for VAP-B) or HA (for Nir proteins). Arrows indicate the fractions with peak intensities of bovine serum albumin(67 kDa; fraction 5) and catalase (232 kDa; fraction 12), while thyroglobulin (669 kDa) appeared in fraction 18. C, models for the formation ofstacked ER membrane arrays in VAP-B/Nir2-expressing cells. VAP-B undergoes cis-oligomerization mediated by its TMD containing the GXXXGmotif (left panel). Binding of Nir2 to the cytoplasmic region of VAP-B induces a conformational change and a head-to-head interaction of VAP-Bproteins on apposing ER membranes. This trans-oligomerization facilitates the formation of stacked membrane arrays (1). Alternatively, ahead-to-head interaction of Nir2 molecules bridges the VAP-B proteins on apposing ER membranes and induces their stacking (2), or binding ofNir2 to VAP-B changes the folding of the VAP-B TMD, which in turn transmits a signal for the formation of stacked membrane arrays (3).

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examined the effect of nocodazole treatment on the microtubulearchitecture in cells that overexpress VAP-B or coexpressVAP-B and Nir3. As shown in Fig. 7E, treatment with nocoda-zole for 3 h caused to complete depolymerization of microtu-bules in the VAP-B overexpressing or in the non-transfectedcontrol cells. However, in cells that coexpress VAP-B and Nir3,some microtubules were still detected following such long treat-ment with nocodazole, suggesting that Nir3-VAP-B interactionenhances the microtubule stability. These results are consist-ent with the proposed function of DVAP-33A in stabilizingmicrotubules (24).

DISCUSSION

Nir1, Nir2, and Nir3 belong to a highly conserved family ofproteins, the Nir/rdgB, which have been implicated in regula-tion of phospholipid trafficking, metabolism, and signaling.Nir2 and Nir3 contain an N-terminal phosphatidylinositol (PI)-transfer domain, followed by a short acidic region, six hydro-phobic stretches, and a long highly conserved C-terminal do-main (Fig. 2D). The N-terminal PI-transfer domain is notpresent in Nir1 or the zebrafish pl-RdgB (31, 40), but all theother family members, including Drosophila retinal degenera-tion B (rdgB), have a functional PI-transfer domain that hasthe ability to transfer PI and phosphatidylcholine (PC) betweenmembrane bilayers in vitro (41).

In this study, we show that the three Nir proteins interactwith VAP-B through their conserved FFAT motif (Fig. 2),which is present within their acidic region. This motif consistsof a conserved EFFDAXE sequence, which acts as an ER-targeting determinant by its direct interaction with VAP pro-teins (35). The FFAT motif has been identified in 17 distincteukaryotic proteins, 14 of which are directly implicated in lipidbinding or lipid sensing, including homologs of OSBP, ho-mologs of Goodpasture’s antigen-binding protein (GPBP), theNir/rdgB proteins, and Opi1p, a transcriptional regulator ofphospholipid synthesis in yeast. The FFAT motif in Opi1pmediates the interaction of Opi1p with Scs2p and therebytargets it to the ER. A similar targeting mechanism has beenshown for the yeast homologs of OSBP, Osh1p, Osh2p, and

Osh3p (35). In mammalian cells, OSBP also interacts withVAP-A protein (28), probably through its FFAT motif. How-ever, in this particular case, the binding affinity of OSBP toVAP-A is largely dependent on its pleckstrin homology (PH)domain. A specific mutation in this domain, W174A, enhancesits binding to VAP-A, and overexpression of this mutant inmammalian cells causes the production of ER inclusions, inwhich OSPB and VAP-A are colocalized (28). Very similarstructures were obtained upon overexpression of Nir2 andVAP-B (Fig. 3B), suggesting that the interaction of FFAT-motif-containing proteins with VAPs can induce the formationof these unusual ER structures. However, our results indicatethat these structures are formed in a specific manner, which islargely dependent on the VAP-interacting protein, as neitherNir1 nor Nir3 induced their formation (Fig. 7), despite theirinteraction with VAP-B protein (Fig. 2E). Electron microscopyanalysis revealed that these structures consist of multiplemembrane arrays that are organized in diverse forms. Thestructures were found either emerging from the NE or periph-erally throughout the cytosol (Fig. 4). Similar structures havebeen previously obtained in different cell types by overexpress-ing HMG-CoA reductase, an integral membrane protein thatcatalyzes the rate-limiting step in cholesterol biosynthesis (42).Detailed analysis using different mutants or chimeric proteinsof HMG-CoA reductase suggests that its catalytic activity is notrequired for stimulation of membrane proliferation; rather, itsmembrane domain appears to be both necessary and sufficientfor the induction of these structures (9, 43). Subsequent studiesusing other transmembrane proteins led to the hypothesis thatthe proper folding of the transmembrane domain is critical fortheir formation, and that this folding is required for the trans-mission of a signal for membrane biogenesis and the productionof stacked membrane arrays (5, 37). In contrast, other studiesproposed that homodimeric interactions between cytoplamicdomains of ER-resident proteins consisting of TMDs is suffi-cient for generating these structures (14). Accordingly, head-to-head interaction between the cytoplasmic domains of theintegral membrane protein on apposing ER membranes zips

FIG. 6. Attenuation of YFP-VSV-G export from the ER of cells coexpressing the Nir2 and VAP-B proteins. Shown are confocal imagesof YFP-VSV-G (ts045) export from the ER of either VAP-B-expressing cells (upper panels) or VAP-B and Nir2-coexpressing cells (lower panels). Thecells were fixed, immunostained with anti-Myc antibody, and analyzed by confocal microscope at the indicated time points following shifting thetemperature from 40 to 32 °C. The localization of YFP-VSV-G in VAP-B-expressing cells is shown in the upper panels. The localization ofYFP-VSV-G in the Nir2/VAP-B-coexpressing cells is shown in the middle panels, along with the localization of VAP-B in the same cells (lowerpanels). Bar, 10 �m.

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the apposing membrane, thereby stacking the ER cisternae(7, 13, 14). This hypothesis prompted us to characterize theoligomerization state of VAP-B in mammalian cells (Fig. 5).

Our finding that VAP-B undergoes dimerization in mamma-lian cells (Fig. 5) is consistent with previous studies performedin yeast and in vitro (27–29). According to the yeast two-hybrid

analysis (28), truncated VAP-A mutants lacking either aminoacids 41–59, the TMD, or the last 84 amino acids (amino acids1–160) interact with wild-type VAP-A but fail to interact withOSBP, suggesting that the C-terminal domain, including theTMD, is not required for VAP-A self-oligomerization. On theother hand, Weir et al. (29) showed that both the N- and

FIG. 7. Nir3-VAP-B interaction leads to rearrangement of the ER and the microtubule network. A, coexpression of Nir1 and VAP-Bhas no effect on VAP-B localization. Shown are confocal images of HeLa cells coexpressing Nir1 (red) and VAP-B (green), along with the mergedimage. Bar, 10 �m. B, Nir3 changes the localization of VAP-B protein. HeLa cells expressing Nir3-HA (left panel), or coexpressing VAP-B-Myc andNir3-HA, were fixed and immunostained with antibodies against the corresponding proteins as indicated. Bar, 10 �m. C, structure of the ER inNir3/VAP-B-expressing cells. Shown are confocal images of HeLa cells coexpressing Nir3-HA and VAP-B-Myc double-immunostained withanti-Myc and PDI antibodies. Bar, 10 �m. High magnification images are shown in the lower panels. Bar, 10 �m. D, coexpression of Nir3 andVAP-B affects the organization of the microtubules. HeLa cells expressing VAP-B-Myc alone (upper panels) or coexpressing VAP-B-Myc andNir3-HA (middle and lower panels) were double-immunostained with anti-�-tubulin and either anti-Myc or anti-HA. Colocalization appears inyellow. Bar, 10 �m. E, effect of nocodazole treatment on depolymerization of the microtubules in VAP-B- (lower panels) and VAP-B/Nir3- (upperpanels) expressing cells. The cells were incubated for 3 h in the presence of nocodazole (5 �M), fixed, and double-immunostained for �-tubulin andVAP-B-Myc. Transfected cells are labeled with an arrow, non-transfected control cells with an arrowhead. Bar, 10 �m.

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C-terminal domains of VAP-A are required for its oligomeriza-tion, and Nishimura et al. (27) suggested that the TMD iscritical for both homo- and hetero-oligomerization of VAP-Aand VAP-B proteins in vitro. Indeed, the TMD of VAP-A/Bcontains a GXXXG motif, which has been identified in a num-ber of TMDs and was proposed to induce strong self-assembly(44). Thus, it could be that several structural domains, includ-ing the TMD, mediate the oligomerization of VAP-A/B. Weshow here that VAP-B expression is insufficient to producestacks of ER membrane arrays unless Nir2 is coexpressed (Fig.3). We also show that VAP-B is found mainly as a dimer inmammalian cells, and that its interaction with Nir2 inducesthe formation of oligomers of various sizes (Fig. 5). These re-sults suggest that the binding of Nir2 to VAP-B induces aconformational change in the VAP-B protein, which either fa-cilitates the trans-oligomerization of VAP-B proteins on appos-ing ER membranes through interaction of their cytoplasmicdomains, or induces specific folding of VAP-B TMD that trans-mits a signal for membrane assembly (Fig. 5C). Although thefirst possibility is consistent with the results of the yeast two-hybrid interaction assays suggesting that the cytoplasmic re-gion of VAP-A is involved in VAP oligomerization, at presentwe cannot exclude the second possibility. We also cannot ex-clude the possibility that a head-to-head interaction of Nir2molecules bridges VAP-B proteins on apposing ER membranes,allowing them to stack together (Fig. 5C). While this is areasonable possibility in light of the sedimentation profile ofNir2 (Fig. 5B), our preliminary studies indicate that overex-pression of a truncated VAP-B protein lacking the highly con-served 16 amino acids within the N-terminal region induces theformation of stacked ER membrane arrays (data not shown).These results suggest that these structures are formed byconformational changes in the VAP-B protein.

In contrast to Nir2, coexpression of Nir1 with VAP-B had noeffect on ER structure (Fig. 7A). Since Nir1 lacks the PI-trans-fer domain, we assumed that this domain is required for induc-tion of the unusual morphology of the ER seen in Nir2/VAP-B-expressing cells. However, deletion of most of the PI-transferdomain (amino acids 1–240), or even its flanking region (1–340), did not abolish the effect of Nir2-VAP-B interaction on theER structure (data not shown). These results suggest thatdespite the high sequence similarity between the three Nirproteins, and their ability to interact with VAP-B via theirFFAT motif (Fig. 2E), they distinctly affect the ER structure inthe presence of VAP-B protein. Indeed, coexpression of Nir3with VAP-B caused a striking rearrangement of the ER andconcomitantly, bundling of microtubules along the altered ERmembranes, to which Nir3 and VAP-B were colocalized (Fig. 7,C and D). Previously, it was shown that the Drosophila homo-log of VAP-A, DVAP-33A, binds microtubules and is involved instabilizing and directing microtubules during the budding ofsynaptic boutons. In DVAP-33A mutant flies, the presynapticmicrotubule architecture is severely compromised, and DVAP-33A was therefore proposed to function as a bridge betweenmicrotubules and the presynaptic membrane (24). Our resultssupport this hypothesis, and suggest that VAP-B bridges theER membrane with the microtubule network. However, over-expression of VAP-B did not induce bundling of microtubulesunless Nir3 was coexpressed. Thus, it could be that the bindingof Nir3 to VAP-B induces a conformational change in the latter,which in this specific case enhances its binding to microtu-bules, or that Nir3 itself serves as a bridge between VAP-B andthe microtubules.

Overexpression of many microtubule-associated proteinsinduces bundling of microtubules and stabilization of theirstructure (45). Indeed, we show that the microtubules in

Nir3/VAP-B-expressing cells are more resistant to nocodazoletreatment than the non-transfected control or VAP-B-trans-fected cells (Fig. 7E), suggesting that this interaction stabi-lizes the microtubules, and is consistent with the proposedfunction of DVAP-33A.

Although, our results were obtained through overexpressionof VAP-B and the different Nir proteins, they imply that VAPproteins play essential roles in the regulation of ER structure.Thus, VAPs can be considered ER-receptors for many FFAT-containing proteins, which are differentially expressed invarious tissue and cell types, and might be differentiallyregulated under specific physiological conditions. These re-ceptors may undergo different conformational changes uponbinding of their cognate ligands, and consequently, distinctlyaffect the ER structure. Thus, our results suggest that theinteraction of VAPs with FFAT-containing proteins is notonly required for targeting of FFAT-containing proteins tothe ER, but is also involved in the regulation of ER organi-zation and positioning.

Acknowledgment—We thank Koret Hirschberg for productivediscussions.

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Regulation of ER Structure5944

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Roy Amarilio, Sreekumar Ramachandran, Helena Sabanay and Sima LevProtein Interaction

Differential Regulation of Endoplasmic Reticulum Structure through VAP-Nir

doi: 10.1074/jbc.M409566200 originally published online November 15, 20042005, 280:5934-5944.J. Biol. Chem. 

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