subcellular localization of neural-specific npdc-1 protein

Subcellular Localization of Neural-SpecificNPDC-1 Protein

C. Evrard* and P. RougetLaboratoire Biologie Moleculaire et Differenciation, Unite de Genetique Oncologique, CNRS-URA 8125,Institut Gustave Roussy, Villejuif, France

NPDC-1 is a gene specifically expressed in neural cellswhen they stop to divide and begin to differentiate. Im-munocytochemical study analysis of differentiated PC12cells transfected with NPDC-tag vectors showed thatNPDC-1 is transported in vesicles from the Golgi appa-ratus to the cell membrane and is then likely internalizedinto endosomes. The protein colocalized, at least par-tially, with synaptic vesicle proteins: synaptophysin, syn-aptobrevin 2, and Rab3 GEP (Rab3 GTP/GDP exchangeprotein). Moreover, subcellular fractionation of rat brainshowed that crude synaptic membrane and crude syn-aptic vesicle fractions were enriched in NPDC-1. Al-though NPDC-1 bound Rab3 GEP in vitro, it seems un-likely to be involved in Ca2�-dependent exocytosis and,thus, in synaptic vesicle trafficking.© 2005 Wiley-Liss, Inc.

Key words: NPDC-1; synaptic vesicle; trafficking; Rab3GEP

NPDC-1 (for neural proliferation differenciationcontrol-1) has been identifed as a neural-specific geneinvolved in the control of cell proliferation and differen-tiation (Galiana et al., 1995). Its expression is developmen-tally regulated and persists in the adult; it increases in theembryonic brain, in distinct, defined regions, and is cor-related with growth arrest and terminal differentiation(Dupont et al., 1998). It has been shown that the mouseNPDC-1 protein could interact in vitro with the tran-scription factor E2F-1 and some cell cycle proteins, such asD cyclins and Cdk2 (Sansal et al., 2000). NPDC-1 andE2F1 were also able to interact in vivo.

For the NPDC-1 encoded protein, different do-mains have been predicted (PSORTII): a long hydropho-bic stretch of amino acids (residues 13–29), a coiled-coilregion (amino acids 93–120), a transmembrane domain(amino acids 191–207), an acidic domain (amino acids277–307), and MAP-kinases consensus sites (amino acids234–244; see Fig. 1). With the aim of gaining some insightinto the function of NPDC-1, we determined the orien-tation of the protein in the membrane and its subcellularlocalization.

The human NPDC-1 cDNA has been cloned (Gen-bank: AF272357 and AF283247), and the human genomicsequence has been mapped to chromosome 9q34-3 (nu-

cleotides 135297948–135291216; Evrard et al., 2004);however, no function has been assigned to humanNPDC-1. However, since the publication by Iwasaki andToyonaga (2000), mouse and human NPDC-1 proteinshave been considered (NCBI, OMIM) to be neural pro-tein counterparts of the Caenorhabditis elegans CAB-1(Genbank Q93249), because this protein presents 41.5%identity in 89 aa overlap with the human NPDC-1C-terminus (amino acids 225–313). CAB-1 has beenidentified by its ability to interact with Aex-3, a homologof rat Rab3 GEP. By regulating the conversion from theGDP-bound inactive form to the GTP-bound activeform, Rab3 GEP is essential for Rab3 action in Ca2�-dependent exocytosis (Tanaka et al., 2001; Yamaguchi etal., 2002). Indeed, Rab3 proteins, particularly Rab3A, areimplicated in the docking and fusion processes of synapticvesicles with the presynaptic plasma membrane and in therelease of neurotransmitters (Shirataki et al., 1994; Wada etal., 1997; Darchen and Goud, 2000). The results obtainedby Iwasaki and Toyonaga (2000) prompted us to study thein vitro interaction of NPDC-1 with rat Rab3 GEP.Mouse cDNA and protein were Genbank X67209 andSwiss-prot Q64322; mouse gene was Genbank AF263513.

MATERIALS AND METHODS

Cell Cultures

Cortical neurons were derived from Wistar rat embryos at17.5 days of gestation (E17). Three hundred thousand cells wereplated in poly-DL-ornithine-coated eight-well chambers(Labtech chamber; Nalge Nunc) and cultivated for 10 daysbefore transfection in Neurobasal medium supplemented withB27 (Invitrogen, La Jolla, CA).

The rat pheochromocytoma PC12 cells were cultivated inhigh-glucose Dulbecco’s modified Eagle’s medium, supple-

Contract grant sponsor: Association Pour la Recherche Contre le Cancer;Contract grant sponsor: CNRS; Contract grant number: UMR 8125.

*Correspondence to: Dr. Claudine Evrard, Laboratoire Biologie Molecu-laire et Differenciation, Unite de Genetique Oncologique, CNRS-URA8125, Institut Gustave Roussy, PR-1, 39 rue Camille Desmoulins, 94805Villejuif Cedex, France. E-mail: [email protected]

Received 1 October 2004; Revised 16 November 2004; Accepted 19November 2004

Published online 27 January 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jnr.20405

Journal of Neuroscience Research 79:747–755 (2005)

© 2005 Wiley-Liss, Inc.

mented with 10% fetal calf serum (FCS) as described elsewhere(Dupont et al., 1998). For immunochemistry, PC12 wereseeded in collagen-coated four-well chambers at a density of20,000 cells per well. The differentiation medium was RPMIsupplemented with Hepes 5 mM, 5% FCS plus 10% horse serum(HS). Human NGF (50 ng/ml; Sigma, St. Louis, MO) wasadded on the following day and maintained throughout theexperiment. GH3 cells (a pituitary cell line) were seeded ongelatin-coated glass coverslips and cultivated in Dulbecco’s me-dium supplemented with 5% FCS plus 15% HS.

Plasmids

The pCMV-NPDC-1-HA plasmid and the different de-leted forms were described previously (Sansal et al., 2000). TheNPDC-green fluorescent protein (GFP) expression vector wasconstructed by cloning the Hind3-Stu1 NPDC-1 fragmentfrom pCMV-NPDC-1-HA, between the Hind3-Xma1 restric-tion sites of the pEGFP-N1 vector (Clontech, Logan, UT). Inthis construct, the 34 NPDC-1 C-terminal amino acids weredeleted.

To insert the Flag tag in the N-terminal region ofNPDC-1, a polymerase chain reaction (PCR) was performedwith the PME vector matrix, encoding NPDC-1 (Galiana et al.,1995). In this construct, the Flag epitope was inserted betweenamino acid 80 and amino acid 81. The sense primer encodes theSty1 restriction site, followed by the Flag tag and the NPDC-15�-end sequence: 5�-GCGTATACCAAGGGACTACAA-GGACGACGATGACAAGAGGAAACACCTATCTTCTGGGGAGGG-3�. The antisense primer encodes theNPDC-1 3�-end sequence (nucleotides 1130–1150). The PCRfragment was digested with Sty1 and cloned in the Sty1-digestedPME vector. The sequence was verified by sequencing. Theconstruct was then digested with Kpn1 and BamH1, and theresulting fragment was inserted in pCDNA3 (Invitrogen, LaJolla, CA).

The GST-Rab3 GEP�Nterm plasmid was constructed bycloning the rat cDNA encoding the 331 C-terminal amino acidsof Rab3 GEP, between the EcoR1 and the BamH1 sites ofpGEX-2T (Pharmacia, Piscataway, NJ). This cDNA was ob-tained by PCR amplification with rat brain total cDNAas a matrix. The sense primer was 5�-GTGGATCCTTAT-GGGACCAAATGCAGTTC-3�, encoding the BamH1 site,followed by the Rab3 GEP 5�-sequence (nucleotide 4005 of thecDNA sequence; Genbank U72995) and the antisense primer:5�-CCGAATTCCCCTCTCTATCAGCTAGAGAC-3�, en-coding the EcoR1 site, followed by the Rab3 GEP 3�-sequenceup to nucleotide 5009. The fragment was then digested withBamH1 and EcoR1, cloned, and sequenced.

Immunological Reagents

The rabbit anti-NPDC-1 polyclonal antibodies were de-scribed previously (Sansal et al., 2000). The Mab anti-Flag(BioM2), the rabbit anti-Flag polyclonal antibodies, and theMab anti-Golgi 58K (clone 58K-9) were purchased fromSigma-Aldrich. Alexafluor 468 and 568 anti-mouse and anti-rabbit antibodies and Alexafluor 568-conjugate transferrin werefrom Molecular Probes (Eugene, OR). Mab anti-HSP60, rabbitanticalreticulin polyclonal antibodies (Stressgen), Mab anti-c-myc Ab2 clone 9E10.3 (Neomarker), Mab anti-Rab3A (Syn-

aptic Systems), and Mab antisynaptophysin (Chemical Inter-national) were purchased. The Mab antisynaptobrevin 2(clone 69-1) was from Dr. R. Jahn (Max Planck Institute). Themouting medium was Fluoromount-G (Southern Biotechnolgy,Birmingham, AL).

Transfection of Construct

Neurons or cells in culture were transfected with Lipo-fectamine 2000 (Invitrogen), according to the manufacturer’sinstructions. Generally, 0.75 �g DNA was mixed with 2 �lLipofectamine in 0.5 ml and added to the cells for 6 hr.

Immunochemistry

Immunochemistry was performed 48 hr after transfection.The cells were fixed with 4% paraformaldehyde for 30 min at4°C and permeabilized with 0.3% Triton X-100 in phosphate-buffered saline (PBS) for 20 min at room temperature (RT).Nonspecific labeling sites were blocked by using 5% FCS plus3% goat serum in PBS for 20 min. Cells were then incubatedwith the first antibody in PBS supplemented with bovine serumalbumin (BSA) and goat serum for 1 hr at room temperature.They were then washed with PBS/BSA, and incubated withAlexafluor-conjugated goat anti-mouse or goat anti-rabbit IgGfor 30 min. After extensive washing, the cells were mounted andexamined via confocal microscopy (LSM 510 Zeiss).

For localization of the Flag epitope at the plasma mem-brane, exponentially growing cells were first incubated for20 min at 4°C with monoclonal anti-Flag antibody in PBS/BSA/goat serum, then washed and incubated with Alexafluoranti-mouse antibody. After several washes, the cells were fixedand the nuclei stained with Hoechst-3342.

Subcellular Fractionation of Rat Brain

Two newborn rat brains were homogenized in 20 ml0.32 M sucrose containing 4 mM Hepes, pH 7.4, 2 mM EDTA,a cocktail of protein inhibitors (Roche, Indianapolis, IN), and10 mM phenylmethylsulfonyl fluoride (PMSF). Subcellular frac-tions were obtained by differential centrifugation, as describedby Ueda et al. (1979). Briefly, after homogenization and elim-ination of nuclei and cell debris, a crude mitochondrial andsynaptosomal pellet (P1) and a supernant (S1) were obtained.Centrifugation of S1 at 100,000g gave a microsomal pellet(P100) and the cytosol (S100). P1 was sedimented on a discon-tinuous gradient, giving an upper myelin fraction (My), a mix-ture of membranes (P2), a synaptosomal fraction (Sy), and acrude mitochondrial pellet (P). The synaptosomal fraction waslysed by osmotic shock and spun twice at 32,000g, giving acrude synaptic membrane (CSM) fraction pellet. The supernantwas finally spun at 78,000g to give a crude synaptic vesicle(CSV) fraction pellet. Protein concentrations were determinedby using the Bio-Rad Dc protein assay kit. Immunoblottingexperiments were as described elsewhere (Sansal et al., 2000).The molecular weight markers were the Multi-Mark Multi-Colored Standart (Invitrogen) or the rainbow mix (Amersham-Pharmacia, Arlington Heights, IL). Transfected cell lysates wereobtained as previously described (Sansal et al., 2000).

748 Evrard and Rouget

Purification of the GST Rab3 GEP�Nterm Protein

GST-Rab3 GEP�Nterm was expressed and purified fromEscherichia coli (Dupont et al., 1998). TNE buffer (20 mM Tris,120 mM NaCl, 1 mM EDTA) plus lysozyme (1 mg/ml) wasadded to the bacterial pellet for 15 min at 4°C, then completedwith 5 mM dithiothrewitol (DTT), protein inhibitors cocktail,and PMSF; sonicated; and centrifuged for 20 min at 10,000g.The resulting pellet was resuspended in the same buffer supple-mented with 1.5% Sarcosyl, agitated for 1 hr at 4°C, thencompleted with Triton X-100 (1% final).

The GST-fusion proteins or the GST protein were im-mobilized on glutathione-sepharose beads for 2 hr at 4°C; thebeads were extensively washed with the same buffer supple-mented with 1% Triton X-100, aliquoted, and eventually fro-zen. For interaction experiments, the loaded beads were firstequilibated with 0.5% NP40 and incubated with NPDC-1(full-length or indicated deleted forms) synthesized in vitrowith 35S-methionine. In vitro translation and in vitro bindingexperiments were performed essentially as previously described(Sansal et al., 2000).

Loading control for each in-vitro-translated product al-lowed us to add the same amount of radioactive proteins to eachsample, to confirm the position of the main band and to estimatethe percentage of protein that bound to the GST-Rab3-GEP.After extensive washing, the bound proteins were resolved by10% SDS-PAGE and detected by fluorography.

RESULTSSubcellular Localization of NPDC-1

The antibodies directed against NPDC-1 did not allowintracellular detection of endogenous NPDC-1, either in situor in cell lines (Dupont et al., 1998). To analyze the subcel-lular distribution of NPDC-1, we transfected neurons andneuroendocrine cells (PC12 and GH3) with NPDC-1 ex-pression vectors carrying a tag. The tag was either the Flagpeptide, inserted into the N-terminal region of the protein,between amino acid 80 and amino acid 81, ahead of thetransmembrane region (amino acids 188–212; see Fig. 1), orthe GFP fused to amino acid 298 of NPDC-1. The expres-sion of NPDC-1 was examined by anti-Flag immunolabelingor by the direct detection of GFP fluorescence.

In primary differentiated embryonic cortical neu-rons, NPDC-1 displayed a punctate staining along and atthe distal part of neuritic processes and accumulated in

small varicosities (Fig. 2a–c). Although the fluorescencewas predominantly vesicular, a small fraction of the pro-tein appeared diffuse in the cytoplasm and might corre-spond to a partial distribution to the cytosol. NPDC-1 wasnot observed in the nuclei.

When immunolabeling was performed on livingcells (PC12 and GH3 cells), the Flag epitope was detectedat the cell surface, showing that the protein was inserted inthe plasma membrane, with its N-terminal region outsidethe cells (Fig. 2d,e). The presence of NPDC-1 at theplasma membrane implies that newly synthesized proteinfollows, at least in part, intracellular secretory pathways.NPDC-1 was also detected in endoplasmic reticulum(ER; partial colocalization with the calreticulin ER pro-tein) and in the Golgi apparatus (partial colocalization withthe 58K Golgi protein; data not shown). The vesicularpattern of NPDC-1 fluorescence in the neurites was rem-iniscent of the tubulovesicular organelles delivering theintegral membrane protein synaptophysin to the cell sur-face (Nakata et al., 1998).

Incubation of living PC12 cells with fluorescenttransferrin (Trn), for 1 hr at 37°C, revealed the presence ofNPDC-1 in Trn-positive punctate structures, correspond-ing to recycling endosomes (Fig. 2f–h). Thus, after trans-port to the plasma membrane, NPDC-1 might be inter-nalized through endosomal elements.

In PC12 differentiated cells, cultivated for 6 days inthe presence of NGF, NPDC-1 partially colocalized, bothin cell body and in neuritic processes, with endogenoussynaptophysin, whose final destination is synaptic-like mi-crovesicle (SLMV; Fig. 3f,h,i). SLMV are highly related tosynaptic vesicles of neurons.

NPDC-1 partially colocalized also with another syn-aptic vesicle protein, synaptobrevin 2 (VAMP2/Sb2),present in SLMV, in large dense core granule (DCG), andon early endosomes (Fig. 3c,g). NPDC-1 was also de-tected in recycling endosomes of the cell bodies labeledwith fluorescent transferrin (Fig. 2g–i), suggesting thatNPDC-1 present at the cell membrane might be internal-ized by endocytosis.

We also observed partial colocalization of NPDC-1protein with C-myc tagged Rab3 GEP. In this experi-ment, the full-length Rab3 GEP vector (pCMV-myc

Fig. 1. Representation of the NPDC-1 protein structure.

NPDC-1 Subcellular Localization 749

Rab3 GEP; Fig. 4c,g,h) or the deletion mutant (pCMV-myc Rab3 GEP�N2, lacking the 1029 N-terminal aminoacids; Tanaka et al., 2001; Fig. 4f,i) was cotransfected withNPDC-1 expression vectors in PC12 cells.

Subcellular Localization of NPDC-1 in Rat BrainPrevious Northern blot analysis showed that

NPDC-1 was expressed predominantly in nervous tissus(Galiana et al., 1995). Newborn Wistar rat brains were

Fig. 2. Expression of NPDC-1 in cultured cortical neurons. Rat embry-onic cortical neurons were cultured for 10 days in Neurobasal medium,transfected with Flag-NPDC (a) or NPDC-GFP (b,c), and fixed 48 hrlater. The Flag epitope was detected with polyclonal anti-Flag antibodiesrevealed with Alexafluor 568 anti-rabbit antibodies (red). Fluorescence wasvisualized via confocal microscopy. Expression at the plasma membrane(d,e). Living cells, PC12 (d) and GH3 (e) cells transfected with Flag-NPDC, were incubated with Mab anti-Flag antibody and then with

Alexafluor 468 anti-mouse antibodies (green). After repeated washes, thecells were fixed, and the nuclei were stained with Hoechst-3342. LivingPC12 cells transfected with NPDC-GFP were incubated with 50 �g/mlAlexafluor transferrin (Trn) in RPMI supplemented with 0.1% BSA for1 hr, washed, and fixed (f–h). NPDC-GFP, green; transferrin, red. Yellowstaining in superimposed images denotes colocalization (h). Figure can beviewed in color online via www.interscience.wiley.com.


homogenized and purified on sucrose gradient accordingto Ueda et al. (1979).

Immunoblotting experiments with polyclonal anti-NPDC-1 antibodies showed rat brain synaptosomes to beenriched in NPDC-1 (Fig. 5A,B). It was present in CSMand CSV derived from synaptosomes after osmotic lysis(Fig. 5B). On the contrary, the mitochondrial HSP60protein, though present in synaptosomes and synaptic ves-

icle membranes, was absent from the CSV pellet (Fig. 5C).Thus, at this step of the purification procedure, the CSVwere free of mitochondrial contamination. As can be seenin Figure 6, the CSV fraction was enriched in synapto-physin, Rab3A, and synaptobrevin 2 proteins (respectiveMWs 38, 24, and 18 kDa), although these proteins werestill present in the CSM fraction, which contains tightlydocked synaptic vesicles.

Fig. 3. Partial colocalization of NPDC-1 with synaptic vesicle markers. PC12 cells were plated incollagen-coated Labtech chambers and grown in differentiation medium supplemented with NGF for6 days before transfection with NPDC-GFP. Detection of NPDC-GFP (green; a,c,d,f–i), and en-dogenous synaptobrevin 2 (red; b,c,g) or endogenous synaptophysin (red; e,f,h,i). Yellow staining insuperimposed images denotes colocalization (c,f–i).


Interaction of NPDC-1 With Rab3 GEPThe interaction of NPDC-1 with Rab3 GEP was

examined, in vitro, by GST pull-down experiments. Be-cause, in the Aex-3 Caenorhabditis elegans protein, theCAB-binding region was localized to the 178 C-terminalamino acid region, the 3� homologous region of Rab3

GEP (from nucleotide 3,816 to the end) was amplified byPCR and cloned in pGEX-2T. As shown in Figure 7, thefusion protein GST-Rab3 GEP-Nterm, including the 331C-terminal amino acids of Rab3 GEP, interacted withfull-length and different deleted forms of NPDC-1 de-scribed by Sansal et al. (2000). Deletion of part of the

Fig. 4. Partial colocalization of NPDC-1 with Rab3 GEP.PCMVmyc-Rab3 GEP (a– c,g,h) and pCMVmyc-Rab3 GEP�N2(d–f,i), were cotransfected with pCMVFlag-NPDC or withpCMVNPDC-GFP in PC12 differentiated cells. The cells werefixed 48 hr later and processed for immunochemistry. The mycepitope was detected with anti-myc monoclonal antibody and re-

vealed either with anti-mouse Alexafluor 468 (green; a– c,g,h) orwith anti-mouse Alexafluor 568 (red; d–f,i). Detection of NPDC-GFP (green; d–f,i) or Flag-NPDC with rabbit polyclonal anti-flagantibodies (red; a– c,g,h). Yellow staining in superimposed imagesdenotes colocalization (c,f–i).


N-terminal region of NPDC-1 (–Nterm), from aminoacid 11 to amino acid 99, abolished this interaction. Underthe same experimental conditions, NPDC-1 did not in-teract with control GST (Fig. 7) nor with the irrelevantGST-p27 fusion protein (data not shown).

DISCUSSIONAs shown by immunochemistry experiments, when

surexpressed in embryonic cortical neurons and in neu-roendocrine cells (PC12 and GH3), NPDC-1 has pre-

dominantly a vesicular distribution and is not detected innuclei. According to these results, the interaction ofNPDC-1 with E2F transcriptional factors and cyclins(Sansal et al; 2000) would occur outside the nuclei; in-deed, these factors are known to be predominantly cyto-plasmic in differentiated cells (Gilla and Hamela, 2000).

The protein is inserted in the plasma membrane withits N-terminal region outside the cell. As expected for anintegral membrane protein, NPDC-1 would be trans-ported from the Golgi membranes to the cell surface bytubulovesicular organelles similar to the structures de-scribed for axons by Nakata et al. (1998).

In PC12 differentiated cells, NPDC-1 partially co-localized with different synaptic vesicle proteins that me-diate fusion of synaptic vesicles with the plasma mem-brane. Indeed, we detected some overlap betweenoverexpressed NPDC-1 protein (after transfection) andendogenous synaptophysin and synaptobrevin 2, whichare both transmembrane proteins; these results were con-firmed by cell fractionation studies in vivo (see below).

We also observed partial colocalization of NPDC-1with Rab3 GEP, a peripheral membrane protein thatcycles between synaptic vesicle membranes and the cyto-plasm. When the 572 C-terminal amino acid region ofmyc-tagged Rab3 GEP was overexpressed in PC12 dif-ferentiated cells, along with Flag-NPDC-1 or NPDC-GFP, we observed partial colocalization of the proteins.The similar distribution of Flag or GFP proteins implies

Fig. 5. Subcellular distribution of NPDC-1 in rat brain. A,B: Thefractions were obtained by differential centrifugation according to Uedaet al. (1979). Aliquots of each fraction containing 20 �g protein (except10 �g for synaptosomes) were subjected to SDS-PAGE, followed byimmunoblotting, using anti-NPDC-1 antibodies. C: The membranefrom B was stripped and incubated with anti-HSP60. S1, supernatant;P1, pellet. Centrifugation of S1 gave S100 (soluble cytosol) and P100(microsomes). P1 was centrifuged on a sucrose gradient giving myelin(My), mixture of ER, Golgi, and plasma membrane (P2), synaptosomes(Sy), sucrose pellet (P). Sy fraction was lysed and centrifuged, givingcrude synaptic membranes (CSM) and crude synaptic vesicles (CSV). T,NPDC-1 transfected cell lysate (1 �g), as a control. NPDC-1 showedan apparent molecular weight of 43 kDa and HSP60 of 60 kDa.

Fig. 6. Subcellular distribution of synaptic proteins in rat brain. Ali-quots of each fraction (see Fig. 5), containing 1 �g protein, weresubjected to SDS-PAGE, followed by immunoblotting, using a mix ofantisynaptophysin, anti-Rab3A, and antisynaptobrevin 2 (respectiveMWs 38, 24, and 18 kDa).

Fig. 7. NPDC-1 interacts with Rab3 GEP�Nterm in GST pull-downexperiments. In vitro translation products (full-lengh and the indicateddeleted forms of NPDC-1) of the same radioactivity were incubatedwith glutathione-sepharose beads loaded with 10 �g of GST (control)or GST-Rab3 GEP. NPDC, full length; �HLH, �aa 100–143; �MB,�aa 193–229; �MAP, �aa 232–298; �Cterm, �aa 149–332; �Nterm,�aa 11–99. The beads were washed and the 35S-labeled bound proteinswere resolved by 10% SDS-PAGE and detected by fluorography.


that there are no specific targeting signals in the 34C-terminal amino acids that are present in the Flag-NPDC-1 protein and deleted in the NPDC-GFP protein.

NPDC-1 was also detected in recycling endosomeslabelled with fluorescent transferrin (Trn). According tosome results (Blagoveshchenskaya et al., 1999; De Wit etal., 1999; Provoda et al., 2000), SLMV-targeted proteinsare transported to the plasma membrane and then inter-nalized in early Trn-positive endosomes before beeingdelivered either to SLMV (Trn-negative) or to lysosomesfor degradation or recycled to the plasma membrane. Thepresence of NPDC-1 in Trn-positive punctate structures,enriched in the perinuclear region and in the region of theGolgi apparatus, could lead to the hypothesis thatNPDC-1, such as synaptophysin, traffics from the trans-Golgi to the cell surface and is then internalized andrecycled through endosomal elements before beeingsorted into SLMV. Immunoelectron microscopic analysisand dynamic labelling studies could allow confirmation ofthese different localizations and the different pathwaysfollowed by the overexpressed NPDC-1 protein (aftertransfection).

Fractionation of rat brain on sucrose gradient, ac-cording to Ueda et al. (1979), showed NPDC-1 to beenriched in synaptosomes and in derived crude synapticvesicles. These results confirmed that the endogenousNPDC-1 protein is partially associated, in vivo, with CSV.Similar association of NPDC-1 with cell membranes wasalso found after fractionation of protein lysates derivedfrom transfected cells (not shown). Hence, it is likely thatoverexpression of NPDC-1 in transfected cells does notlead to major mislocalization (except the accumulation inlysosomes) and to immunolabeling artefacts.

We wondered whether NPDC-1 protein presentsspecific organelle targeting signals. Indeed, the transport oftransmembrane proteins to post-Golgi destinations hasbeen shown to be controlled by specific sequences. In Sb2,a single amphipathic helix has been demonstrated to beinvolved in SLMV targeting (Grote et al., 1995), but othersynaptic vesicle membrane proteins do not present a helixwith the characteristics of helix 1, and it is unlikely thatthere is a single structure for targeting all synaptic vesicles.However, in NPDC-1, a coiled-coil structure has beenpredicted between amino acid 93 and amino acid 120(PSORT II). Deletions in this region should demonstratewhether it is implicated in this transport. Other organelletargeting signals are described to be localized in the cyto-plasmic domain. The most common are tyrosine- anddileucine-containing motifs (Blagoveshchenskaya et al.,1999; Blagoveshchenskaya and Cutler, 2000). Sequenceanalysis of the NPDC-1 cytoplasmic tail does not revealsuch signals.

The partial colocalization of NPDC-1 with synapticvesicle proteins, particularly with Sb2, a key proteininvolved in exocytic membrane fusion, prompted usto examine whether NPDC-1 was involved in Ca2�-dependent exocytosis. Full-length NPDC-1 or the N-terminal-deleted mutant (�Nterm) was coexpressed with a

plasmid encoding the human growth hormone (hGH) inPC12 cells, and high-K�-induced hGH release was mea-sured, according to Oishi et al. (1998). The basal hGHrelease was not affected (F. Darchen, data not shown).Thus, at present, the results do not allow us to concludethat NPDC-1 is involved in synaptic vesicle trafficking.

The interaction of NPDC-1 and Rab3 GEP wasobserved, in vitro, in GST pull-down experiments. In-deed, the fusion protein GST-Rab3 GEP�Nterm (330C-terminal amino acid region) was able to interact withfull-length NPDC-1 and different deleted forms, exceptwith the N-terminal-deleted form (�Nterm, aminoacids 11–99). Under the same experimental conditions,NPDC-1 did not interact with control GST, with GST-p27, with different GST-E2F1-deleted forms (�2, �4, �6;see Sansal et al., 2000), or with cyclin A, suggesting thatthe observed interactions were specific. Such results sup-port the idea that the interaction of NPDC-1 with Rab3GEP occurs through its N-terminal region. However, werather expected that the C-terminal region of NPDC-1(amino acids 210–332) would be involved in this inter-action. Indeed, the interactive region should present somesequence homology with CAB-1 and should be cytoplas-mic, insofar as Rab3 GEP is a peripheral membrane pro-tein. Possibly, the observed in vitro interactions resultedfrom artefacts due to abnormal in vitro protein conforma-tions. The in vitro interaction between CAB-1 and AEX-3 had been confirmed in a yeast two-hybrid system, andcab-1 mutants displayed presynaptic defects (Iwasaki andToyonaga, 2000). For NPDC-1 and Rab3 GEP, a two-hybrid approach in mammalian cells should help in resolv-ing this question. At the present time, however, it may bepremature to assume that NPDC-1 and CAB-1 belong tothe same family.

ACKNOWLEDGMENTSWe are grateful to F. Darchen for Ca2�-dependent

exocytosis analysis, to M. Mallat for neuron cultures, andto A. Jalil for confocal microscopy. We are indebted to Y.Takai for sending myc-tagged Rab3 GEP vectors. Wethank T. Galli, M. Gunther, and A. Prochiantz for helpfuldiscussions.

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subcellular localization of neural-specific npdc-1 protein

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