Journal of Cell Science 113, 2725-2735 (2000) 2725 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1362

The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus

Frank J. M. Opdam1, Arnaud Echard2, Huib J. E. Croes1, José A. J. M. van den Hurk3, Rinske A. van de Vorstenbosch1, Leo A. Ginsel1, Bruno Goud2 and Jack A. M. Fransen1,* 1Department of Cell Biology, Institute of Cellular Signalling, University of Nijmegen, PO Box 9101, 6500 HB, Nijmegen, The Netherlands 2UMR CNRS 144, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France 3Department of Human Genetics, University Hospital Nijmegen, Nijmegen, The Netherlands *Author for correspondence (e-mail: [email protected])

Accepted 23 May; published on WWW 10 July 2000

SUMMARY

Members of the Rab subfamily of small GTPases play an cell line SK-N-SH. In brain, Rab6B was found to be important role in the regulation of intracellular transport specifically expressed in microglia, pericytes and Purkinje routes. Rab6A has been shown to be a regulator of cells. Endogenous Rab6B localises to the Golgi apparatus membrane traffic from the Golgi apparatus towards the and to ERGIC-53-positive vesicles. Comparable studies endoplasmic reticulum (ER). Here, we report on the between Rab6A and Rab6B revealed distinct biochemical identification of a Rab6 isoform, termed Rab6B. The and cellular properties. Rab6B displayed lower GTP- corresponding full-length cDNA was isolated from a Caco- binding activities and in overexpression studies, the protein 2 cell library. The deduced amino acid sequence showed is distributed over Golgi and ER membranes, whereas 91% identity with the Rab6A protein and revealed that Rab6A is more restricted to the Golgi apparatus. Since the sequence divergence is dispersed over a large region of GTP-bound form of Rab6B (Rab6B Q72L) does interact the COOH-terminal domain. Rab6B is encoded by an with all known Rab6A effectors, including Rabkinesin-6, independent gene which is located on chromosome 3 region the results suggest a cell-type specific role for Rab6B in q21-q23. In contrast to Rab6A whose expression is retrograde membrane traffic at the level of the Golgi ubiquitous, northern blot analysis, immunohistochemistry, complex. and immunofluorescence demonstrated that Rab6B is expressed in a tissue and cell-type specific manner. Rab6B is predominantly expressed in brain and the neuroblastoma Key words: Golgi apparatus, Rab, Rabkinesin-6, Transport carrier

INTRODUCTION Rab33 (Armstrong et al., 1996; Barbosa et al., 1995; Chen et al., 1996, 1997b; Tisdale et al., 1992). Some Rab isoforms, Transfer of cargo between intracellular membrane organelles such as Rab1A and Rab1B (Nuoffer et al., 1994) may be takes place via carrier vesicles. The correct delivery of these functionally redundant. However, distinct expression patterns vesicles to the appropriate membranes is regulated by Rab of others suggest possible differences in function. This appears proteins. This subfamily of small GTPases triggers the to be the case, for instance, for the four Rab3 isoforms (Rab3A- recruitment of specific docking complexes (Christoforidis et D). Although they are all expressed in cells with regulated al., 1999; McBride et al., 1999), and thereby might facilitate secretory pathways, Rab3A and Rab3C are preferentially the formation of SNARE complexes leading to membrane expressed in neurons and neuroendocrine cells, whereas fusion between the donor and acceptor membrane (Aridor and Rab3D is found on granules of gastric chief cells and of Balch, 1996; Chavrier and Goud, 1999; Rothman and Sollner, peritoneal mast cells (Fischer von Mollard et al., 1990, 1994; 1997; Schimmoller et al., 1998). Recently, Rab proteins have Roa et al., 1997; Tang et al., 1996). Rab3D is also the most also been found to be involved in the movement of transport abundant Rab3 isoform in adipocytes (Baldini et al., 1998). carriers along actin or microtubule cytoskeleton (Echard et al., Rab3B is highly expressed in epithelial cells (Weber et al., 1998; Nielsen et al., 1999; Walch-Solimena et al., 1997). 1994). Rab3A and Rab3B appear to have distinct functional To date, the Rab family consists of approximately 50 family properties in PC12 cells, although the four isoforms are present members. Several members have been shown to be part of on secretory granules in PC12 cells and each of them inhibits subgroups based on structural relationships and high sequence secretion (Iezzi et al., 1999; Chung et al., 1999). identities (80%-95%). Such isoforms have been described for In addition to Rab6A, involved in retrograde membrane Rab1, Rab3, Rab4, Rab5, Rab8, Rab11, Rab22, Rab27 and traffic between the Golgi complex and the endoplasmic 2726 F. J. M. Opdam and others reticulum (ER) (Martinez et al., 1994, 1997; White et al., RNA isolation and northern blotting 1999), two additional Rab6 subfamily (Rab6A′ and Rab6B) HT-29 cells were grown with (+) or without D-glucose (−) to compare members have now been identified. Rab6A′ was recently found nondifferentiated versus differentiated cells (Darmoul et al., 1992). to be generated by alternative splicing of a duplicated exon Total RNAs from cultured cell lines and various human tissues within the Rab6A gene. Rab6A and Rab6A′ are ubiquitously (rectum, jejunum, stomach, oesophagus) were prepared following the expressed and differ in only three amino acid residues (F. J. M. guanidium isothiocyate-phenol-chloroform extraction method µ Opdam et al., unpublished). Interestingly, Rab6A′ does not (Chirgwin et al., 1979). Samples of total RNA (15 g) were separated interact with the Rab6A effector Rabkinesin-6, a kinesin-like by electrophoresis on a 1% formamide agarose gel and transferred to nylon membrane according to standard procedures (Sambrook et al., protein associated with the Golgi apparatus (Echard et al., ′ ′ 1989). Blots were probed with a 0.7 kb 3 PstI/EcoRI fragment of 1998). In addition, the GTPase-deficient mutant Rab6A Q72L Rab6B (Fig. 1A, 530-1270), and a 1.35 kb glyceraldehyde-3- does not induce the redistribution of Golgi resident proteins phosphate dehydrogenase (GAPDH) cDNA probe was used to enable into the ER, as Rab6A Q72L does (Martinez et al., 1997; F. J. comparison of RNA loading. The human tissue blot with 2 µg M. Opdam, unpublished). Here, we isolated the full length poly(A)+ RNA samples was purchased from Clontech. cDNA encoding Rab6B, whose partial sequence has previously been reported (Chen et al., 1997a). Rab6B displays 91% Expression plasmid construction identity with Rab6A and is encoded by a different gene. Like Constructs for the expression of GST fusion and epitope-tagged Rab6A/A′, the bulk of Rab6B localises to the Golgi apparatus. versions of Rab6B were generated. Using the complete cDNA clone However, Rab6B shows a cell-type specific expression pattern as template the open reading frame of Rab6B including the initiator AUG codon and the stop codon for proper termination of translation in brain and GTP-binding properties distinct from that of ′ was PCR amplified with specific primers. One set of primers (forward; Rab6A/A . Despite these differences, Rab6B interacts with the 5′-GGAATTCCGGATGTCCGCAGGGGGAGA-3′ introducing an same Rab6A effector molecules, including Rabkinesin-6. Our EcoRI site in front of the start codon, and reverse; 5′- results suggest a cell type-specific role for Rab6B. CCGCTCGAGCGGTTAGCAGGA-3′ introducing an XhoI site downstream of the stop codon) was used to subclone Rab6B in-frame into an EcoRI/XhoI digested pMyc vector. pMyc is a pCDNA3- ′ MATERIALS AND METHODS derived vector in which at the 5 end of the pCDNA3 (Invitrogen) multiple cloning site a synthetic DNA fragment was introduced that entails an initiator AUG codon followed by the cMyc-epitope tag and RT-PCR and library screening an EcoRI site. An other forward primer (5′-CGGGATCCATGTCC- Total RNA (1.5 µg) from polarised Caco-2 cells was reverse GCAGGGGGAGA-3′, containing a BamHI site) was used to subclone transcribed using random hexamers (2 µg, Pharmacia) and Rab6B in-frame into a BamHI/XhoI digested eukaryotic expression Superscript reverse transcriptase (100 U, Gibco/BRL). One-sixth of vector pSG5 (Green et al., 1988), which was modified to generate an the cDNA was subsequently analysed by PCR using degenerate ‘Rab- N-terminal VSV epitope tag, or into the multiple cloning site of the specific’ primers (forward; 5′-GGCGGCGGCTCGAGGGI(A0.2/G0.8) prokaryotic expression vector PGEX (Pharmacia) to produce GST- (G0.2/A0.8)II(A0.2/G0.2/C0.6)IIII(A0.2/T0.2/G0.6)(G0.2/C0.2/T0.6)(A0.2/ Rab6B fusion protein in Escherichia coli following manufacturer’s T0.8)GGIAA(A0.5/G0.5)(A0.5/T0.5)C-3′ containing a XhoI restriction instructions (Pharmacia). Similar constructs were prepared for Rab6A site, and reverse; 5′-GGCGGCGGATCCTTC(C0.5/T0.5)TGICC(A0.5/ following the exact cloning procedures of Rab6B. All PCR constructs T0.5) GCIGT(A0.5/G0.5)TCCCA-3′ containing a BamHI restriction were checked for absence of mutations by DNA sequencing. site] matching conserved domains PM1 and PM3 of GTP-binding; see Fig. 1B). Purified BamHI/XhoI-digested PCR products were ligated Antibodies into the pBluescript vector KS+. Library cloning and subsequent Purified GST-Rab6B fusion protein was used to immunise a rabbit. preparations and screening of replica filters were carried out following Affinity-purified polyclonal antibodies were obtained by applying standard procedures (Sambrook et al., 1989). whole serum to Affigel-10-immobilised (Bio-Rad, Richmond, CA) The partial Rab6B sequence was radiolabeled by random GST-Rab6B fusion protein and eluting bound antibodies. For oligonucleotide priming (Feinberg and Vogelstein, 1983) and used as elimination of cross-reactivity on western blot, affinity-purified a probe to screen a human Caco-2 λgt11 cDNA library consisting of antibodies were purified three times over a GST-Rab6A bound approximately 4×106 independent clones (Lacey et al., 1989). The glutathione Sepharose 4B column and flow through was used for the insert sequences of isolated clones were subcloned and sequenced by incubation step. the method of Sanger (T7 sequencing kit, Pharmacia). Predicted polypeptides were compared with database entries using the BLAST Immunoblotting program (Altschul et al., 1997). Transient transfected COS-1 cells (electroporation; 300 V, 125 µF) were plated on 10-cm dishes. After 48 hours cells were lysed in 50 Radiation hybrid mapping µl 2× sample buffer (100 mM Tris-HCl, pH 6.8, 200 mM Mapping of the Rab6B gene was performed by PCR analysis of the dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol), and Stanford G3 Radiation Hybrid Panel (Research Genetics). Primers 15 µl of each lysate was subjected to SDS-PAGE and transferred onto (forward, 5′-GGCTAGCTTCCTAAGGGGGG-3′, see Fig. 1A, bases nitro-cellulose by western blotting. After blocking with 5% non-fat 1023-1042; reverse, 5′-GCAAAAAATTGTATACACATG-3′, bases dry milk in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% 1246-1267) were designed to amplify a 244 bp product from the 3′ Tween-20 (TBST), the blot was incubated with cell culture UTR of the Rab6B gene. Each PCR reaction contained 25 ng of supernatant of the anti-cMyc hybridoma 9E10 (dilution 1:100) (Kari genomic DNA, 25 ng of both primers, 250 µM of each dNTP, and 0.4 et al., 1986). For characterisation of the antibodies, GST-Rab6A U Taq DNA polymerase (Gibco/BRL) in 10 µl of 10 mM Tris-HCl, purified anti-Rab6B antibodies (1:5000) were additionally incubated pH 8.6, 50 mM KCl, 5 mM DTT, and 2 mM MgCl2. PCR for one hour with 40 µg GST-Rab6A prior to blot incubation. One amplification was carried out for 30 cycles of 94°C for 20 seconds, hour incubations with primary and secondary antibodies (horseradish 58°C for 20 seconds, and 72°C for 30 seconds. PCR products were peroxidase-conjugated AffiniPure goat anti-mouse or anti-rabbit IgG; analysed on a 2% agarose gel. Scores were submitted to the Stanford 0.06 µg/ml) and subsequent washes were carried out in TBST at room Human Genome Centre website (http://www-shgc.stanford.edu). temperature. Immunoreactive bands were visualised using freshly Rab6B, a cell-type specific Rab6 isoform 2727 prepared chemiluminescent substrate (100 mM Tris-HCl, pH 8.5, 1.25 mM dithiothreitol, and 0.1% Triton X-100. To each sample 30 µl of mM p-coumaric acid (Sigma), 0.2 mM luminol (Sigma), and 0.009% GTPγS-binding mix (20 mM Tris buffer, pH 8.0, 1 mM EDTA, 2 mM 7 35 H2O2). dithiothreitol, 2 µM GTPγS, and approx. 1.5×10 cpm of [ S]GTPγS) was added. Non-specific binding was assayed with samples Immunohistochemistry containing 0.1 mM unlabeled GTPγS. Samples were incubated at Cryo-sections of 2% paraformaldehyde-fixed adult human tissues 30°C for 0, 5, 15, 30, 60, or 120 minutes and incubation was from cerebrum or cerebellum (6 µm) were cut and mounted on terminated by addition of 2 ml of ice-cold washing buffer (20 mM Superfrost/Plus slides (Menzel Gläser, Germany). All incubation steps Tris-HCl, pH 8.0, 25 mM MgCl2,100 mM NaCl). The samples were were carried out at room temperature. After thawing, sections were filtered through nitro-cellulose membranes (NC45, Schleicher & incubated with 1% cold water fish skin gelatin in PBS for 30 minutes Schuell), subsequently washed four times in ice-cold washing buffer, followed by incubation for 1 hour with primary antibodies. For air dried, and counted in a water-compatible scintillation mixture examination of Rab6B-specific labelling, affinity-purified anti-Rab6B (Opti-Fluor, Packard). As a control for the calculation of the amount (1:10 dilution) was directly used or pre-incubated with 10 µg of GST- of GTPγS bound to the proteins, 15 µl of the GTPγS-binding mix was Rab6A or GST-Rab6B fusion protein for 1 hour at room temperature counted in duplicate. All samples were assayed in duplicate. before incubating the sections in PBS/0.05% Tween-20 (PBST). Other primary antibodies used here were mouse monoclonal anti-NF 160 Two hybrid experiments kDa antibody (Neural Cell Typing Set, Boehringer Mannheim, The yeast reporter strain L40, which contains the reporter genes HIS3 dilution 1:1), and 1 mg/ml mouse monoclonal anti-CD11b IgG and LacZ, was cotransformed with either pLexA-Rab6A Q72L, (Celltech, UK, dilution 1:200). Sections were incubated with biotin- pLexA-Rab6B Q72L and pGADGH-GAPCenA/Rab6 interacting conjugated AffiniPure Donkey anti-rabbit or anti-mouse IgG (Jackson domain (Cuif et al., 1999), pGADGH-Rabkinesin-6 (Echard et al., ImmunoResearch, diluted 1:250) in PBST for 1 hour. Specific 1998), or pGADGH-clone 1. After 3 days at 30°C on selective media, labelling was detected using the Vectastain ABC system (Vector cotransformants were patched on DO W- L- and replicated on DO W- Laboratories, USA). Sections were subsequently incubated in AEC L- and DO W- L- H-. Transformation, analysis and media are as (0.33 mg/ml 3-amino-9-ethyl-carbazole, 6% dimethylformamide, previously described by Janoueix Lerosey et al. (1995). 0.03% H2O2, 0.1 M NaAc, pH 5.0) for 5 minutes. Hematoxylin staining was prepared according to standard histological procedures. After thorough washing in MQ, sections were mounted in Kaiser’s RESULTS glycerol gelatin (Merck). Immunofluorescence analyses Molecular cloning of human Rab6B Transient transfected COS-1 cells (grown in DMEM, supplemented To investigate the spectrum of Rab proteins that are expressed with 10% FCS, 1% NEAA, L-glutamine, sodium pyruvate (Gibco in human Caco-2 cells, we performed an RT-PCR with primers BRL) and 0.001% β-mercaptoethanol) and SK-N-SH cells (grown in corresponding to the conserved domains PM1 and PM3 DMEM/10% foetal calf serum) were cultured in 24-well plates on (nomenclature following Valencia et al., 1991) involved in glass coverslips. After 48 hours, cells were fixed in 1% GTP-binding (Opdam et al., 2000). Among several distinct Rab paraformaldehyde and permeabilised with 0.1% saponine/20 mM cDNAs, we identified a partial sequence (Fig. 1A, bp 407-557) glycine. COS-1 cells were incubated with polyclonal anti-VSV alpha that showed high similarity with human Rab6A cDNA (F. J. M. P4 (dilution 1:1000) (Kreis, 1986) and monoclonal anti-cMyc Opdam et al., unpublished). We used the partial sequence to antibody 9E10 (dilution 1:1), or monoclonal anti-VSV P5D4 (ascites, screen a Caco-2 cDNA library, which resulted in the isolation dilution 1:1000) (Kreis, 1986) and polyclonal anti-PDI (dilution 1:100) (Freedman et al., 1989) for 1 hour. SK-N-SH cells were of three corresponding clones of approximately 1.3 kb (Fig. directly incubated with affinity-purified anti-Rab6B antibody (1:10 1A). All three clones showed identical integrated cDNA dilution), or the antibody was pre-incubated in a 30 µl volume with sequences, which contained no poly(A) sequence. In the open 10 µg of purified GST-Rab6B or GST-Rab6A protein for 1 hour at reading frame, the Rab6A-like nucleotide sequence shows 79% room temperature prior to incubation of the cells. Here, monoclonal similarity with Rab6A. The deduced amino acids contain the antibody CTR 433 (dilution 1:10) (Jasmin et al., 1989) and six conserved protein motifs involved in GTP-binding, and a monoclonal anti-ERGIC-53 antibody G1/93 (dilution 1:100) cysteine-motif (CXC) necessary for proper prenylation to (Schindler et al., 1993) were used in co-labeling studies. For both cell membranes (Fig. 1A). Since the sequence is 91% identical to lines, specific labelling was detected by incubation with FITC or Rab6A, we termed the protein Rab6B, a novel Rab6 subfamily Texas Red-conjugated goat anti-rabbit IgG and Texas Red or FITC- member. conjugated goat anti-mouse IgG (dilution 1:75, 10 µg/ml; Jackson The amino acid alignment of human Rab6B and Rab6A is ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 hour. All − washing steps and incubations with primary or secondary antibodies shown in Fig. 1B, together with Rab6( like) sequences from were carried out in PBST. Finally, coverslips were rinsed in PBS and other species. The majority of amino acid differences between water and cells were mounted on glass slides by inversion over 5 µl human Rab6B and Rab6A are mainly dispersed over a large C- Mowiol (Sigma). Cells were examined using a confocal laser scanning terminal region of the proteins (Fig. 1B, indicated by asterisks). microscope (MRC 1000, Bio-Rad). For C. elegans, two Rab6-like sequences were previously α 32 γ identified (Fig. 1B, U43283 and P34213). However, Rab6B [ - P]GTP blot overlay assay and GTP S binding assay does not represent a human homologue for one of them since µ GST-Rab fusion protein samples (1 g/sample) were separated in it shares more unique sequence features and is more closely duplicate on 12.5% polyacrylamide gels containing SDS (SDS- related to human Rab6A. PAGE). One gel was stained with Coomassie Blue (LKB), whereas the second was electrophoretically transferred onto nitro-cellulose Chromosomal localisation of human Rab6B gene membranes. The blot was incubated with 1 nM [α-32P]GTP (1 µCi [α-32P] GTP/ml) as previously described (Celis, 1998). To exclude the possibility that Rab6B is an alternative product GST-(fusion) protein samples (500 ng/assay) to be analysed were from the Rab6A gene we examined the chromosomal diluted to 30 µl with 20 mM Tris buffer (pH 8.0), 1 mM EDTA, 1 localisation of the Rab6B gene. The Rab6A gene was 2728 F. J. M. Opdam and others previously mapped to chromosome 2 in region q14-q21 by in Rab6B gene to chromosome 3, placing it 49 cR10000 from the situ hybridisation (Rousseau Merck et al., 1991). Radiation SHGC-1180 marker within the interval flanked by anchor hybrid mapping with Rab6B-specific primers assigned the markers D3S3606 and D3S3554 (146.0-156.2 cM). A 1 gcgcgccgtc cctgcgcccc gctccgcccg cgcctctctc ccagcgccgc gcctcgggcg taaagagcgc 71 gcccctcgca ccgcagccag tgccgaccgc agcagcccag ccccagcctt cctccgcctc cgcctctctc 141 tcctcctcct cctccgccgc cggacacgca gccgcagccg ggaccgggac gcagctgggg agtcagggac 211 gcgcgcagcc agcccttccc cctccggctc ccgcaccgcc ggccgcctcc cctcgccctc ctactctccc 281 ctccctgctc cttcgctttt tcctcctcct cctctcccgg ccccggctgc cagcacc M S A G G D F G N P L R K F K L V F L 19 338 atg tcc gca ggg gga gat ttt ggg aat cca ctg aga aaa ttc aag ttg gtg ttc ttg G E Q S V G K T S L I T R F M Y D S F 38 395 ggg gag cag agc gtc ggg aag acg tct ctg att acg agg ttc atg tac gac agc ttc D N T Y Q A T I G I D F L S K T M Y L 57 Fig. 1. Nucleotide and 452 gac aac aca tac cag gca acc att ggg att gac ttc ttg tca aaa acc atg tac ttg deduced amino acid sequence E D R T V R L Q L W D T A G Q E R F R 76 509 gag gac cgc acg gtg cga ctg cag ctc tgg gac aca gct ggt cag gag agg ttc cgc of human Rab6B and S L I P S Y I R D S T V A V V V Y D I 95 sequence alignment with 566 agc ctg atc ccc agc tac atc cgg gac tcc acg gtg gct gtg gtg gtg tac gac atc Rab6(−like) sequences from T N L N S F Q Q T S K W I D D V R T E 114 623 aca aat ctc aac tcc ttc caa cag acc tct aag tgg atc gac gac gtc agg aca gag various species. (A) The R G S D V I I M L V G N K T D L A D K 133 complete cDNA of Rab6B 680 agg ggc agt gat gtt atc atc atg ctg gtg ggc aac aag acg gac ctg gct gat aag (accession number R Q I T I E E G E Q R A K E L S V M F 152 737 agg cag ata acc atc gag gag ggg gag cag cgc gcc aaa gaa ctg agc gtc atg ttc AF166492) was isolated from I E T S A K T G Y N V K Q L F R R V A 171 a Caco-2 cDNA library with 794 att gag acc agt gcg aag act ggc tac aac gtg aag cag ctt ttt cga cgt gtg gcg a probe (approx. 150 bp) S A L P G M E N V Q E K S K E G M I D 190 851 tcg gct cta ccc gga atg gag aat gtc cag gag aaa agc aaa gaa ggg atg atc gac encoding the sequences I K L D K P Q E P P A S E G G C S C * 208 between GTP-binding 908 atc aag ctg gac aaa ccc cag gag ccc ccg gcc agc gag ggc ggc tgc tcc tgc taa domains PM1 and PM3. 965 tgcagagccg acctgtggct tcccatgaca ctccttgctt gttgtgttgc ttcctattgg ctagcttcct 1035 aaggggggag ggaaccgagt tatcaagatg ggaggatttt tcttttctct ctgtctttag gagtagggtg Amino acids are given in 1105 ggatggggag ggaggctggg catcagggat cacatcactc ttaacggctg ttacttaaac aactattttt one-letter code above the 1175 tggtttggtt gtaatatatt gtactttatt aagattgcca aaaactgtta aaatttaaaa aaaatttaaa respective codons. The six 1245 tcatgtgtat acaatttttt gc conserved consensus motifs involved in GTP binding are boxed. The CXC prenylation B motif is underlined. * __PM1__ G1 PM2 __PM3__ HsRab6B 1 MSAGGDF.GNPLRKFKLVFLGEQSVGKTSLITRFMYDSFDNTYQATIGIDFLSKTMYLEDRTVRLQLWDTAGQERFRSLI (B) Alignment of the amino HsRab6A 1 MSTGGDF.GNPLRKFKLVFLGEQSVGKTSLITRFMYDSFDNTYQATIGIDFLSKTMYLEDRTVRLQLWDTAGQERFRSLI acid sequences of human HsRab6A' 1 MSTGGDF.GNPLRKFKLVFLGEQSVGKTSLITRFMYDSFDNTYQATIGIDFLSKTMYLEDRTIRLQLWDTAGQERFRSLI − MmRab6A 1 MSAGGDF.GNPLRKFKLVFLGEQSVGKTSLITRFMYDSFDNTYQATIGIDFLSKTMYLEDRTVRLQLWDTAGQERFRSLI Rab6B with Rab6( like) DmRab6 1 MSS.GDF.GNPLRKFKLVFLGEQSVGKTSLITRFMYDSFDNTYQATIGIDFLSKTMYLEDRTVRLQLWDTAGQERFRSLI sequences from various CeU43283 1 MS...DF.GNPLKKFKLVFLGEQSVGKTSLITRFMYDSFDNTYQATIGIDFLSKTMYLEDRTVRLQLWDTAGQERFRSLI species. Amino acid changes CeP34213 1 ~~~MADFTNNALKKFKLVFLGEQSVGKTSIITRFMYDSFDNTYQATIGIDFLSKTMYLEDRTIRLQLWDTAGQERFRSLI SpRyh1 1 MSENYSFS...LRKFKLVFLGEQSVGKTSLITRFMYDQFDNTYQATIGIDFLSKTMYLEDRTVRLQLWDTAGQERFRSLI between Rab6A and Rab6B ScYpt6 1 MS....RSGKSLTKYKIVFLGEQGVGKTSLITRFMYDTFDDHYQATIGIDFLSKTMYLDDKTIRLQLWDTAGQERFRSLI are highlighted by asterisks. PfRab6 1 ~~~MDEFQNSGLNKYKLVFLGEQAVGKTSIITRFMYDTFDNNYQSTIGIDFLSKTLYLDEGPVRLQLWDTAGQERFRSLI The conserved motifs for NtRab6 1 ~~~~~MAPVSALAKYKLVFLGDQSVGKTSIITRFMYDKFDNTYQATIGIDFLSKTMYLEDRTVALQLWDTAGQERFRSLI AtRab6 1 ~~~~~MAPVSALAKYKLVFLGDQSVGKTSIITRFMYDKFDNTYQATIGIDFLSKTMYLEDRTVRLQLWDTAGQERFRSLI phosphate/magnesium binding (PM1-3) and switch I switch II guanine-nucleotide binding (G1-3) are depicted above the * * _G2_ ** ** * __G3_ HsRab6B 80 PSYIRDSTVAVVVYDITNLNSFQQTSKWIDDVRTERGSD.VIIMLVGNKTDLADKRQITIEEGEQRAKELSV.MFIETSA sequences. Identical amino HsRab6A 80 PSYIRDSTVAVVVYDITNVNSFQQTTKWIDDVRTERGSD.VIIMLVGNKTDLADKRQVSIEEGERKAKELNV.MFIETSA acid residues are boxed in HsRab6A' 80 PSYIRDSAAAVVVYDITNVNSFQQTTKWIDDVRTERGSD.VIIMLVGNKTDLADKRQVSIEEGERKAKELNV.MFIETSA black, conservative changes MmRab6A 80 PSYIRDSTVAVVVYDITNVNSFQQTTKWIDDVRTERGSD.VIIMLVGNKTDLADKRQVSIDEGERKAKELNV.MFIETSA DmRab6 79 PSYIRDSTVAVVVYDITNTNSFHQTSKWIDDVRTERGSD.VIIMLVGNKTDLSDKRQVSTEEGERKAKELNV.MFIETSA are given in grey. Hs, Homo CeU43283 77 PSYIRDSTVAVVVYDITNSNSFHQTSKWIDDVRTERGSD.VIIMLVGNKTDLSDKRQVTTDEGERKAKELNV.MFIETSA sapiens (acc. no. M28212, CeP34213 78 PSYIRDSSVAVVVYDITNANSFHQTTKWVDDVRNERGCD.VIIVLVGNKTDLADKRQVSTEDGEKKARDLNV.MFIETSA AF198616); Mm, Mus SpRyh1 78 PSYIRDSSVAIIVYDITNHNSFVNTEKWIEDVRAERGDD.VIIVLVGNKTDLADKRQVTQEEGEKKAKELKI.MHMETSA ScYpt6 77 PSYIRDSRVAIIVYDITKRKSFEYIDKWIEDVKNERGDENVILCIVGNKSDLSDERQISTEEGEKKAKLLGAKIFMETST musculus (acc. no. PfRab6 78 PSYIRDSAAAIVVYDITNRQSFENTTKWIQDILNERGKD.VIIALVGNKTDLGDLRKVTYEEGMQKAQEYNT.MFHETSA AA645214); Dm, Drosophila NtRab6 76 PSYIRDSSVAVIVYDVASRQSFLNTSKWIEEVRTERGSD.VIIVLVGNKTDLVEKRQVSIEEAEAKARELNV.MFIETSA melanogaster (acc. no. AtRab6 76 PSYIRDSSVAVIVYDVASRQSFLNTTKWIDEVRTERGSD.VIVVLVGNKTDLVDKRQVSIEEAEAKARELNV.MFIETSA D84314); Ce, Caenorhabditis switch II elegans (acc. no.U43283, P34213); Sp, * * ** ** * * * * * HsRab6B 158 KTGYNVKQLFRRVASALPGMENVQEKSKEGMIDIKL.DKPQE....PPAS.E..GGCSC Schizosaccharomyces pombe HsRab6A 158 KAGYNVKQLFRRVAAALPGMESTQDRSREDMIDIKL.EKPQE....QPVS.E..GGCSC (acc. no. X52475); Sc, HsRab6A' 158 KAGYNVKQLFRRVAAALPGMESTQDRSREDMIDIKL.EKPQE....QPVS.E..GGCSC Saccharomyces cerevisiae MmRab6A 158 KAGYNVKQLFRRVAAA DmRab6 157 KAGYNVKQLFRRVAAALPGMDSTENKPSEDMQEVVLKDSPNE....TKDP.E..GGCAC (acc. no. Q99260); Pf, CeU43283 155 KAGYNVKQLFRRIAGALPGI..IKDDPVEPPNVVTMDPIRQR....QIVTDE..GSCWC Plasmodium falciparum (acc. CeP34213 155 KAGYNVKQLFRKIATALPGI..VQEETPEQPNIVIMNP.PKD....AEESQG..RQCPC no. X92977); Nt, Nicotinia SpRyh1 156 KAGHNVKLLFRKIAQMLPGMENVETQSTQ.MIDVSIQPNENE....S...... SCNC ScYpt6 157 KAGYNVKALFKKIAKSLPEFQNSESTPLDSENANSANQNKPGVIDISTAEEQEQSACQC tabacum (acc. no. L29273); PfRab6 156 KAGHNIKVLFKKTASKLPNLDNTNNNEA.NVVDIQLTNNSNKN...... DKNMLSKCLC At, Arabidopsis thaliana NtRab6 154 KAGFNIKPLFRKIAAALPGMETLSSAKQEDMVDVNLKSSNAN....ASQSQAQSGGCAC (acc. no. CAB38902). AtRab6 154 KAGFNIKALFRKIAAALPGMETLSSTKQEDMVDVNLKSSNAN....ASLAQQQSGGCSC Rab6B, a cell-type specific Rab6 isoform 2729

Cytogenetically, this links the Rab6B gene to region q21-q23 and stained with Coomassie Blue (Fig. 3A) or transferred onto of chromosome 3, a gene-rich area that also exhibits the Rab7 nitro-cellulose and incubated with GST-Rab6A pre-adsorbed gene. Thus, the two Rab6 isoforms are expressed from separate anti-Rab6B antibodies (Fig. 3B). GST-Rab6B fusion protein genes, which are located on different chromosomes. appeared as a band migrating at an apparent molecular mass of approximately 49 kDa (Fig. 3A), which corresponds with Tissue- and cell-type specific expression of Rab6B the calculated sum of the masses of GST (26 kDa) and Rab6B gene (23 kDa). As can be observed from Fig. 3B, only GST-Rab6B Previous studies showed that Rab6A/A′ are ubiquitously showed immunoreactivity indicating that cross-reactivity was expressed small-GTPases (Goud et al., 1990; F. J. M. Opdam completely blocked by the adsorption method. et al., unpublished). To compare this expression pattern with We made use of the above described procedure to that of Rab6B, we performed northern blot hybridisation on specifically localise Rab6B in cryo-sections from brain tissue adult human tissues and various human cell lines with a 3′ (Fig. 4). In cerebral sections we could establish that Rab6B is Rab6B-specific probe. Three transcripts of approximately 1.35 specifically expressed in microglia (Fig.4A, arrowheads), kb, 3.1 kb and 5.6 kb were detected (Fig. 2). Rab6B RNA levels based on the density and shape of the stained cells and the appeared to be high in brain tissue and SK-N-SH cells, a neuroblastoma cell line from human brain (ATCC no. HTB-11). These expression levels were ten times more pronounced than in any other tissue or cell line, as determined by phosphoimaging (data not shown). Expression, albeit low, was also detected in heart and Caco-2 cells (Fig. 2), the latter reflecting the source from which the cDNA was generated The Rab6B messages do not correspond to Rab6A/A′ mRNA that are expressed as a single transcript of 3.6 kb (Zahraoui et al., 1989). The existence of three transcripts for Rab6B may reflect alternative splicing and/or different usage of promoter or polyadenylation signal. The 1.35 kb transcript probably reflects the form we isolated from the Caco-2 cDNA library. Taken together, northern blot analysis Fig. 2. Northern blot analysis reveals a tissue- and cell-type specific expression of Rab6B. revealed a tissue- and cell-type specific Poly(A)+ RNA (2 µg/lane, left panel) or total RNA (15 µg/lane, right panel) of the indicated expression pattern for Rab6B mainly in human tissues and cell lines were hybridised with a probe generated from a cDNA fragment neuronal tissue and cells. comprising the coding- and 3′ UTR region of Rab6B. A GAPDH probe was used to enable comparison of RNA loading (right lower panel). Cell-type specific expression of Rab6B in human brain The specific expression pattern of the Rab6B gene prompted us to investigate its putative cell-type specific expression in human brain tissue. Since the amino acid changes between Rab6B and Rab6A are scattered over a large region of the proteins (see Fig. 1B), we raised polyclonal antibodies against GST-Rab6B fusion protein that were purified over a GST-Rab6A column after affinity-purification. We used an additional excess of GST- Rab6A protein in a pre-incubation step to ensure elimination of cross- reactivity. This set-up was validated on Fig. 3. Characterisation of anti-Rab6B antibodies. Indicated GST-Rab fusion proteins were western blot. GST protein and several expressed in E. coli, and purified over a glutathione-Sepharose column. The (fusion) proteins GST-Rab fusion proteins, including were subjected to SDS-PAGE and stained with Coomassie Blue (A), or blotted onto nitro- Rab6A, Rab6B, Rab21, Rab22A, and cellulose membrane (B). The blot was incubated with affinity purified anti-Rab6B antibody, Rab22B, were subjected to SDS-PAGE which was pre-adsorbed with an excess of GST-Rab6A fusion protein. 2730 F. J. M. Opdam and others labelling of CD11b integrin (Fig. 4B), a specific marker for the neuroblastoma cell line SK-N-SH. As is shown in Fig. 5A, microglial cells (Akiyama and McGeer, 1990). In addition, GST-Rab6A pre-adsorbed anti-Rab6B antibodies stained a pericytes surrounding blood vessels showed immunoreactivity perinuclear region, reminiscent of the distribution of the Golgi for Rab6B (Fig. 4A, arrows). The Rab6B-specific expression complex. The localisation of Rab6B to the Golgi apparatus was pattern was confirmed on sections from cerebellar tissue (Fig. supported by double-staining with CTR 433 (Fig. 5B), a medial 4C,D). Again, staining was detected in pericytes and microglia Golgi marker (Jasmin et al., 1989). Despite the fact that both (Fig. 4C, arrows and arrowheads, respectively) with an proteins were detected at distinct levels of fluorescent additional staining in Purkinje cells (Fig. 4C, asterisks). The intensities, they clearly showed colocalisation. Concomitant majority of microglial cells was localised and stained in the with this restricted Golgi staining, vesicular structures were white matter as is shown by staining with anti-CD11b (Fig. observed to be Rab6B-positive (Fig. 5A). To further investigate 4D). Without pre-adsorption, the anti-Rab6B antibodies this additional localisation, cells were double-stained with stained a variety of cell-types including neuronal cells (Fig. 4E) and the expression pattern resembled staining with the neuronal marker anti- neurofilament 160 kDa (Fig. 4F). This ubiquitous expression pattern presumably reflects antibody recognition of Rab6B together with Rab6A and Rab6A′, proteins that have recently been shown to be ubiqitously expressed (F. J. M. Opdam et al., unpublished). Rab6B-specific staining was observed in cerebral and cerebellar sections from several individuals and could be eliminated by an excess of GST-Rab6B protein (Fig. 4G,H). Thus, elimination of cross-reactivity extensively reduced the ubiquitous expression pattern of the antibodies and revealed a cell-type specific expression of Rab6B in microglial cells, pericytes, and Purkinje cells (compare Fig. 4E with Fig. 4A). Rab6B is localised to the Golgi complex in SK-N-SH cells Next, we examined the intracellular localisation of endogenous Rab6B in

Fig. 4. Cell-type specific expression of Rab6B in human brain tissue. Cryo- sections of cerebral (C,D,H) and cerebellar (A,B,E-G) tissue were fixed in 2% paraformaldehyde. Anti-Rab6B antibodies (1:10) were directly used for incubation on the sections (E), or pre-incubated for 1 hour with an excess (10 µg) of GST-Rab6A protein (A and C) or GST-Rab6B protein (G and H). A neuronal (F, monoclonal anti- NF 160 kDa) and a microglial marker (B and D, monoclonal anti-CD11b) were used to compare staining patterns. Staining of immune-reactive antigen was visualised by incubation with AffiniPure donkey anti- rabbit or anti-mouse IgG and was followed by haematoxylin staining of the sections. Arrows indicate capillaries, arrowheads show staining of microglial cells, and Purkinje cells are indicated by asterisks. ML, molecular layer; GL, granular layer; WM, white matter. Bar, 50 µm. Rab6B, a cell-type specific Rab6 isoform 2731

Fig. 5. Endogenous expression of Rab6B at the Golgi apparatus. SK- N-SH cells were grown on coverslips and fixed in 1% paraformaldehyde. Cells were double-stained with GST-Rab6A pre-adsorbed anti-Rab6B antibodies (A and C) and monoclonal CTR 433 (B) or monoclonal anti-ERGIC-53 antibody (D). As a control, cells were directly incubated with anti- Rab6B antibodies (E) or specific labelling was blocked by pre- incubation with an excess of GST- Rab6B for 1 hour (F). Immunoreactive antigen was visualised by incubation with FITC-conjugated goat anti-rabbit and/or Texas Red-conjugated goat anti-mouse IgG, and detected by a confocal laser scanning microscope. Arrows indicate vesicular structures that are positive for ERGIC-53 and Rab6B. Bar, 10 µm.

Rab6B (Fig. 5C) and anti-ERGIC-53 (Fig. 5D), a protein that Second, a GTP-binding assay was performed with the use recycles between cis-Golgi and the ER/Golgi intermediate of nonhydrolysable radiolabeled GTPγS (Fig. 6C). The compartment (Schindler et al., 1993). As is shown in Fig. recombinant Rab6A and Rab6B proteins bound the 5C,D, Rab6B colocalised with peripheral extra-Golgi staining radiolabeled nucleotide in a time-dependent, saturable fashion of ERGIC-53 (arrows). Interestingly, colocalisation with at 30°C (approximately 0.2 pmol [35S]GTPγS per pmol GST- ERGIC-53 positive vesicles was never observed for Rab6A in Rab6A and GST-Rab6B). For Rab6A, binding reached its other cell-types (data not shown). Finally, we confirmed the maximum already after 5 minutes. This binding appeared much specificity of the antibodies. Without pre-adsorption, the faster than that for Rab6B, with maximal [35S]GTPγS-binding antibodies already showed a specific Golgi localisation (Fig. after 30 minutes. Binding of [35S]GTPγS to Rab6B could be 5E), similar to Rab6B-specific staining. Competition of the competed by an excess of unlabeled GTP (Fig. 6C). GST failed immunoreactive antigen with an excess of GST-Rab6B to bind any tracer. The results indicate that under two distinct completely blocked staining (Fig. 5F). These data indicate that GTP-binding conditions, Rab6B shows a lower GTP-binding native Rab6B, like Rab6A/A′, is localised to the Golgi activity than Rab6A. apparatus, but shows an extra-Golgi localisation to ERGIC-53- positive vesicular structures. Distribution of Rab6B and Rab6A in COS-1 cells To address the mutual cellular localisation of Rab6A and GTP-binding properties of Rab6A and Rab6B Rab6B, we prepared constructs that were differentially tagged We compared the GTP-binding properties of GST-Rab6A and with VSV epitope (Rab6B) and cMyc epitope (Rab6A). GST-Rab6B in two distinct GTP-binding assays. First, the Expression of both proteins was examined in COS-1 cells by proteins were subjected to SDS-PAGE and stained with co-transfecting the tagged versions in mammalian expression Coomassie Blue (Fig. 6A) or transferred onto nitro-cellulose vectors and detecting specific labelling using polyclonal and incubated with [α-32P]GTP (Fig. 6B). The blot overlay anti-VSV antibodies for Rab6B and monoclonal anti- assay demonstrated that both Rab6A and Rab6B bind [α- cMyc antibodies for Rab6A. As is shown in Fig. 7A,B, 32P]GTP, but under the same conditions Rab6A bound immunofluorescence of double-stained cells demonstrated that significantly higher amounts of [α-32P]GTP. As a control, GST both proteins colocalised at the Golgi complex. Remarkably, protein alone did not bind radiolabeled GTP (Fig. 6B). when immunofluorescence intensities of both proteins at the 2732 F. J. M. Opdam and others

and anti-PDI (Fig. 7D), the latter recognizing ER-resident protein disulphide isomerase (Freedman et al., 1989). The network-like ER structure of Rab6B overlapped with that of PDI. To further show that gel migration properties of Rab6A and Rab6B were similar, we independently expressed cMyc- epitope tagged versions in COS-1 cells. As is shown in Fig. 7E, both Rab6A and Rab6B appeared as two bands of approximately 25 kDa. The appearance of two bands likely accounts for the absence (or presence) of geranylgeranyl groups at the C terminus of the proteins (Yang et al., 1993). The results indicate that in overexpression studies Rab6B completely colocalises with Rab6A at the Golgi complex and ER peripheral membranes, but Rab6B is differentially distributed over the organelles with a more pronounced staining at the ER than Rab6A. GTP-bound form of Rab6B interacts with Rab6A effector molecules We next examined the interaction of Rab6B with known Rab6A partners in order to get information about Rab6B function. In recent studies, Rab6A has been reported to interact with several effector molecules: Rabkinesin-6, a kinesin-like protein associated with the Golgi apparatus (Echard et al., 1998), GAPCenA, a Rab6 GTPase activating protein (GAP) (Cuif et al., 1999), and ‘clone 1’, a 150 kDa cytosolic protein with an extensive coiled-coil domain whose exact function is still unknown (F. Jollivet, I. Janoueix-Lerosey and B. Goud, unpublished results). The three proteins preferentially interact with the GTP-bound conformation of Rab6A (Rab6A Q72L). The interaction pattern of the GTP-bound form of Rab6B (Rab6B Q72L) with these three Rab6A effectors was monitored using the yeast two hybrid assay. As shown in Fig. 8, Rab6B interacts with the three effector molecules to a Fig. 6. Rab6A and Rab6B show distinct GTP-binding properties. similar extent as Rab6A. In addition, on X-gal plates, Rab6A and Rab6B open reading frames were subcloned in the comparable deep blue color staining was observed for all prokaryotic expression vector PGEX and transformed to E. coli cells. interactions examined (data not shown). µ Samples (1 g/lane) of GST (fusion) proteins were subjected to The interaction of Rab6B with Rabkinesin-6 suggested that SDS-PAGE and stained with Coomassie Blue (A) or transferred to Rab6B may regulate a transport route through a molecular nitro-cellulose membranes (B). The blot was incubated with 1 nM [α-32P]GTP as described. For time-course studies of [35S]GTPγS- machinery comparable to that of Rab6A. We therefore looked binding (C), 500 ng of GST-Rab6A (᭡) or GST-Rab6B (᭺) were for the expression of Rabkinesin-6 in brain. A ubiquitous incubated with [35S]GTPγS at 30°C. At indicated times, the binding expression pattern with high expression in protoplasmic and/or activities were measured as described. As a control, GTPγS-binding fibrillar astrocytes, pericytes, microglia and Purkinje cells was was measured to GST (×) alone or to GST-Rab6B in the presence of found for Rabkinesin-6, indicating that Rab6B-positive cell- an excess of unlabeled GTPγS (᭢). The results show the means of types also express Rabkinesin-6 (data not shown). two independently performed assays.

Golgi complex were equalised in cells expressing higher DISCUSSION amounts of myc-tagged Rab6A, significant staining of Rab6B was found at ER-like structures (Fig. 7A) whereas Rab6A Here, we characterized a new isoform of Rab6, Rab6B, showed a distribution that was more restricted to the Golgi encoded by a separate gene than the one encoding Rab6A/A′. complex (Fig. 7B). Interestingly, expression of Rab6B was also Rab6B was found to be preferentially expressed in brain, found to be concentrated at specific sites in the ER periphery especially in microglial cells, pericytes and Purkinje cells. and colocalised with Rab6A expression (Fig. 7A,B, arrows). It Rab6B is also abundant in SK-N-SH cells. Previous studies was recently reported in live cells that the retrograde cargo have shown that subclones with distinct phenotypes can be Shiga toxin B-fragment enters the ER via specialised obtained from primary cultures SK-N-SH cells. In particular, peripheral regions that accumulate Rab6A (White et al., 1999). these cells can either differentiate into cells with a neuronal Similar results were obtained when Rab6A and Rab6B were phenotype, or in cells exhibiting properties common to glial exchanged for the epitope-tag (data not shown). The general cells (Sano et al., 1990; Shinohara et al., 1997). Thus, the high ER localisation of Rab6B was confirmed by a single expression levels found in SK-N-SH cells may be correlated to transfection of VSV-Rab6B in COS-1 cells. Cells were double- the expression pattern of Rab6B in brain. Rab6B may also be stained with monoclonal anti-VSV antibody P5D4 (Fig. 7C) abundant in human melanocytes, as partial cDNA sequence of Rab6B, a cell-type specific Rab6 isoform 2733

Fig. 7. Colocalisation of Rab6A and Rab6B in COS-1 cells. COS-1 cells were co-electroporated with VSV epitope-tagged Rab6B (A) and cMyc epitope-tagged Rab6A (B) in their corresponding mammalian expression vectors as described or transfected with VSV- Rab6B alone (C). Cells were grown on coverslips, fixed in 1% paraformaldehyde, and double-stained with polyclonal anti-VSV alpha P4 (A) or monoclonal anti-VSV P5D4 (B) and monoclonal anti-cMyc 9E10 (B) or polyclonal anti-PDI antibody (D), respectively. Specific labelling was visualised by incubating the cells with FITC- or Texas Red-conjugated goat anti-mouse and FITC or Texas Red-conjugated goat anti-rabbit IgG, and detected by a confocal laser scanning microscope. Arrows indicate specific sites in the ER periphery, which are highly concentrated for Rab6A and Rab6B. Bar, 10 µm. (E) Rab6A and Rab6B encoding constructs were cloned into the mammalian expression vector pMyc and transfected to COS-1 cells. Cell lysates, together with a control cell lysate, were subjected to western blotting. For detection of cMyc-tagged Rab6A and Rab6B, the blot was incubated with monoclonal anti-cMyc antibody 9E10 (1:100).

Fig. 8. GTP-bound form of Rab6B (Q72L) interacts with Rab6A effector molecules. Two hybrid experiment of GTP-bound forms of Rab6B and Rab6A with known Rab6A effector molecules. The Saccharomyces cerevisiae reporter strain L40 was cotransformed with either pLexA-Rab6A Q72L or pLexA-Rab6B Q72L and pGADGH- GAPCenA/Rab6 interacting domain III (amino acids 750-1013), pGADGH-Rabkinesin-6, or pGADGH-clone 1. Transformants were patched for 3 days at 30°C on a selective medium lacking tryptophan and leucine (left) or lacking tryptophan, leucine, and histidine (right). Growth in the right panel indicates an interaction between the encoded proteins. encoding Rab6B has first been isolated from these cells (Chen In addition, endogenous Rab6B was found to be present in et al., 1997a). ERGIC-53 positives structures in SK-N-SH cells. Finally, The fact that isoforms of Rab proteins display different Rab6B displayed a lower capacity to bind GTP than Rab6A. expression patterns has previously been described. For We do not know at the moment the exact function of Rab6B. instance, Rab33B shows a ubiquitous expression pattern, Rab6A is involved in a COPI-independent retrograde pathway whereas Rab33A is exclusively expressed in brain and cells between Golgi and ER (White et al., 1999). This pathway is from the immune system (Zheng et al., 1997, 1998). However, used by Shiga toxin to enter the ER and possibly by Golgi to our knowledge, the precise localization, or the biochemical enzymes to recycle through this compartment (White et al, properties of differentially expressed Rab isoforms have not 1999; Girod et al., 1999). Based on the localisation of Rab6B, been addressed. In this study, we noticed differences in the it is likely that this protein is also involved in a retrograde localisation of Rab6A and Rab6B isoforms. Although the bulk pathway between Golgi and ER. In support of this hypothesis, of both proteins localises to the Golgi complex, Rab6B it should be pointed out that peripheral structures positive for appeared to be more abundant in ER membranes than Rab6A. both Rab6A and Rab6B, which may represent ER entry sites 2734 F. J. M. Opdam and others of retrograde cargoes (White et al., 1999), were observed in Celis, J. E. (1998). Cell Biology. A Laboratory Handbook. San Diego: COS cells (Fig. 7). However, the presence of Rab6B in ERGIC- Academic Press. 53 containing structures is puzzling, since ERGIC-53 uses a Chavrier, P. and Goud, B. (1999). The role of ARF and rab GTPases in membrane transport. Curr. Opin. Cell Biol. 11, 466-475. COP-I dependent pathway to cycle back to the ER. Chen, D., Guo, J., Miki, T., Tachibana, M. and Gahl, W. A. (1996). Accordingly, microinjection of a plasmid encoding dominant Molecular cloning of two novel rab genes from human melanocytes. Gene negative form of Rab6A (Rab6A T27N) has no effect on 174, 129-134. cycling of ERGIC-53 (Girod et al., 1999). Another interesting Chen, D., Guo, J. and Gahl, W. A. (1997a). RAB GTPases expressed in human melanoma cells. Biochim. Biophys. Acta 1355, 1-6. result of this study is that Rab6B appears to interact with all Chen, D., Guo, J., Miki, T., Tachibana, M. and Gahl, W. A. (1997b). known putative effectors of Rab6A, including Rabkinesin-6 Molecular cloning and characterization of rab27a and rab27b, novel human thought to be involved in the movement of Rab6A containing rab proteins shared by melanocytes and platelets. Biochem. Mol. Med. 60, tubular structures between Golgi and ER. To address the 27-37. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. and Rutter, W. J. function of Rab6B, we performed preliminary pilot (1979). Isolation of biologically active ribonucleic acid from sources experiments in which the GTPase deficient mutant Rab6B enriched in ribonuclease. Biochemistry 18, 5294-5299. Q72L was overexpressed in HeLa cells. In contrast to Rab6A Christoforidis, S., McBride, H. M., Burgoyne, R. D. and Zerial, M. (1999). Q72L (Martinez et al., 1997), Rab6B Q72L was not able to The Rab5 effector EEA1 is a core component of endosome docking. Nature redistribute Golgi resident proteins into the ER (data not 397, 621-625. Chung, S. H., Joberty, G., Gelino, E. A., Macara, I. G. and Holz, R. W. shown). This experiment would suggest that Rab6B regulates (1999). Comparison of the effects on secretion in chromaffin and PC12 cells a transport pathway different from the one regulated by Rab6A. of Rab3 family members and mutants. Evidence that inhibitory effects are However, one have to keep in mind that endogenous expression independent of direct interaction with Rabphilin3. J. Biol. Chem. 274, of Rab6B was not detected in HeLa cells and that all Rab6B 18113-18120. Cuif, M. H., Possmayer, F., Zander, H., Bordes, N., Jollivet, F., Couedel effector molecules may not be present in these cells. Courteille, A., Janoueix Lerosey, I., Langsley, G., Bornens, M. and It should be pointed out that changes in biochemical Goud, B. (1999). Characterization of GAPCenA, a GTPase activating properties of Rab6A and Rab6B may as well contribute to protein for Rab6, part of which associates with the centrosome. EMBO J. differences in the way Rab6A and Rab6B regulate membrane 18, 1772-1782. Darmoul, D., Lacasa, M., Baricault, L., Marguet, D., Sapin, C., Trotot, P., traffic. Indeed, the cycle between GDP- and GTP-bound state, Barbat, A. and Trugnan, G. (1992). Dipeptidyl peptidase IV (CD 26) gene as well as the steady state level of Rab-GTP inserted into expression in enterocyte-like colon cancer cell lines HT-29 and Caco-2. membranes, is crucial for Rab function. These events are Cloning of the complete human coding sequence and changes of dipeptidyl regulated through interactions with nucleotide exchange peptidase IV mRNA levels during cell differentiation. J. Biol. Chem. 267, factors (GEFs) and GTPase activating proteins (GAPs) 4824-4833. Echard, A., Jollivet, F., Martinez, O., Lacapere, J. J., Rousselet, A., (Horiuchi et al., 1997; Rybin et al., 1996). Both Rab6A and Janoueix Lerosey, I. and Goud, B. (1998). Interaction of a Golgi- Rab6B appear to interact with GAPCenA, a GAP for Rab6 associated kinesin-like protein with Rab6. Science 279, 580-585. (Cuif et al., 1999), but it is tempting to speculate that Rab6A Feinberg, A. P. and Vogelstein, B. (1983). A technique for radiolabeling DNA and Rab6B may not be activated by the same GEF protein. restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13. In conclusion, our results suggest that Rab6B may exert a Fischer von Mollard, G., Mignery, G. A., Baumert, M., Perin, M. S., cell-type specific function in neuronal cells. Further studies are Hanson, T. J., Burger, P. M., Jahn, R. and Sudhof, T. C. (1990). rab3 is necessary to determine whether Rab6B regulates membrane a small GTP-binding protein exclusively localized to synaptic vesicles. Proc. trafficking between Golgi and ER in these cells, as Nat. Acad. Sci. USA 87, 1988-1992. ubiquitously expressed Rab6A does. Fischer von Mollard, G., Stahl, B., Khokhlatchev, A., Sudhof, T. C. and Jahn, R. (1994). Rab3C is a synaptic vesicle protein that dissociates from synaptic vesicles after stimulation of exocytosis. J. Biol. Chem. 269, 10971- We thank Wiep Scheper for providing us with the SK-N-SH cell 10974. line. We are grateful to Irene Otte-Höller for the gift of human brain Freedman, R. B., Bulleid, N. J., Hawkins, H. C. and Paver, J. L. (1989). tissue and Wiljan Hendriks for critical reading of the manuscript. Role of protein disulphide-isomerase in the expression of native proteins. Biochem. Soc. Symp. 55, 167-192. Girod, A., Storrie, B., Simpson, J. C., Johannes, L., Goud, B., Roberts, L. M., Lord, J. M., Nilsson, T. and Pepperkok, R. (1999). Evidence for copI- REFERENCES independent transport routes from the Golgi complex to the endoplasmic reticulum. Nature Cell Biol. 1, 423-430. Akiyama, H. and McGeer, P. L. (1990). Brain microglia constitutively Goud, B., Zahraoui, A., Tavitian, A. and Saraste, J. (1990). Small GTP- express beta-2 integrins. J. Neuroimmunol. 30, 81-93. binding protein associated with Golgi cisternae. Nature 345, 553-556. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, Green, S., Issemann, I. and Sheer, E. (1988). A versatile in vivo and in vitro W. and Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new eukaryotic expression vector for protein engineering. Nucl. Acids Res. 16, generation of protein database search programs. Nucl. Acids Res. 25, 3389- 369. 3402. Horiuchi, H., Lippe, R., McBride, H. M., Rubino, M., Woodman, P., Aridor, M. and Balch, W. E. (1996). Membrane fusion: timing is everything Stenmark, H., Rybin, V., Wilm, M., Ashman, K., Mann, M. et al. (1997). [news; comment]. Nature 383, 220-221. A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links Armstrong, J., Thompson, N., Squire, J. H., Smith, J., Hayes, B. and nucleotide exchange to effector recruitment and function. Cell 90, 1149- Solari, R. (1996). Identification of a novel member of the Rab8 family from 1159. the rat basophilic leukaemia cell line, RBL. 2H3. J. Cell Sci. 109, 1265- Iezzi, M., Escher, G., Meda, P., Charollais, A., Baldini, G., Darchen, F., 1274. Wollheim, C. B. and Regazzi, R. (1999). Subcellular distribution and Baldini, G., Wang, G., Weber, M., Zweyer, M., Bareggi, R., Witkin, J. W. function of Rab3A, B, C, and D isoforms in insulin-secreting cells. Mol. and Martelli, A. M. (1998). Expression of Rab3D N135I inhibits regulated Endocrinol. 13, 202-212. secretion of ACTH in AtT-20 cells. J. Cell Biol. 140, 305-313. Janoueix Lerosey, I., Jollivet, F., Camonis, J., Marche, P. N. and Goud, B. Barbosa, M. D., Johnson, S. A., Achey, K., Gutierrez, M. J., Wakeland, E. (1995). Two-hybrid system screen with the small GTP-binding protein K., Zerial, M. and Kingsmore, S. F. (1995). The Rab protein family: Rab6. Identification of a novel mouse GDP dissociation inhibitor isoform genetic mapping of six Rab genes in the mouse. Genomics 30, 439-444. and two other potential partners of Rab6. J. Biol. Chem. 270, 14801-14808. Rab6B, a cell-type specific Rab6 isoform 2735

Jasmin, B. J., Cartaud, J., Bornens, M. and Changeux, J. P. (1989). Golgi Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A apparatus in chick skeletal muscle: changes in its distribution during end Laboratory Manual. New York: Cold Spring Harbor Laboratory Press. plate development and after denervation. Proc. Nat. Acad. Sci. USA 86, Sano, K., Nakamura, H., Mabuchi, S., Tanaka, T., Nakagawara, A. and 7218-7222. Takai, Y. (1990). Expression of the smg p25A (a ras p21-like GTP-binding Kari, B., Lussenhop, N., Goertz, R., Wabuke Bunoti, M., Radeke, R. and protein) gene in human neuroblastoma cell lines and tumor tissues. Cancer Gehrz, R. (1986). Characterization of monoclonal antibodies reactive to Res. 50, 7242-7245. several biochemically distinct human cytomegalovirus glycoprotein Schimmoller, F., Simon, I. and Pfeffer, S. R. (1998). Rab GTPases, directors complexes. J. Virol. 60, 345-352. of vesicle docking. J. Biol. Chem. 273, 22161-22164. Kreis, T. E. (1986). Microinjected antibodies against the cytoplasmic domain Schindler, R., Itin, C., Zerial, M., Lottspeich, F. and Hauri, H. P. (1993). of vesicular stomatitis virus glycoprotein block its transport to the cell ERGIC-53, a membrane protein of the ER-Golgi intermediate compartment, surface. EMBO J. 5, 931-941. carries an ER retention motif. Eur. J. Cell Biol. 61, 1-9. Lacey, S. W., Sanders, J. M., Rothberg, K. G., Anderson, R. G. and Shinohara, T., Nagashima, K. and Major, E. O. (1997). Propagation of the Kamen, B. A. (1989). Complementary DNA for the folate binding protein human polyomavirus, JCV, in human neuroblastoma cell lines. Virology 228, correctly predicts anchoring to the membrane by glycosyl- 269-277. phosphatidylinositol. J. Clin. Invest. 84, 715-720. Tang, L. H., Gumkowski, F. D., Sengupta, D., Modlin, I. M. and Jamieson, Martinez, O., Schmidt, A., Salamero, J., Hoflack, B., Roa, M. and Goud, J. D. (1996). rab3D protein is a specific marker for zymogen granules in B. (1994). The small GTP-binding protein rab6 functions in intra-Golgi gastric chief cells of rats and rabbits. Gastroenterology 110, 809-820. transport. J. Cell Biol. 127, 1575-1588. Tisdale, E. J., Bourne, J. R., Khosravi Far, R., Der, C. J. and Balch, W. E. Martinez, O., Antony, C., Pehau Arnaudet, G., Berger, E. G., Salamero, (1992). GTP-binding mutants of rab1 and rab2 are potent inhibitors of J. and Goud, B. (1997). GTP-bound forms of rab6 induce the redistribution vesicular transport from the endoplasmic reticulum to the Golgi complex. of Golgi proteins into the endoplasmic reticulum. Proc. Nat. Acad. Sci. USA J. Cell Biol. 119, 749-761. 94, 1828-1833. Valencia, A., Chardin, P., Wittinghofer, A. and Sander, C. (1991). The ras McBride, H. M., Rybin, V., Murphy, C., Giner, A., Teasdale, R. and Zerial, protein family: evolutionary tree and role of conserved amino acids. M. (1999). Oligomeric complexes link Rab5 effectors with NSF and drive Biochemistry 30, 4637-4648. membrane fusion via interactions between EEA1 and syntaxin 13. Cell 98, Walch-Solimena, C., Collins, R. N. and Novick, P. J. (1997). Sec2p Mediates 377-386. Nucleotide Exchange On Sec4p and Is Involved in Polarized Delivery of Nielsen, E., Severin, F., Backer, J. M., Hyman, A. A. and Zerial, M. (1999). Post Golgi Vesicles. J. Cell Biol. 137, 1495-1509. Rab5 regulates motility of early endosomes on microtubules. Nature Cell Weber, E., Berta, G., Tousson, A., St John, P., Green, M. W., Biol. 1, 376-382. Gopalokrishnan, U., Jilling, T., Sorscher, E. J., Elton, T. S., Nuoffer, C., Davidson, H. W., Matteson, J., Meinkoth, J. and Balch, W. E. Abrahamson, D. R. et al. (1994). Expression and polarized targeting of a (1994). A GDP-bound of rab1 inhibits protein export from the endoplasmic rab3 isoform in epithelial cells. J. Cell Biol. 125, 583-594. reticulum and transport between Golgi compartments. J. Cell Biol. 125, 225- White, J., Johannes, J., Mallard, F., Girod, A., Grill, S., Reinsch, S., Keller, 237. P., Echard, A., Goud, B. and Stelzer, E. (1999). Rab6 coordinates a novel Opdam, F. J. M., Kamps, G., Croes, H., van Bokhoven, H., Ginsel, L. A. Golgi to ER retrograde transport pathway in live cells. J. Cell Biol. 147, and Fransen, J. A. M. (2000). Expression of Rab small GTPases in 743-759. epithelial Caco-2 cells: Rab 21 is an apically located GTP-binding protein Yang, C., Mollat, P., Chaffotte, A., McCaffrey, M., Cabanie, L. and Goud, in polarised intestinal epithelial cells. Eur. J. Cell Biol. 79, 308-316. B. (1993). Comparison of the biochemical properties of unprocessed and Roa, M., Paumet, F., Le Mao, J., David, B. and Blank, U. (1997). processed forms of the small GTP-binding protein, rab6p. Eur. J. Biochem. Involvement of the ras-like GTPase rab3d in RBL-2H3 mast cell exocytosis 217, 1027-1037. following stimulation via high affinity IgE receptors (Fc epsilonRI). J. Zahraoui, A., Touchot, N., Chardin, P. and Tavitian, A. (1989). The Immunol. 159, 2815-2823. human Rab genes encode a family of GTP-binding proteins related to yeast Rothman, J. E. and Sollner, T. H. (1997). Throttles and dampers: controlling YPT1 and SEC4 products involved in secretion. J. Biol. Chem. 264, the engine of membrane fusion. Science 276, 1212-1213. 12394-12401. Rousseau Merck, M. F., Zahraoui, A., Touchot, N., Tavitian, A. and Zheng, J. Y., Koda, T., Arimura, Y., Kishi, M. and Kakinuma, M. (1997). Berger, R. (1991). Chromosome assignment of four RAS-related RAB Structure and expression of the mouse S10 gene. Biochim. Biophys. Acta genes. Hum. Genet. 86, 350-354. 1351, 47-50. Rybin, V., Ullrich, O., Rubino, M., Alexandrov, K., Simon, I., Seabra, C., Zheng, J. Y., Koda, T., Fujiwara, T., Kishi, M., Ikehara, Y. and Kakinuma, Goody, R. and Zerial, M. (1996). GTPase activity of Rab5 acts as a timer M. (1998). A novel Rab GTPase, Rab33B, is ubiquitously expressed and for endocytic membrane fusion. Nature 383, 266-269. localized to the medial Golgi cisternae. J. Cell Sci. 111, 1061-1069.