The EMBO Journal vol.15 no.16 pp.4262-4273, 1996 p619, a giant protein related to the chromosome condensation regulator RCC1, stimulates guanine nucleotide exchange on ARFi and Rab proteins
Jose Luis Rosa1, Ricardo P.Casaroli-Marano2, by Rothman, 1994). One of these families of proteins, Alan J.Buckler3, Senen Vilaro2 and designated as ARFs, was identified initially as co-factors Mariano Barbacid4 required for ADP-ribosylation of the trimeric G protein o-chain by cholera toxin (Kahn and Gilman, 1986). More Department of Molecular Oncology, Bristol-Myers Squibb, recently, ARF proteins have been found to be involved in Pharmaceutical Research Institute, Princeton, NJ 08543, USA, intracellular vesicular transport and phospholipid metabol- 2Departament de Biologia Cellular, Universitat de Barcelona, 08028 Barcelona, Spain and 3Molecular Genetics Laboratory, ism (reviewed by Kahn et al., 1993; Donaldson and Massachusetts General Hospital, Charlestown, MA 02129, USA Klausner, 1994). In agreement with these observations, 'Present address: Unitat de Bioquimica, Campus Bellvitge, Universitat ARFI, the best characterized member of this protein de Barcelona, Barcelona, Spain family, appears to be localized primarily on coated vesicles and Golgi membranes. Binding of ARFI to membranes 4Corresponding author is essential for its biological activity. This interaction We report the identification of a novel human gene, requires addition of a myristoyl group to its amino- designated p619, that encodes a polypeptide of 4861 terminus, a post-translational modification unique among amino acid residues, one of the largest human proteins small GTP binding proteins which are modified by the known to date. The p619 protein contains two regions addition of isoprenyl derivatives to their carboxy-terminus of seven internal repeats highly related to the cell cycle (reviewed by Nuoffer and Balch, 1994). regulator RCC1, a guanine nucleotide exchange factor Another group of small GTP binding proteins involved for the small GTP binding protein, Ran. In addition, in membrane trafficking is the Rab family (reviewed by p619 possesses seven 13-repeat domains characteristic Novick and Brennwald, 1993; Zerial and Stenmark, 1993; of the p-subunit of heterotrimeric G proteins, three Pfeffer, 1994). The members of this large family of putative SH3 binding sites, seven polar amino acid- proteins (>30) are localized to the surfaces of various rich regions, a putative leucine zipper and a carboxy- membrane-bound organelles involved in both exocytic and terminal HECT domain characteristic of E3 ubiquitin- endocytic pathways. Rab proteins play a role in those protein ligases. p619 is expressed ubiquitously in mouse processes by which transport vesicles dock and/or fuse and human tissues and overexpressed in several human with their cognate target membranes. Association of Rab tumor cell lines. Subcellular localization studies indi- proteins with membranes, a process mediated by the cate that p619 is located in the cytosol and in the Golgi addition of geranyl-geranyl groups to their carboxy- apparatus. Localization of p619 in the Golgi is altered terminal cysteine motif, is essential for their function by Brefeldin A. The carboxy-terminal RCC1-like (reviewed by Clarke, 1992). domain ofp619 interacts specifically with myristoylated The activity of the ARF and Rab proteins, like those ARF1, a small GTP binding protein also located in the of all known small GTP binding proteins, is regulated by Golgi. Moreover, the second RCC1-like motif located binding of guanine nucleotides (reviewed by Boguski and at the amino-terminus of p619 stimulates guanine McCormick, 1993). The GDP-bound forms of ARF and nucleotide exchange on ARF1 and on members of the Rab proteins are inactive and remain located mostly in related Rab proteins, but not on other small GTP the cytosol. Exchange of GDP by GTP is mediated by binding proteins such as Ran or R-Ras2/TC21. These guanine nucleotide exchange factors (GEF) which, for the observations suggest that p619 is a Brefeldin A-sensitive most part, remain to be identified. Several reports have Golgi protein that functions as a guanine nucleotide described the presence of ARFI GEF activity in Golgi exchange factor for ARF1 and, possibly, for members membranes (Donaldson et al., 1992; Helms and Rothman, of the Rab family of proteins. 1992). More recently, Tsai et al. (1994) have reported the Keywords: Golgi apparatus/guanine nucleotide exchange partial purification of a protein from bovine brain with factors/membrane trafficking/small GTP binding proteins ARFI GEF activity. In yeast, the DSS4-1 gene product, a small protein of 17 kDa, functions as a GEF for the yeast Sec4 protein and, with lesser efficiency, for the mammalian Introduction Rab3A (Moya et al., 1993). Similar activities have been observed in its mammalian homolog, MSS4p (Burton Intracellular protein transport between organelles is funda- et al., 1993). A Rab3A-GEF protein of -300 kDa has mental to eukaryotic cell function. This transport has to been partially purified from bovine brain (Burstein and be tightly regulated in order to deliver specific molecules Macara, 1992). Finally, Rab5 and Rab9 GEF activities to their target acceptor organelles. Membrane trafficking associated with endosomes and clathrin-coated vesicles, is believed to be regulated by a series of small (20- have been described recently (Soldati et al., 1994; Ullrich 25 kDa) GTP binding proteins that confer the necessary et al., 1994; Horiuchi et al., 1995). specificity and directionality to this transport (reviewed ARF and Rab proteins are representative examples of
426246©Oxford University Press p619 has GEF activity for ARF1 and Rab proteins a class of small GTP binding proteins whose GTP binding gene (> 150 kbp) with a complex arrangement of sequences and hydrolysis appear to be strictly coupled to a rapid derived from at least three distinct loci, only two of which membrane-cytosol localization cycle. Binding of GTP to were found to be of human origin (T.Koda, E.Sakai and ARF proteins results in their biochemical activation and M.Barbacid, unpublished observations). Exon trapping of subsequent association with their cognate membranes oncH genomic sequences yielded a 382 bp long cDNA (Rothman, 1994). However, in the case of the Rab proteins, fragment derived from one of the two human genes present it has been postulated that membrane association precedes in oncH. Nucleotide sequence analysis of this DNA GTP binding (Stenmark et al., 1995). Moreover, activation fragment revealed significant sequence homology with the of Rab proteins is regulated by cytosolic proteins known gene encoding the regulator of chromosome condensation, as GDIs (guanine nucleotide dissociation inhibitors) and RCC1 (Ohtsubo et al., 1987). REPs (Rab escort proteins) that prevent the exchange Considering the potential biological relevance of mem- of GDP by GTP by forming stochiometric complexes bers of the RCC1 gene family, we decided to isolate a with the Rab proteins (Alexandrov et al., 1994; Soldati full-length cDNA clone of this novel gene from a human et al., 1994; Ullrich et al., 1994). These complexes fetal brain cDNA library. After seven rounds of screening, dissociate upon binding to their target membranes, eight overlapping cDNA clones encompassing a linear allowing the GTP-mediated activation of Rab proteins sequence of 15 171 nucleotides were obtained. Sequences (Ullrich et al., 1994; Horiuchi et al., 1995). To date, no derived from these overlapping clones were combined to GDI or REP factors have been found associated with generate a single cDNA clone (pJLR75) which encom- ARF proteins. passes 96 nucleotides of 5' non-coding sequences with During the course of our efforts aimed at characterizing translational terminator codons in all possible reading human sequences with oncogenic activity, we identified a frames, a single open reading frame (ORF) of 14 586 novel oncogene that contains sequences derived from a nucleotides whose first codon is in an optimal context for novel human locus, designated p619, related to the regu- translation initiation and a 3' non-coding region of 492 lator of chromosomal condensation, RCC1. RCC1 was nucleotides with a polyadenylation signal and a 20 nucleo- first identified as the product of a hamster gene whose tide long poly(A) tail. The single ORF of this cDNA clone function was required to prevent chromosome condensa- predicts a 4861 amino acid long polypeptide with an Mr tion before completion of DNA replication (Ohtsubo et al., of 619 062 Da (pl = 5.62), the second largest human 1987). Subsequently, RCC1 has been shown to be a protein known to date (Figure 1). We have designated this GEF for Ran, a small GTP binding protein located novel human gene as p619 to predominantly in the nucleus (Bischoff and Ponstingl, reflect the molecular mass 1991). Ran has been implicated in the nuclear import of of its gene product. proteins with nuclear localization signals (Ren et al., 1995; Analysis of the amino acid sequence of the p619 protein Schlenstedt et al., 1995). Therefore, it is believed that revealed several structural domains including two regions loss of RCC1 may lead to suppression of nuclear import of high internal homology (48% identity and 64% of proteins needed for proper regulation of the cell cycle similarity) each consisting of seven internal repeats of (Tachibana et al., 1994). RCC1 and Ran may also be 50-56 amino acid residues located at both ends of the involved in RNA processing and in its export to the molecule (residues 377-735 and 4002-4360). The overall cytosol (Cheng et al., 1995; Ren et al., 1995; Schlenstedt structure of these motifs is highly reminiscent of the et al., 1995). RCC 1 protein. More importantly, these p619 domains, Based on the structural similarities between this novel designated as RLD (RCC1 like domains), have significant human gene and RCC 1, we set about to determine whether sequence homology (30% and 29% identity and 51% and its gene product, p619, had guanine nucleotide exchange 49% similarity, respectively) with RCCI (Figure 1). activity for small GTP binding proteins. Here we describe p619 also contains seven degenerated ,8-repeat domains the molecular cloning, structural analysis and initial func- (also known as WD repeats) characteristic of the n-subunit tional characterization of p619. Our results indicate that of heterotrimeric G proteins (reviewed by Neer et al., p619 co-localizes, binds and has GEF activity for ARFI. 1994) (Figure 2B). This type of domain has been found In addition, p619 can also activate guanine nucleotide in proteins implicated in distinct cellular functions and it exchange on Rab proteins. These observations suggest is believed to play a role in protein-protein interactions that p619 is a GEF for ARFI, and possibly for certain (Neer et al., 1994). The carboxy-terminus of p619 displays Rab proteins, and thus plays an important role in the a well conserved HECT domain characteristic of E3 regulation of membrane trafficking. ubiquitin-protein ligases (Huibregtse et al., 1995) (Figure 2C). These enzymes catalyze the transfer of ubiquitin to specific proteins through a conserved cysteine residue, an Results essential step for the subsequent degradation of those Isolation of cDNAs encoding p619 proteins through the ubiquitin-mediated proteolytic path- Transfection of DNA isolated from a human breast adeno- way (Scheffner et al., 1993; Huibregtse et al., 1995). p619 carcinoma in the nude mouse tumorigenicity assay (Fasano possesses other structural motifs, including three proline- et al., 1984) led to the identification of a novel oncogene, rich sequences that resemble the class II consensus designated as oncH (T.Koda, E.Sakai and M.Barbacid, sequences for SH3 binding sites (Feng et al., 1994), several unpublished observations). oncH did not induce morpho- stretches of polar amino acids consisting preferentially of logic transformation of NIH3T3 cells in culture. However, acidic residues, a leucine zipper-like motif and several it conferred tumorigenic properties on them in nude mice. potential phosphorylation sites for cAMP-dependent Molecular characterization of oncH revealed a very large protein kinases (Kennelly and Krebs, 1991) (Figure 1). 4263 J.L.Rosa et al.
A PR Polar stretches h-repeats PR PR HECT NH2 1 RLD:-1. -^-| HRLD-2-t l l }lCOOlH§t i ffi Z LZ B .WTP-.LF WL F1-L &S _ E A': R FG'VAV1.Y`FK .'NK11iV' PQQA'-;C:LNGC Qi, PFIFERa-)L S SDEAQDH YL AL SS L.A
Fig. 1. Predicted amino acid sequence and structural motifs of p619. (A) Schematic diagram. Structural motifs include two regions of homology to the RCCl cell cycle regulator (RLD-1 and RLD-2), a putative leucine zipper (LZ), seven regions of highly polar (mostly acidic) residues (black boxes), seven 5-repeats related to those found in the ,B-subunit of heterotrimeric G proteins, three proline-rich regions (PR) and a HECT domain found in a class of functionally related E3 ubiquitin-protein ligases. (B) Predicted amino acid sequence of the 14 586 bp long open reading frame identified in overlapping cDNA clones of the human p619 gene (GeneBank accession No. U50078). Shaded boxes correspond to the RLD-l, seven f-repeats, RLD-2 and HECT domains as indicated. Stretches of polar amino acid residues are underlined by a solid line. The leucine zipper-like motif is underlined;i'i_g2iY,.AY by a broken line. Proline-rich regions are boxed. Threonine and serine residues that serve as potential phosphorylation sites for the cAMP-dependent.;iZ.' protein kinase are circled.
Expression pattern human cells, we performed Northern blot analysis using Northern blot analysis of total RNA isolated from a variety total RNAs isolated from either normal (CCD32SK and of mouse tissues and probed with mouse p619 cDNA CCD45SK skin fibroblasts) or tumor cell lines derived from sequences revealed a single band of ~15 kb (Figure 3A). a variety of human malignancies including carcinomas The size of this transcript is in good agreement with (COLO201, DU145, MCF7, ME180 and T24), sarcomas that predicted from the isolated human cDNA clones (A204 and RDES), melanoma (A375), gliomas and neuro- (15 171 bp), indicating that most, if not all, transcribed blastomas (HS683, U87MG and HT230) and leukemias and p619 sequences are represented in our composite cDNA lymphomas (K562, RAJI and CEM). As illustrated in Figure clone, pJLR75. This 15 kb long transcript was detected 3B, all tumor cell lines displayed the same 15 kb long in all tissues examined. Comparison of densitometric transcript detected above in mouse tissues. Visualization of signals obtained from each of the lanes with those the 15 kb long p619 transcript in the non-tumor CCD32SK measured upon rehybridization of the same blot with a and CCD45SK cell lines required overexposure of the auto- ,B-actin probe indicated some variability in expression radiogram. Analysis of poly(A)-containing mRNA isolated levels among the different tissues, with the highest levels from two of the highest expressing cell lines, RDES and found in testis and brain and the lowest in liver (detection ME 180, also revealed a single 15 kb transcript (not shown). in liver could be observed upon longer exposure of the Densitometric analysis of the relative levels of expression autoradiogram) (Figure 3A). A similar distribution pattern of p619 transcripts in tumor versus non-tumor human cell was found in human tissues (not shown). These results lines using the levels of ,-actin expression as a reference suggest that the p619 gene may be distributed ubiquitously indicated that the p619 gene is overexpressed in all tested in mammalian tissues. human tumor cell lines, independently of their develop- To determine the expression levels ofp619 transcripts in mental lineage (Figure 3B).
4264 p619 has GEF activity for ARFM and Rab proteins A
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Fig. 2. Sequence alignment of the RLD, ,B-repeats and HECT domains of p619. (A) Alignment of the seven internal repeats of the RLD-1 and RLD-2 motifs with the corresponding repeats of RCC1. Conserved residues are shaded. The consensus sequence for the seven repeat structure is indicated at the bottom. 0 denotes any of the following hydrophobic residues: A. F, I, L, M, V or W. (B) Alignment of the seven 5-repeat domain of p619 with the 1-repeat consensus sequence (Neer et al., 1994). h denotes any of the following hydrophobic residues: A, C, F, I, L, M or V. (C) Alignment of the HECT domains of p619 and E6-AP. Identical amino acids are shaded. Amino acid residues conserved in other proteins containing HECT domains are indicated by asterisks (Huibregtse et al., 1995). The conserved cysteine presumably involved in ubiquitin transfer is indicated by an arrow. Alignments were performed using the GCG and GeneWork programs.
Identification of the p619 protein RLD-2 antibodies (#410 antiserum) resulted in the detec- p619 cDNA sequences corresponding to amino acid tion of a single band migrating with the same mobility as residues 3684-3745, 4001-4385 and 4408-4861 were the high molecular weight (>500 kDa) protein detected expressed as indicated in Materials and methods and the in the MCF-7 immunoprecipitates (Figure 4A). This band resulting polypeptides used as antigens to raise polyclonal was not observed either in mock Sf9-infected cells or antibodies. Antibodies were also raised against a peptide when the infected cells were incubated with pre-immune corresponding to the 15 carboxy-terminal residues of p619. serum (Figure 4A). These results indicate that the protein As illustrated in Figure 4A, an antiserum (#410) raised identified in MCF7 cells is the product of the p619 gene against the RLD-2 domain (residues 4001-4385) readily and that the composite p619 cDNA clone described in immunoprecipitated a protein of >500 kDa from 35S- this study is likely to contain the entire coding sequence labeled MCF-7 cells, a human breast adenocarcinoma cell for the p619 protein. line known to contain high levels of p619 mRNA (Figure Additional evidence supporting the concept that the 3B). In addition to this high molecular weight protein, >500 kDa band corresponds to the product of the p619 an additional band of 180 kDa (p1 80) was observed gene was provided by Western blot analysis of MCF7 consistently in these immunoprecipitates (Figure 4A). immunoprecipitates obtained with anti-RLD-2 antibodies To determine whether any of these bands correspond (#410 antiserum). Fractionation of these immunoprecipit- to the product of the p619 gene, the cDNA insert of ates by SDS-PAGE followed by blotting with either the pJLR75 was subcloned into a baculovirus expression same antiserum or with antibodies raised against two vector and the resulting recombinant baculovirus used to different regions of the p619 molecule (#363 and #417 infect Sf9 insect cells. These cells expressed extremely antisera) consistently revealed the same >500 kDa high low levels of protein, thus preventing us from identifying molecular weight polypeptide (Figure 4B). Interestingly, the p619 protein by Coomassie staining. However, none of these antisera recognized the p180 band detected immunoprecipitation of 35S-labeled Sf9 cells with anti- in the 35S-labeled immunoprecipitates (Figure 4A), 4265 J.L.Rosa et al suggesting that this polypeptide is likely to be a p619- cDNA insert of pJLR75 into the pMEXneo mammalian associated protein rather than a degradation and/or expression vector (Martin-Zanca et al., 1989). The cleavage product of p619. resulting expression plasmid subsequently was transfected Since p619 was found to be part of an oncogene, we into NIH3T3 cells. No detectable levels of transforming examine whether this normal protein had transforming activity were observed. Moreover, cells expressing p619 properties. To this end, we subcloned the full-length failed to induce tumors when injected into nude mice. These results suggest that the normal p619 protein does not have transforming properties. However, the levels of A o tYtf'+ +/, t/ expression of the human p619 protein in NIH3T3 cells kb were very low, comparable with those of the endogenous p619 protein in these cells (not shown). Therefore, these --w ..- M% 4 ., h, pal 9 *00 . ".s Om #0 not rule out the that I. observations do possibility p619 may - 9.5 have some tumorigenic properties in those cells in which L - 6.2 it is overexpressed.
- 3.9 Subcellular distribution of p619 To determine the subcellular distribution of p619, MCF-7 10-~i_ cells were fractionated into plasmatic and internal mem- brane, cytosolic and nuclear fractions according to standard protocols (see Materials and methods). Equivalent amounts of each of these fractions were analyzed by Western blot with antisera raised against either cx-tubulin (mainly B O located in the cytosol), p53 (mainly located in the nucleus) gb,o 0'Oi* e ,. * "s, +s i$ $ soA/#6 h ' or Hsp7O (ubiquitously distributed) to control for the o * a qwO .o -. W p619 - effectiveness of the fractionation procedure. As depicted in Figure 4C, p619 was detected exclusively in those fractions containing internal membranes and cytosolic
Ilactin proteins. No detectable amounts of p619 were observed associated with either the nucleus or the plasma membrane (Figure 4C). Fig. 3. Expression pattern of the p619 gene. (A and B) Northern blot To define further the subcellular localization of p619, analysis of total RNA (20 .tg) isolated from either (A) adult mouse we performed indirect immunofluorescence microscopy tissues or (B) human cell lines with a partial cDNA probe derived using human skin fibroblasts (CCD45SK cells) with from the (A) mouse and (B) human p619 gene. Blots were stripped affinity-purified polyclonal antibodies elicited and reprobed with a f-actin cDNA probe. The migration of p619 and against P-actin mRNAs is indicated by arrows and arrowheads, respectively. p619. As shown in Figure 5a, immunofluorescence staining Molecular size markers are indicated. of permeabilized CCD45SK cells revealed extensive
A yc e .1 B C MCF-7 P P P kDa P p P ka N PM UM C V.. p619 *, F M.P0.. p61!19 *'-p619 (x-Tubulin _
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Fig. 4. Identification and subcellular distribution of the p619 protein. (A) 35S-Labeled lysates from either Sf9 insect cells not infected (mock) or infected with a recombinant baculovirus encoding p619 or from MCF-7 human cells were immunoprecipitated with either pre-immune (P) or immune (I) anti-p619 antibodies (#410 antiserum). The resulting immunocomplexes were subjected to SDS-PAGE (4-8% gradient with a ratio of acrylamide:bis-acrylamide of 80:1). The migration of p619 and p180 (see text) is indicated by an arrow and arrowhead, respectively. Co- electrophoresed molecular weight markers including myosin (200 000) and phosphorylase b (97 000) are indicated. (B) Lysates from MCF-7 cells were immunoprecipitated (IP) with either pre-immune (P) or immune (I) anti-p619 antibodies (#410 antiserum) and the resulting immunocomplexes submitted to Western blot (WB) analysis using three different anti-p619 antibodies (#363; #410 and #417 antisera) as indicated. The migration of p619 is indicated by an arrow. Molecular weight markers are those described above. (C) Lysates from MCF-7 cells were fractionated as indicated in Materials and methods. Equivalent amounts of nuclear (N), plasma membrane (PM), intracellular membrane (IM) and cytosolic (C) fractions were immunoprecipitated and analyzed by Western blot using anti-p619 antibodies (top) as described in (B) or directly submitted to Western blot analysis using anti-a-tubulin, anti-p53 or anti-Hsp7O antibodies.
4266 p619 has GEF activity for ARF1 and Rab proteins
Helms and Rothman, 1992). To determine whether p619 is also sensitive to BFA, we used confocal immunofluores- cence analysis to localize this protein in normal rat kidney (NRK) cells before and after treatment with BFA. As control, we used p58, a well known BFA-sensitive Golgi protein (Bloom and Brashear, 1989; Donaldson et al., 1990). As shown in Figure 6, the Golgi apparatus of NRK cells was clearly labeled with antibodies against the p58 and p619 proteins. Moreover, overlaying of these antibodies clearly shows co-localization of these proteins in the Golgi (Figure 6, right panels). However, and in agreement with the results depicted in Figure 5, p619 is expressed in other subcellular compartments, including vesicular-like structures (J.L.Rosa, R.P.Casaroli-Marano and S.Vilaro, unpublished observations). Treatment of these cells with BFA (5 gg/ml) altered the localization of p58 and p619 in the Golgi apparatus in a time-dependent manner (Figure 6). The effect of BFA was more evident after 30 min of exposure where both proteins were distributed dispersely throughout the cytoplasm, losing their co-localization. Similar results were obtained when we used human fibroblasts (not shown). These observations demonstrate the localization of p619 in the Golgi apparatus and define p619 as a new BFA-sensitive Golgi protein. Specific association of the RLD-2 domain of p619 with ARF1 RCC1 has been shown to interact with and to function as a GEF for Ran, a small GTP binding protein localized primarily in the nuclear fraction (Bischoff and Ponstingl, 1991). The structural similarities between RCC1 and p619 raised the possibility that this protein may also associate Fig. 5. Co-localization of p619 and ARFI in the Golgi region. Indirect with, and perhaps serve as a GEF for, other small GTP immunofluorescence staining of CCD45SK human fibroblasts (a and binding proteins associated with Golgi structures such as b) with affinity-purified rabbit anti-p619 antibodies (#410 antiserum) the members of the ARF and Rab families. To this end, followed by Texas red-labeled donkey anti-rabbit antibodies in (a) the recombinant baculoviruses encoding ARFI, Rab3A and absence or (b) the presence of 10 tg of immunizing antigen. Double Rab5 as well as the RLD-1 staining using (c, e and g) anti-p619 antibodies [#407 antiserum in (c proteins (residues 365-794) and e) and #410 antiserum in (g)] and mouse (d) anti-p58 or (f and h) and RLD-2 (residues 4001-4385) domains of p619 were anti-ARFI antibodies followed by (c, e and g) Texas red-labeled generated. Each of these proteins carried the Flag epitope donkey anti-rabbit antibodies and (d, f and h) fluorescein-labeled sheep to facilitate their detection. To avoid interference with anti-mouse antibodies. post-translational modifications, the Flag peptide was placed at the amino-terminus of the Rab proteins and at labeling of a perinuclear structure along with faint and the carboxy-terminal end of ARFI. Sf9 cells infected with diffuse cytoplasmic staining, probably representing the a baculovirus containing the entire p619 cDNA clone soluble pool of p619. As a control, pre-absortion of expressed extremely low levels of p619, even lower than the anti-p619 antibodies with the immunizing antigen those found in MCF7 cells (see Figure 4A), thus effectively completely blocked all detectable staining (Figure Sb). preventing us from utilizing the full-length p619 protein Perinuclear staining similar to that displayed by the anti- in these experiments. As an alternative, we generated a p619 antibodies has been shown to be characteristic of baculovirus encoding an amino-terminus truncated p619 proteins associated with the Golgi apparatus, such as protein, p619AN, that encompasses the 1354 carboxy- p58 (Bloom and Brashear, 1989). Double labeling of terminal residues of p619. Additional baculoviruses encod- CCD45SK cells with anti-p619 and anti-p58 antibodies ing RCC 1 and Ran proteins were also obtained for control revealed identical immunostaining, indicating that p619 is experiments. also localized in the Golgi region (Figure Sc and d). Lysates from either non-infected (mock) Sf9 cells, Sf9 cells singly infected with baculoviruses encoding p619AN, Brefeldin A alters the localization of p619 in the ARFI, Ran, Rab3A and Rab5, or Sf9 cells doubly infected Golgi apparatus with p619AN/ARF1, p619AN/Ran, p619AN/Rab3A and The fungal metabolite Brefeldin A (BFA) has a profound p619AN/RabS were immunoprecipitated with anti-p619 effect on the structure of the Golgi apparatus, causing antibodies (#363 antiserum) and submitted to Western blot Golgi proteins to dissociate minutes after BFA treatment analysis with either a different anti-p619 antiserum (#410) (Lippincott-Schwartz et al., 1989). Moreover, the ARFI to determine the amount of p619AN protein present in GEF activity associated with Golgi membranes has been each immunoprecipitate or with anti-Flag and anti-Ran shown to be sensitive to BFA (Donaldson et al., 1992; antibodies to determine whether any of the above small
4267 J.L.Rosa et al.
pb 8 p,6 i fL Time (ni i n )
0
3
15
30
Fig. 6. Confocal immunofluorescence analysis of p619 in NRK cells treated with Brefeldin A. NRK cells were treated with 5 ,ug/ml of BFA for the indicated times, fixed, permeabilized with saponin and stained by double-label immunofluorescence with anti-p58 monoclonal (left panels) and anti- p619 affinity-purified polyclonal (middle panels) antibodies followed by fluorescein-labeled sheep anti-mouse antibodies and Texas red-labeled donkey anti-rabbit antibodies, respectively. Overlays are shown in yellow (right panels) indicating co-localization of p58 (green) and p619 (red). Scale bar, 25 ,um.
GTP binding proteins were associated with the co- (mock) or infected with baculovirus encoding ARFI. An expressing p619AN protein (Figure 7A, left panel). To equivalent amount of beads alone was used as negative control for the amount of protein present in each sample, control. As shown in Figure 7B, the RLD-2 motif, but total cellular extracts were submitted directly to Western not RLD-1 nor beads alone, binds to ARF1. Moreover, blot analysis with the same antibodies (Figure 7A, right this interaction appears to require proper myristoylation panel). of the ARFI protein since RLD-2 failed to bind to a fast As illustrated in Figure 7A, ARFI, but not Rab3A, migrating form of ARFI that corresponds to the non- Rab5 or Ran, was capable of interacting with p619. myristoylated protein, as determined by direct labeling To illustrate further the specificity of this interaction, with [3H]myristic acid (not shown). These results, taken baculoviruses encoding RLD-1 and RLD-2 proteins were together, illustrate a specific interaction between ARFI purified from lysates of Sf9 insect cells infected with and the RLD-2 domain of p619. the corresponding baculoviruses using Ni2+-NTA agarose We next examined whether ARFI and p619 protein co- beads. Beads containing the purified proteins were incuba- localized inside the cell. Double immunofluorescence ted with lysates of Sf9 insect cells either non-infected labeling of CCD45SK cells with two different anti-p619
4268 p619 has GEF activity for ARF1 and Rab proteins A Immunocomplexes Total cell lysates
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(i Flag
Fig. 7. Association of ARFI with the RLD-2 domain of p619. (A) Lysates of Sf9 insect cells either not infected (mock), singly infected with recombinant baculoviruses encoding p619AN (residues: 3508-4861), Ran, Rab3A, Rab5 and ARFI or doubly infected with recombinant baculoviruses encoding p619AN/Ran, p619AN/Rab3A, p6I9AN/Rab5 and p619AN/ARFl were analyzed by Western blot either (left panel) after immunoprecipitation with anti-p619 antibodies or (right panel) directly from total cell lysates using (top) anti-p619, (middle) anti-Flag or (bottom) anti-Ran antibodies. The migration of p619, Rab3A, Rab5, ARF1 and Ran proteins is indicated by arrows. (B) Ten tg of Ni2> beads containing purified RLD-1 or RLD-2 proteins were incubated with lysates of Sf9 insect cells either (M) not infected or (A) infected with baculoviruses encoding ARFI. An equivalent amount of beads alone was used as control. (Left panel) The resulting complexes as well as (right panel) total cell lysates (70 ,ug) were fractionated by SDS-PAGE (12% gels) and analyzed by Western blot using anti-Flag antibodies. The migration of the RLD motifs as well as the myristoylated (myrARFI) and non-myristoylated (ARFI) forms of ARFI is indicated by arrows.
antibodies (Figure 5e and g) and with an anti-ARF (Figure the labeled nucleotide. Exchange activity by RLD- 1 was 5f and h) antiserum revealed that these proteins co-localize not specific for GDP. In agreement with previous observ- within the Golgi apparatus structures, thus suggesting that ations, [35S]GTP-yS also became incorporated onto ARFI, these proteins may also become associated inside the cell. albeit with a lower incorporation rate of 0.005 pmol/,g/ min. Incubation of [35S]GTP-yS-ARFl with RLD-1, but The RLD-1 motif of p619 stimulates guanine not with RLD-2, in the presence of excess GTP also nucleotide exchange on ARF1 and Rab proteins resulted in the stimulation of [35S]GTP-yS release (Figure Next, we examined whether the RLD domains of p619 8B). In both cases, the rate of guanine nucleotide exchange could stimulate guanine nucleotide exchange on ARFI. was dependent on the concentration of RLD-1 present in To this end, RLD-1, RLD-2 and ARFI proteins were the reaction mixture (not shown). Considering the distinct purified from infected Sf9 insect cells as indicated in activities of the two RLD domains of p619, we examined Materials and methods. The purity of each protein was whether RLD-2 may modulate the GEF activity of RLD- 1. found to be >90% as determined by SDS-PAGE and Addition of increasing amounts of purified RLD-2 protein Coomassie staining (Figure 8A). The purified ARFI to the guanine nucleotide exchange reaction catalyzed by protein incorporated [3H]GDP at a rate of 0.027 pmol/,ug/ RLD- 1 did not affect the rate of [3H]GDP release from min in a time-dependent fashion for at least 60 min. The ARF1 (not shown). loaded [3H]GDP-ARFI protein was incubated with cold To determine the specificity of these observations, the GTP (100 gM) in the absence or presence of RLD-1 or RLD-1 motif was incubated with a variety of small GTP RLD-2 for the indicated periods of time. As shown in binding proteins including Rab3A, Rab5, Ran and R-Ras2/ Figure 8B, RLD- 1, but not RLD-2, stimulated the exchange TC21 (see Materials and methods). As shown in Figure of GDP bound to ARFI as determined by the release of 8C, RLD- 1 was also capable of stimulating the release of
4269 J.L.Rosa et al. A B C kOa 0 200- c 97- U. S - CL 48- CZa. 1- 1X0 _"*: 0 30- 0.20 0 21 - IK 140 14- U. (5-2 I- 0
20 20 400 20 Time (min) Time (min) Time (min)
Fig. 8. The RLD-1 domain of p619 stimulates guanine nucleotide exchange on ARFI and Rab proteins. (A) SDS-PAGE (12% gels) analysis and Coomassie staining of purified RLD-1, RLD-2 and ARFI proteins (1-3 gg). Co-electrophoresed molecular weight markers included myosin (200 000) phosphorylase b (97 000), BSA (69 000), ovalbumin (46 000), carbonic anhydrase (30 000), trypsin inhibitor (21 500) and lysozyme (14 300). (B and C) (B, top) [3H]GDP-ARFI; (B, bottom) [35S]GTP-yS-ARF1; (C, top left) [3H]GDP-Rab3A; (C, top right) [3H]GDP-Rab5; (C, bottom left) [3H]GDP-Ran and (C, bottom right) [3H]GDP-R-Ras2/TC21 were incubated either with buffer alone (lI) or with 1 tg of RLD-1 (0) or RLD-2 (0) proteins for the indicated times and analyzed as indicated in Materials and methods. Data is shown as mean ± SE from 3-4 independent experiments.
[3H]GDP from [3H]GDP-loaded Rab3A and Rab5 proteins gene Tiam-1 functions as a Rho/Rac GEF involved in but not from [3H]GDP-loaded Ran or R-Ras2/TC2 1. inducing membrane ruffling that presumably contributes As a control, baculovirus-expressed RCC1 induced the to the metastatic process (Habets et al., 1994; Michiels efficient exchange of [3H]GDP from [3H]GDP-loaded Ran et al., 1995). Finally, the oncogene ost catalyzes guanine under conditions in which neither RLD- 1 or RLD-2 nucleotide exchange on RhoA and Cdc42, and interacts displayed any detectable activity. These observations, with Rac 1, thus linking the Rho and Rac signaling taken together, suggest that p619 is a GEF for ARFI and pathways (Horii et al., 1994). Interestingly, the normal possibly for certain Rab proteins. p619 allele is expressed at higher levels in all tested human tumor cell lines than in normal human skin fibroblasts. However, transfection of an expression vector Discussion encoding the normal p619 protein into rodent fibroblasts In this study, we report the identification of a novel human did elicit tumorigenic properties. Therefore, it is possible gene, p619, which encodes a polypeptide of 4861 amino that the observed overexpression of the p619 gene in acid residues, one of largest proteins known to date. The human tumor cells is a consequence, rather than a cause, predicted amino acid sequence of p619 revealed the of their enhanced proliferating activity. presence of two regions (RLDs) of seven internal repeats Our results suggest a model in which the RLD-2 region that display a remarkable resemblance to RCC1, a cell of p619 functions as a recruitment domain to present the cycle regulatory protein that acts as a GEF for the nuclear GDP-bound ARFI protein to the catalytic center contained small GTP binding protein Ran (Bischoff and Ponstingl, within RLD-1. The differential roles of the two RLD 1991). We demonstrate here that p619 also stimulates motifs of p619 in promoting ARFI guanine nucleotide guanine nucleotide exchange activity when incubated in exchange activity are at variance with the ability of the the presence of certain members of the Ras superfamily RCC 1 protein to bind and activate Ran with a single seven of proteins such as ARFI, Rab3A and Rab5, but not Ran repeat domain. This specialization might reflect the need nor the highly transforming R-Ras2/TC21. of the cell to reach higher levels of specificity in membrane Sequences derived from the p619 gene were first identi- trafficking (mediated by multiple ARF and Rab proteins) fied as a component of an oncogene generated during than in nuclear/cytoplasmic transport (apparently carried transfection of human tumor DNA in the nude mouse out by a single Ran protein) (reviewed by Moore and tumorigenicity assay (T.Koda and M.Barbacid, unpub- Blobel, 1994). If so, the activity of the RLD-l motif lished observations). Preliminary observations indicate promoting nucleotide exchange on Rab proteins might be that only a small subset of p619 sequences became restricted in vivo by the binding specificity provided by incorporated into this oncogene. To date, we do not have the RLD-2 domain. evidence as to whether the p619 sequences present in this Helms et al. (1993) have proposed a model in which a novel oncogene contribute to its transforming properties. putative ARFI GEF mediates the association of myristoyl- Several GEFs have been found to be associated with ated ARFI to Golgi membranes by a process that requires human genetic diseases as well as with the development the exchange of GDP (cytoplasmic ARFI) by GTP (mem- of neoplasia and metastatic invasiveness. For instance, the brane bound ARFI). According to this model, activation faciogenital dysplasia (FGDY) gene codes a putative Rho/ of the ARFI protein as a result of GTP binding only Rac GEF (Pasteris et al., 1994). The invasion-inducing results in a loose interaction with Golgi membranes.
4270 p619 has GEF activity for ARF1 and Rab proteins
Strong binding of ARFI proteins to their target membranes facilitate the access of the RLDs to other p619 substrates. is mediated by a specific 'receptor'. The interaction The presence of a carboxy-terminal HECT domain also between the p619 and ARFI proteins as w'ell as their co- raises the possibility that p619 may catalyze the transfer localization in the Golgi region raises the possibility that of ubiquitin to specific proteins in a fashion similar to p619 may play both roles. If so, the RLD-1 motif of p619 that reported for other HECT domain-containing proteins may mediate the guanine nucleotide exchange necessary such as E6-AP (Scheffner et al., 1993; Huibregtse et al., for the initial association of ARF1 with Golgi membranes, 1995). Indeed, preliminary results suggest that the HECT and the RLD-2 domain may provide the membrane domain of p619 possesses intrinsic ubiquitin-protein ligase anchoring site postulated by Helms et al. (1993). activity (our unpublished observations). If so, it is possible The possible role of p619 as a GEF for other members that some of the above protein-protein interaction motifs of the Ras superfamily of proteins is likely to be restricted may serve to bring putative ubiquitination substrate(s) to by their respective subcellular localization. For instance, p619. Additional studies will be required to ascertain the Rab5 and Rab3, two substrates for p619 in vitro, are physiological role of the other structural motifs of p619 located on endosomes and synaptic vesicles, respectively. and to determine whether p619 is indeed a multifunctional Preliminary observations using confocal microscopy indi- protein with multiple roles in membrane trafficking cate that p619 has a vesicular-like distribution (J.L.Rosa, processes. R.P.Casaroli-Marano and S.Vilaro, unpublished observa- tions). In addition, it is possible that p619 may stimulate guanine nucleotide exchange in vivo on those Rab proteins Materials and methods localized in the Golgi. Unlike ARFI, Rab proteins do not cDNA cloning form stable complexes, at least with the isolated RLD-2 A human 382 bp long cDNA probe (pTK78-5) isolated by exon trapping domain of p619. It is possible that the entire p619 protein (Buckler et al., 1991) from genomic DNA clones of a novel human interacts with Rab proteins. Alternatively, the interaction transforming gene (T.Koda and M.Barbacid, unpublished observations) was used to screen a human fetal brain cDNA library (Stratagene). between p619 and these GTP binding proteins could be Fourteen positive clones were isolated, sequenced and their 5'-most mediated by other molecules such as the GDI or REP sequences used to generate probes for subsequent screenings. After proteins known to be required for Rab activation seven rounds of screening, eight overlapping cDNA clones encompassing (Alexandrov et al., 1994; Soldati et al., 1994; Ullrich a linear sequence of 15 171 nucleotides with a single ORF of 14 586 bp were subcloned into pBluescript (Stratagene) to generate a single cDNA et al., 1994). These putative interactions could be mediated clone (pJLR75; GeneBank accession No. U50078). All cDNA clones by some of the additional protein-protein recognition were sequenced at least twice in both orientations by fluorescent DNA motifs present in p619 (see below). Generation of signific- sequencing (Applied Biosystems). Nucleotide sequences were compiled ant amounts of purified p619 protein (instead of RLD using Sequencher 2.1 software (Gene Codes Corporation. MI). domains) should facilitate additional biochemical studies other small Northern blot analysis using GTP binding proteins known to co- Total RNA was isolated from a variety of human cell lines and localize with p619 inside the cell. mouse tissues by the RNAzol method (Tel-Test, Inc.), fractionated by The ARFI GEF activity associated with Golgi mem- electrophoresis in formaldehyde-agarose gels (0.8%), transferred to branes has been shown to be sensitive to the fungal nitrocellulose membranes and submitted to hybridization analysis follow- metabolite BFA (Donaldson et al., 1992; Helms and ing standard protocols (Sambrook et al., 1989). RNAs derived from human cells were probed with the human 382 bp cDNA insert of Rothman, 1992). BFA inhibited the ARFI GEF activity pTK78-5 described above. RNAs isolated from mouse tissues were associated with Golgi membranes, thereby preventing the hybridized with a probe derived from the same region of the mouse ARF1-dependent recruitment of cytosolic coat proteins p619 gene. To this end, DNA from a mouse brain cDNA library (Rothman, 1994). Preliminary experiments indicate that (Stratagene) was submitted to PCR-aided amplification using two primers derived from sequences located at both ends of the human pTK78-5 BFA does not affect the ARFI GEF activity of the clone (5'-ATGTCTTGTGGCTTCAAGCAC-3' and 5'-TTGACCAAA- RLD-1 domain of p619 in vitro (J.L.Rosa and M.Barbacid, GGTATACACATG-3'). The amplified 382 bp long DNA fragment, unpublished observations). However, BFA alters the which exhibited 92% identity to the pTK78-5 insert, was subcloned into localization of p619 in the Golgi apparatus, possibly pBluescript (pJLR9). disrupting its GEF activity. BFA may act Alternatively, Antibodies directly on p619 by interacting with a putative regulatory Polypeptides corresponding to various domains of p619 fused to either region. Interestingly, Tsai et al. (1994) have described an glutathione S-transferase (GST) (residues 3684-3745), the His6 tag ARF1 GEF in bovine brain that migrated as a large BFA- sequences (residues 4001-4385) or the MS2 phage polymerase (residues sensitive complex of -700 kDa, thus possibly correspond- 4408-4861) were injected intraperitoneally into rabbits to generate ing to p619. polyclonal antibodies 363, 410 and 417, respectively. A synthetic peptide (residues 4847-4861) coupled to keyhole limpet hemocyanin (KLH) In addition to its RLD domains, p619 has seven was also used to generate anti-p619 antibodies (antiserum #407). ,-repeats, three proline-rich motifs, seven regions rich in Antibodies were immunoaffinity purified as previously described (Bustelo polar amino acid residues, a leucine zipper and a carboxy- et al., 1995) from #407 and #410 antisera. A polyclonal rabbit antiserum terminal HECT domain. Some of these structural motifs, was raised against a GST-Ran fusion protein. Anti-ARF mouse mono- clonal lD9 antibody was kindly provided by R.Kahn. Anti-Flag M2 (IBI such as the seven n-repeats, the proline-rich sequences Technology), anti-p58 (Sigma), anti-ax-tubulin, anti-p53 and anti-Hsp70 and the leucine zipper, are likely to mediate the interaction (Oncogene Science) mouse monoclonal antibodies were also utilized. of p619 with one or more cellular partners. For instance, Texas red-labeled donkey anti-rabbit or fluorescein-labeled sheep anti- each RLD region is followed by a proline-rich domain mouse antibodies were purchased from Amersham. whose amino acid sequence matches that of the class II Baculovirus expression consensus SH3 binding sites (Feng et al., 1994). Thus, it cDNAs encoding human Rab3A, RabS, ARF1, Ran and RCCI proteins is likely that these domains mediate the interaction of as well as the p619 RLD-l (residues 365-794) and RLD-2 (residues p619 with SH3-containing adaptor proteins that may 4001-4385) domains were isolated by PCR-aided amplification from 4271 J.L.Rosa et al. pJLR75 DNA (RLD-1 and RLD-2) or from human cDNA libraries sucrose, 10 jg/ml aprotinin, 10 jig/ml leupeptin, 5 jg/ml pepstatin and (Stratagene) from human fetal brain (Rab3A, Rab5 and ARFI) or HeLa 0.5 mM PMSF), disrupted with 15 strokes of a motor-driven Teflon- cells (Ran and RCC1) and subcloned in the proper orientation in either glass homogenizer and centrifuged at 1000 g for 10 min at 4°C. The pVL1393 (ARFI), a pVL1393 derivative containing a His6 leader pellet (nuclear fraction) was washed once in homogenization buffer and sequence (RLD-1, RLD-2 and RCCI) or pBlueBacHis (Rab3A, Rab5 disrupted with Iysis buffer (10 mM Tris-HCI, pH 8.0, 150 mM NaCl, and Ran) (Invitrogen). Some of these proteins were tagged with the Flag 10% glycerol, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, epitope (DYKDDD) located either at their amino- (Rab3A and Rab5) 10 jig/ml aprotinin, 10 jg/mI leupeptin, 5 jig/ml of pepstatin and 0.5 mM or carboxy- (ARF1, RCC1, RLD-l and RLD-2) terminus. A baculovirus PMSF). The supernatant from the 1000 g spin was centrifugated at containing the human R-Ras2/TC21 oncogene fused to GST sequences 16 000 g for 20 min at 4°C. The pellet (plasma membrane fraction) was was generated by subcloning R-Ras2/TC21 sequences from pGEX-TC21 solubilized with lysis buffer. The supernatant from the 16 000 g spin (Lopez-Barahona et al., 1996) into pVL 1393. Plasmids were sequenced was centrifuged at 150 000 g for 30 min at 4°C. The pellet (internal and used to generate recombinant baculoviruses following standard membrane fraction) was solubilized with lysis buffer. A 1/10 (v/v) procedures (Summers and Smith, 1987). volume of a lOx lysis buffer solution was added to the supernatant Protein and in vitro from the 150 000 g spin (cytosolic fraction). Immunoprecipitations and purification binding assays subsequent Western blot analysis were carried out as described above. Sf9 insect cells (2X 109) were infected with recombinant baculoviruses containing the His6-tagged sequences and harvested 48 h later by centrifugation at 4°C for 5 min. The cell pellet was lysed in 50 ml of Immunofluorescence microscopy cold buffer A [10 mM Tris-HCl pH 8.0, 5 mM KCI, 2 mM MgCl,, CCD45SK human fibroblasts or NRK cells were growth on coverslips 10% glycerol, 0.5% NP-40, 5 mM f-mercaptoethanol, 1 mM benzamid- or chamber slides (Nunc Inc.), fixed with 4% paraformaldehyde in ine, 10 tg/ml aprotinin, 10 jg/ml leupeptin, 10 jg/ml pepstatin and phosphate-buffered saline (PBS) for 30 min at room temperature, washed 0.5 mM phenylmethylsulfonyl fluoride (PMSF)I. After lysis, NaCl and three times with PBS for 5 min, permeabilized with 0.1% saponin in imidazole were added to a final concentration of 500 and 1 mM, PBS for 10 min, washed twice with PBS and incubated with 1% BSA respectively. Insoluble material was removed by centrifugation at in PBS for 1 h. Coverslips or slides were incubated with primary 15 000 g for 20 min at 4°C. The resulting supernatant was incubated antibody [#407 (6 jig/ml); #410 (4 jig/ml); anti-p58 (0.7 mg/ml) and with 200 PI of a Ni2+-NTA-agarose suspension (Qiagen) for 2 h at 4°C anti-ARF (70 jg/ml)] in 1% BSA in PBS for 2 h, washed five times with rocking. The beads were washed three times at 4°C for 5 min with with 1% BSA in PBS, incubated with secondary antibody (Texas red- 15 ml of buffer B (buffer A containing 1 mM imidazole). Bound proteins labeled anti-rabbit or fluorescein-labeled anti-mouse antibodies; 1:200) were eluted with 1.5 ml of buffer A containing 100 mM imidazole. in 1% BSA in PBS for 1 h, washed five times with PBS and finally Fractions containing the purified protein were pooled and dialyzed mounted with fluoromount G (Southern Biotechnologies). Microscopy against 20 mM Tris-HCl pH 7.5, 5 mM MgCl2, 10 mM EDTA and 10% (Carl Zeiss, Inc.) was performed with 63x and lOOx oil lenses. Confocal glycerol. Purified proteins were quantified, aliquoted and stored at immunofluorescence analysis was performed using a confocal microscope -20°C. In the case of the ARF1 protein, which lacks the His6 tag, at the excitation wavelengths of 476 and 529 nm as previously described cells were disrupted with buffer A containing 0.6% CHAPS {3-[(3- (Pagan et al., 1996). cholamidopropyl)dimethylammonio]- I -propanesulfonate; Sigma) as described above. The resulting supernatant was incubated with I ml of anti-Flag M2 affinity gel (IBI Technology). The bound ARFI protein was eluted with 0.6 mg/ml of Flag peptide. For in vitro binding, 10 jig Acknowledgements of RLD- 1 and RLD-2 bound to beads were incubated Ni2+-NTA-agarose We are indebted to T.Koda for the human with of Sf9 insect cells which were either non-infected or infected providing genomic DNA lysates clones of oncH for the exon trapping experiments. We also thank E.Sakai with baculovirus encoding ARFI as previously described (Bustelo et al., 1995). for the 3' cDNA clone of p619 and D.Carrasco for his help in immunofluorescence experiments. We are also grateful to R.A.Kahn for Immunoprecipitation and Western blot analysis anti-ARF antibodies, N.Thomson and T.Nelson for their invaluable help Cells were labeled with Trans35S-label (50 ,uCi/ml; 1037 Ci/mmol; ICN) in nucleotide sequencing, E.O'Rourke for generating the recombinant for 3 h and submitted to immunoprecipitation analysis as previously baculoviruses, and X.R.Bustelo, E.Nogueira, F.Ventura and R.Bartrons described (Bustelo et al., 1995). For experiments using Sf9 insect for helpful discussions. J.L.R. was supported by a postdoctoral fellowship cells, cells were collected 48 h after infection with the corresponding from the Ministerio de Educaci6n y Ciencia of Spain. recombinant baculovirus. After washing once with protein-free medium, cells were resuspended in 10 ml of buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM benzamidine, 10 jg/ml aprotinin, 10 jg/ml leupeptin, 10 tg/ml pepstatin and 0.5 mM References PMSF), disrupted by vortexing and centrifuged at 15 000 g for 30 min Alexandrov,K., Horiuchi,H., Steele-Mortimer,O., Seabra,M. and at 4°C. Supernatants (1 ml) were immunoprecipitated with the appropriate Zerial,M. (1994) Rab escort protein- lis a multifunctional protein that antibodies, fractionated by SDS-PAGE and submitted to Western blot accompanies newly prenylated Rab proteins to their target membranes. analysis as previously described (Bustelo et al., 1995). Immunoprecipit- EMBO J., 5262-5273. ation and 13, Western blot analysis of human MCF7 cells was performed Bischoff,F.R. and Ponstingl,H. (1991) Catalysis of guanine nucleotide as described et previously (Bustelo al., 1995). 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