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).