p619, a giant protein related to the chromosome condensation regulator RCC1, stimulates guanine nucleotide exchange on ARF1 and Rab proteins.

Abstract

We report the identification of a novel human gene, designated p619, that encodes a polypeptide of 4861 amino acid residues, one of the largest human proteins known to date. The p619 protein contains two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP binding protein, Ran. In addition, p619 possesses seven beta‐repeat domains characteristic of the beta‐subunit of heterotrimeric G proteins, three putative SH3 binding sites, seven polar amino acid‐rich regions, a putative leucine zipper and a carboxy‐terminal HECT domain characteristic of E3 ubiquitin‐protein ligases. p619 is expressed ubiquitously in mouse and human tissues and overexpressed in several human tumor cell lines. Subcellular localization studies indicate that p619 is located in the cytosol and in the Golgi apparatus. Localization of p619 in the Golgi is altered by Brefeldin A. The carboxy‐terminal RCC1‐like domain of p619 interacts specifically with myristoylated ARF1, a small GTP binding protein also located in the Golgi. Moreover, the second RCC1‐like motif located at the amino‐terminus of p619 stimulates guanine nucleotide exchange on ARF1 and on members of the related Rab proteins, but not on other small GTP binding proteins such as Ran or R‐Ras2/TC21. These observations suggest that p619 is a Brefeldin A‐sensitive Golgi protein that functions as a guanine nucleotide exchange factor for ARF1 and, possibly, for members of the Rab family of proteins.

A giant protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins forms a cytosolic ternary complex with clathrin and Hsp70

“…RPGR (retinitis pigmentosa GTPase regulator) has recently been identi®ed by positional cloning as a locus responsible for X-linked retinitis pigmentosa (Meindl et al, 1996). Finally, we have identi®ed two structural domains highly related to RCC1 in a giant human protein of 4861 amino acid residues that, at least in vitro, stimulates guanine nucleotide exchange on ARF1 and certain members of the Rab family of small GTPases (Rosa et al, 1996a). We chose to name this gene as p532 based on the predicted molecular mass of its product (Rosa et al, 1996b).…”

“…Finally, we have identi®ed two structural domains highly related to RCC1 in a giant human protein of 4861 amino acid residues that, at least in vitro, stimulates guanine nucleotide exchange on ARF1 and certain members of the Rab family of small GTPases (Rosa et al, 1996a). We chose to name this gene as p532 based on the predicted molecular mass of its product (Rosa et al, 1996b). It should be noted, however, that this locus was erroneously designated as p619 in the original publication (Rosa et al, 1996a).…”

“…We chose to name this gene as p532 based on the predicted molecular mass of its product (Rosa et al, 1996b). It should be noted, however, that this locus was erroneously designated as p619 in the original publication (Rosa et al, 1996a).…”

“…Nucleotide sequence analysis of these cDNA clones which encompassed a linear sequence of 15 171 nucleotides revealed a single open reading frame corresponding to a protein of 4861 amino acid residues. This protein displays an array of structural motifs including two RCC1-like domains located at both ends of the molecule, seven b-repeat domains characteristic of the b subunit of heterotrimeric G proteins, three putative SH3 binding sites, seven polar amino-acid rich regions, a putative leucine-zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases (Rosa et al, 1996a).…”

“…Subcellular localization experiments show that the p532 protein is localized in the Golgi apparatus, although signi®cant levels of p532 were also observed in the cytoplasm. The localization of p532 in the Golgi was altered by Brefeldin A. Biochemical studies indicated that the carboxy-terminal RCC1-like domain of p532 (RLD-2) binds myristoylated ARF1 and the amino-terminal RCC1-like domain (RLD-1) stimulates guanine nucleotide exchange on ARF1 and certain members of the Rab family of proteins (Rosa et al, 1996a).…”