Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

The human Ras-related nuclear protein Ran/TC4 (refs 1-4) is the prototype of a well conserved family of GTPases that can regulate both cell-cycle progression and messenger RNA transport. Ran has been proposed to undergo tightly controlled cycles of GTP binding and hydrolysis, to operate as a GTPase switch whose GTP- and GDP-bound forms interact differentially with regulators and effectors. One known regulator, the protein RCC1 (refs 12, 13), interacts with Ran to catalyse guanine nucleotide exchange, and both RCC1 and Ran are components of an intrinsic checkpoint control that prevents the premature initiation of mitosis. To test and extend the GTPase-switch model, we searched for a Ran-specific GTPase-activating protein (GAP), and for putative effectors (proteins that interact specifically with Ran/TC4-GTP). We report here the identification of a Ran GAP and its use to characterize the GTP-hydrolysing properties of mutant Ran proteins, and the identification and cloning of a binding protein specific for Ran/TC4-GTP.

A mixed-oligonucleotide probe was used to identify four ras-like coding sequences in a human teratocarcinoma cDNA library. Two of these sequences resembled the rho genes, one was closely related to H-, K-, and N-ras, and one shared only the four sequence domains that define the ras gene superfamily. Homologs of the four genes were found in genomic DNA from a variety of mammals and from chicken. The genes were transcriptionally active in a range of human cell types.Mammalian ras genes (8,11,36) encode a family of proteins that show low but significant homology to the Go, subunits of G proteins (18). A number of genes encoding proteins with Mrs of 20,000 to 25,000 that share significant homology with the ras proteins have been isolated. The homology is greatest in four domains that have been shown through both mutagenic (see reference 2 for a review) and X-ray crystallographic (9, 17, 28) studies to be involved in the binding and hydrolysis of guanine nucleotides. Many ras-related proteins also contain a fifth conserved domain at their carboxy termini that, in H-, K-, and N-ras, is required for membrane localization and biological activity (13, 40). These ras-related proteins are found in a variety of eucaryotic organisms and appear to be well conserved over evolutionary time.The ras gene superfamily can be divided into several major groups on the basis of amino acid sequence: (i) the H-ras, K-ras, and N-ras proto-oncogenes (H, K, and N genes); (ii) the ral genes, which share about 50% homology with H-, K-, and N-ras (4, 5); (iii) the rap genes (29, 30) and R-ras (21), which differ significantly from each other but all share about 50 to 55% homology with the ras proteins, including strict conservation of the ras effector domain (amino acids 32 to 40 of H-ras); (iv) the rho genes, a more distantly related group that exhibits only about 35% identity with the ras proteins (23, 24); and (v)

Abstract. Ran/TC4, first identified as a well-conserved gene distantly related to H-RAS, encodes a protein which has recently been shown in yeast and mammalian systems to interact with RCC1, a protein whose function is required for the normal coupling of the completion of DNA synthesis and the initiation of mitosis. Here, we present data indicating that the nuclear localization of Ran/TC4 requires the presence of RCC1. Transient expression of a Ran/TC4 protein with mutations expected to perturb GTP hydrolysis disrupts host cell DNA synthesis. These results suggest that Ran/TC4 and RCC1 are components of a GTPase switch that monitors the progress of DNA synthesis and couples the completion of DNA synthesis to the onset of mitosis. AN/TC4, was initially described as a RAS-related transcript of unknown function. It was identified in a human teratocarcinoma cell line but is abundant in a variety of cultured cell lines, and is of interest because it defines a new, evolutionarily well-conserved branch of the RAS gene superfamily (Drivas et al., 1990(Drivas et al., , 1991a. Recent genetic and biochemical analyses (Matsumoto and Beach, 1991;Bischoff and Ponstingl, 1991a) suggest that Ran/TC4 also plays a key role in the regulation of cell cycle progression in eukaryotes, and that this role depends on its nuclear localization and interaction with the product of a second gene, RCC1, defined by the tsBN2 mutation of BHK cells (Nishimoto et al., 1978;Uchida et al., 1990) and the piml mutation of the fission yeast Schizosaccharomyces pombe (Matsumoto and Beach, 1991). The compound name used here reflects this: a teratocarcinoma-derived eDNA clone that encodes a Ras-related nuclear (Ran) protein.S. pombe piml (premature initiation of mitosis) mutants enter mitosis without completing chromosomal DNA replication. Overexpression of the wild-type allele of a second gene, spil (suppressor ofpiml), suppresses the piml mutant phenotype. The predicted amino acid sequences of spil (yeast) and Ran/TC4 (human) are 80% identical. The fact that spil overexpression cannot rescue null mutants and the existence of a cold-sensitive mutation in spil suggest direct interaction between Piml and Spil proteins (Matsumoto and Beach, 1991).The mammalian homolog of piml is RCC1 (regulator of chromosomal condensation), a gene originally defined by the tsBN2 mutation of BHK cells (Uchida et al., 1990;Nishitani et al., 1991). The wild-type activity of RCC1 is required both to initiate DNA synthesis (Dasso et al., 1992) and to prevent chromosome condensation until the completion of S phase (Uchida et al., 1990;Nishitani et al., 1991;Enoch and Nurse, 1991;Dasso and Newport, 1990). piml is predicted to encode a larger protein (539 aa) than RCC1 (421 aa), but over the region shared by the two proteins, their sequences are 30 % identical and share a sequence motif repeated seven times in each protein (Matsumoto and Beach, 1991).RCC1 protein can bind DNA and is associated with chromatin (Ohtsubo et al., 1989). It is present in Xenopus egg extracts in amounts...