The Ran/TC4 GTPase-binding domain: identification by expression cloning and characterization of a conserved sequence motif.

Ran/TC4 is an essential, nuclear GTPase implicated in the initiation of DNA replication, entry into and exit from mitosis, and in nuclear RNA and protein transport through the nuclear pore complex. This diversity of functions suggests that Ran interacts with a large number of down-stream targets. Using an overlay assay, we detected a family of putative target proteins that associate with GTP-bound Ran. The sequence of only one such protein, HTF9a/RanBP1, is known. We have now cloned two additional Ran-binding proteins, allowing identification of a distinctive, highly conserved sequence motif of approximately 150 residues. This motif represents a minimal Ran-binding domain that stabilizes the GTP-bound state of Ran. The isolated domain also functions as a coactivator of Ran-GTPase-activating protein. Mutation of a conserved residue within the Ran-binding domain of HTF9a protein drastically reduced Ran binding. Ran-binding proteins coimmunoprecipitated with epitope-tagged Ran from cell lysates, suggesting that these proteins may associate in vivo. A previously uncharacterized Caenorhabditis elegans gene could encode a protein (96 kDa) possessing two Ran-binding domains. This open reading frame also contains similarities to nucleoporins, suggesting a functional link between Ran and nuclear pore complexes.

Isolated Mammalian and Schizosaccharomyces pombe Ran-binding Domains Rescue S. pombe sbp1 (RanBP1) Genomic Mutants

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein–mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1− yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1− yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin β (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

A protein required for nuclear-protein import, Mog1p, directly interacts with GTP–Gsp1p, the Saccharomyces cerevisiae Ran homologue

We previously isolated 25 temperature-sensitive gsp1 alleles of Saccharomyces cerevisiae Ran homologue, each of which possesses amino acid changes that differ from each other. We report here isolation of three multicopy suppressors—PDE2, NTF2, and a gene designated MOG1—all of which rescued a growth defect of these gsp1 strains. The gsp1 suppression occurred even in the absence of GSP2, another S. cerevisiae GSP1-like gene. Previously, NTF2 was reported to suppress gsp1 but not PDE2. Mog1p, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W. Both MOG1 and NTF2 suppressed a series of gsp1 alleles with similar efficiency, and both suppressed gsp1 even with a single gene dose. Consistent with the high efficiency of gsp1 suppression, Mog1p directly bound to GTP, but not to GDP-Gsp1p. The disruption of MOG1 made yeast temperature-sensitive for growth. Δmog1, which was suppressed by overexpression of NTF2, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export. Thus, Mog1p, which was localized in the nucleus, is a Gsp1p-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p. Furthermore, the finding that PDE2 suppressed both gsp1 and rna1–1 indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway.

Transforming Growth Factor-β Induces Nuclear Import of Smad3 in an Importin-β1 and Ran-dependent Manner

Smad proteins are cytoplasmic signaling effectors of transforming growth factor-β (TGF-β) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-β type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. By focusing on the R-Smad Smad3 we demonstrate that 1) only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. 2) Smad3, via its N-terminal domain, interacts specifically with importin-β1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-β1. 3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-β1. 4) Importin-β1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-β receptor leads to enhanced interaction with importin-β1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains.

A family of Ran binding proteins that includes nucleoporins.

Ran, a small nuclear GTP binding protein, is essential for the translocation of nuclear proteins through the nuclear pore complex. We show that several proteins, including the Saccharomyces cerevisiae Nup2p and Caenorhabditis elegans F59A2.1 nucleoporins, contain domains similar to the previously characterized murine Ran binding protein (RBP, termed RBP1). To test the significance of this similarity, we have used the corresponding domains of Nup2p and a putative S. cerevisiae RBP in Ran binding assays and the yeast two-hybrid system. Both proteins bind S. cerevisiae Ran, but only the putative S. cerevisiae RBP binds human Ran. Two-hybrid analysis revealed Ran-Ran interactions and that yeast and human Rans can interact. These data identify Nup2p as a target for Ran in the nuclear pore complex, suggesting a direct role for it in nuclear-cytoplasmic transport. We discuss the possibility that proteins harboring Ran binding domains link the Ran GTPase cycle to specific functions in the nucleus.

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10–20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

Protein export from the nucleus requires the GTPase Ran and GTP hydrolysis.

Nuclei of digitonin-permeabilized cells that had been preloaded with a model transport substrate in a cytosol-dependent import reaction were subsequently incubated to investigate which conditions would result in export of transport substrate. We found that up to 80% of the imported substrate was exported when recombinant human Ran and GTP were present in the export reaction. Ran-mediated export was inhibited by nonhydrolyzable GTP analogs and also by wheat germ agglutinin but was unaffected by a nonhydrolyzable ATP analog. Moreover, a recombinant human Ran mutant that was deficient in its GTPase activity inhibited export. These data indicate that export of proteins from the nucleus requires Ran and GTP hydrolysis but not ATP hydrolysis. We also found that digitonin-permeabilized cells were depleted of their endogenous nuclear Ran, thus allowing detection of Ran as a limiting factor for export. In contrast, most endogenous karyopherin alpha was retained in nuclei of digitonin-permeabilized cells. Unexpectedly, exogenously added, fluorescently labeled Ran, although it accessed the nuclear interior, was found to dock at the nuclear rim in a punctate pattern, suggesting the existence of Ran-binding sites at the nuclear pore complex.

The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin β-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.

Nucleus-associated pools of Rna1p, the Saccharomyces cerevisiae Ran/TC4 GTPse activating protein involved in nucleus/cytosol transit.

Rna1p is the GTPase activating enzyme for Ran/TC4, a Ras-like GTPase necessary for nuclear/cytosolic exchange. Although most wild-type Rna1p is located in the cytosol, we found that the vast majority of the mutant Rna1-1p and, under appropriate physiological conditions, a small portion of the wild-type Rna1p cofractionate with yeast nuclei. Subnuclear fractionation studies show that most of the Rna1p is tightly associated with nuclear components, and that a portion of the active protein can be solubilized by treatments that fail to solubilize inactive Rna1-1p. To learn the precise nuclear locations of the Rna1 proteins, we studied their subcellular distributions in HeLa cells. By indirect immuno-fluorescence we show that wild-type Rna1p has three subcellular locations. The majority of the protein is distributed throughout the cytosol, but a portion of the protein is nucleus-associated, located at both the cytosolic surface and within the nucleoplasm. Mutant Rna1-1p is found at the outer nuclear surface and in the cytosol. We propose that a small pool of the wild-type Rna1p is located in the nuclear interior, supporting the model that the same components of the Ran/TC4 GTPase cycle exist on both sides of the nuclear membrane.

Nucleotide-specific interaction of Ran/TC4 with nuclear transport factors NTF2 and p97.

The use of permeabilized cell models to study nuclear protein import has led to the identification of cytosolic components of the import machinery, including the NLS receptor, p97, Ran/TC4, and nuclear transport factor 2 (NTF2). These proteins are required to reconstitute docking of transport ligand at the nuclear pore complex and subsequent translocation through the nuclear pore. However, a detailed molecular understanding of how these factors mediate protein import is lacking. Here we describe the results of solution and solid phase binding assays, which demonstrate that the small GTPase Ran/TC4 interacts directly with the cytosolic transport factors p97 and NTF2. By preloading recombinant Ran/TC4 with [gamma-32P]GTP or [3H]GDP, we show that the interactions with p97 and NTF2 are specific for the GTP- and GDP-bound forms, respectively. These data together with previous studies lead us to suggest that the interaction of the GTP-bound form of Ran/TC4 with p97 is linked to an early step in the nuclear protein import pathway and that the association of the GDP-bound form of Ran/TC4 with NTF2 helps define vectorial transport.

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

The alpha subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of karyopherin beta's binding sites both for Ran-GTP and for karyopherin alpha. We discovered that karyopherin beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and karyopherin alpha bind to overlapping sites on karyopherin beta. Having a higher affinity to the overlapping site, Ran-GTP displaces karyopherin alpha and binds to karyopherin beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for karyopherin beta to a cluster of basic residues analogous to those previously shown to constitute karyopherin alpha's binding site to karyopherin beta.

A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export

Yeast cells mutated in YRB2, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus. Unlike Xpo1p/Crm1p/exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus. However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin/karyopherin β superfamily. Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export. Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.

RanGTP-mediated nuclear export of karyopherin α involves its interaction with the nucleoporin Nup153

Using binding assays, we discovered an interaction between karyopherin α2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of karyopherin β1, termed β1*, that contains a determinant for binding to the peptide repeat containing nucleoporin Nup98. In an in vitro assay in which export of endogenous nuclear karyopherin α from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-karyopherin α antibodies, we found that karyopherin α export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin. RanGTP-mediated export of karyopherin α was inhibited by peptides representing the interacting domains of Nup153 and karyopherin α2, indicating that the binding reactions detected in vitro are physiologically relevant and verifying our mapping data. Moreover, β1*, although it inhibited import, did not inhibit export of karyopherin α. Hence, karyopherin α import into and export from nuclei are asymmetric processes.

Complementation of sporulation and motility defects in a prokaryote by a eukaryotic GTPase

The complex prokaryote, Myxococcus xanthus, undergoes a program of multicellular development when starved for nutrients, culminating in sporulation. M. xanthus makes MglA, a 22-kDa, soluble protein that is required for both multicellular development and gliding motility. MglA is similar in sequence to the Saccharomyces cerevisiae SAR1 protein, a member of the Ras/Rab/Rho superfamily of small eukaryotic GTPases. The SAR1 gene, when integrated into the M. xanthus genome, complements the sporulation defect of a ΔmglA strain. A forward, second-site mutation on the M. xanthus chromosome, rpm, in combination with SAR1, restores fruiting body morphogenesis and gliding motility to a ΔmglA strain. The result that the rpm mutation suppresses the substitution of SAR1 for mglA suggests that Sar1p interacts with other M. xanthus proteins to control the motility-dependent aggregation of cells during development.