Ran-binding Domain Research Articles

Interconversion of red opsin isoforms by the cyclophilin-related chaperone protein Ran-binding protein 2.

Ran-binding protein 2 (RanBP2) (type II) is a retinal cyclophilin-related protein that binds Ran-GTPase. Type I cyclophilin is a shorter, alternatively spliced isoform of RanBP2. Recently, we showed that the Ran-binding domain 4 (RBD4)/cyclophilin (CY) supradomain of RanBP2 acts both in vitro and in vivo as a specific chaperone for bovine red/green opsin (R/G opsin). R/G opsin undergoes a stable modification of its electrophoretic mobility upon binding to RanBP2. This modification is likely due to cis-trans isomerization of one or more proline residues in the opsin protein. Here, we show that expression of human red opsin in Escherichia coli and COS cells results in the production of still a third electrophoretic variant of this protein. This variant was converted to the RBD4 binding-competent form of opsin through direct interaction with RBD4/CY, both in vivo and in vitro. We suggest that these distinct opsin species may represent kinetically or thermodynamically trapped prolyl conformers that can be interconverted by concerted action of the RBD4 and CY domains of RanBP2. We also show that the C-terminal half of RBD4 is the binding domain for bovine R/G opsin and that coexpression of human red opsin with type I cyclophilin in vivo enhances the production of functional visual pigment. These observations imply that prolyl isomerization may have importance beyond its role in protein folding, possibly as a molecular switch modulated by cyclophilin for the loading of opsin onto RanBP2 during visual pigment processing in cones.

Mutations within the Ran/TC4 GTPase: EFFECTS ON REGULATORY FACTOR INTERACTIONS AND SUBCELLULAR LOCALIZATION

Ran, a member of the Ras superfamily of GTPases, is predominantly localized in the nucleus and is a necessary component in the active transport of proteins through nuclear pores. Disruption of Ran function affects the regulation of mitosis, DNA synthesis, and RNA processing and export. To explore the mechanisms of Ran function, mutants of the Ran GTPase were characterized, several of which are capable of dominantly interfering with nuclear protein import. Unlike wild-type Ran, the putative gain-of-function mutant (G19V Ran) was not sensitive to the exchange factor, RCC1. In addition the G19V Ran and effector domain mutants (L43E and E46G Ran) were not sensitive to the GTPase-activating protein, Fug1. Epitope-tagged G19V Ran and L43E Ran isolated from transfected BHK21 cells were each about 50% GTP-bound, whereas the wild-type and a C-terminal deletion mutant (Delta-DE Ran) were primarily bound to GDP. While G19V Ran interacted with known Ran-binding proteins and with an isolated Ran-binding domain, the T24N Ran did not, and binding by L43E Ran was substantially reduced. Wild-type HA1-tagged Ran expressed in BHK21 cells was nuclear, whereas the G19V, T24N, L43E, and E46G forms of Ran were predominantly localized at the nuclear envelope, and Delta-DE Ran was primarily cytosolic. Similar results were observed when permeabilized BHK21 cells were incubated with extracts of COS cells expressing the mutants. Thus mutations that affect the interaction of Ran with regulatory proteins and effectors can disrupt the normal subcellular localization of Ran, lending support for the current model of Ran-mediated nuclear import.

Cyclophilin-related protein RanBP2 acts as chaperone for red/green opsin.

Cyclophilins are ubiquitous and abundant proteins that exhibit peptidyl prolyl cis-trans isomerization (PPlase) activity in vitro. Their functions in vivo, however, are not well understood. Two new retinal cyclophilin isoforms, types I and II, are highly expressed in cone photoreceptors of the vertebrate retina. Type-II cyclophilin is identical to RanBP2, a large protein that binds the GTPase Ran. Here we report that two contiguous domains in RanBP2, Ran-binding domain 4 (RBD4) and cyclophilin, act in concert as a chaperone for the opsin molecule of the red/green-sensitive visual pigment of a dichromatic vertebrate. In Drosophila, the cyclophilin NinaA is expressed in all photoreceptors and is required for the expression of only a subset of opsins. The molecular basis of these photoreceptor class-specific effects and the functions of NinaA and other cyclophilins in vivo remain unclear. Unlike NinaA, which forms a stable complex with opsin from retinular cells R1-6, we find that the cyclophilin domain of RanBP2 does not bind opsin directly; rather, it augments and stabilizes the interaction between red/green (R/G) opsin and the RBD4 domain. This involves a cyclophilin-mediated modification of R/G opsin, possibly involving proline isomerization. The RBD4-cyclophilin supradomain of RanBP2, therefore, is a form of vertebrate chaperone of defined substrate specificity, which may be involved in the processing and/or transport of long-wavelength opsin in cone photoreceptor cells.

A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1.

RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and beta-importin /beta-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways.

Ran Binding Domains Promote the Interaction of Ran with p97/β-Karyopherin, Linking the Docking and Translocation Steps of Nuclear Import

Nuclear protein import is accomplished by two sequential events; docking at the nuclear pore complex followed by ATP-dependent translocation across the nuclear envelope. Docking of nuclear targeted proteins requires a 56-kDa nuclear localization signal receptor (alpha-karyopherin, importin-alpha, SRP1 alpha) and a 97-kDa protein (beta-karyopherin, importin-beta). Components necessary for translocation include the Ran/TC4 GTPase and NTF2/B-2. The functions of these factors at a molecular level remain unclear. We have now found that a complex of Ran, in the GTP-bound state, with either the Ran binding protein, RanBP1, or an isolated Ran binding domain binds with high affinity and specificity to beta-karyopherin to form a ternary complex. We find that a C-terminal truncation mutant of Ran, delta-DE Ran, also binds to beta-karyopherin and that delta-DE Ran can associate with a cytosolic, multiprotein complex that contains beta-karyopherin and another delta-DE Ran binding protein of 115/120 kDa. These data suggest a physical link between docking and translocation mediated by a Ran GTPase-Ran binding protein complex.

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.

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.