Facilitated Nucleocytoplasmic Shuttling of the Ran Binding Protein RanBP1

Abstract

The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B, and collapse of the nucleocytoplasmic Ran:GTP gradient leads to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)-RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin ␤. These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.The defining feature of eukaryotic cells is the compartmentalization of DNA replication and transcription within the nucleus. Access to the nuclear compartment is provided by pores that are plugged through the double membrane of the nuclear envelope (for reviews, see references 15 and 66). A multitude of soluble transport receptors and accessory proteins controls the transit of protein and nucleic acid cargo through these pores (for recent reviews, see references 24, 40, 42, and 64). The vectoriality of cargo transport is in many cases dependent on the asymmetric distribution of Ran, a small GTP binding protein, and of its regulatory factors (8,17). Ran is predominantly nuclear and is believed to be maintained in the GTPbound state (Ran:GTP) by a guanine nucleotide exchange factor, RCC1, which is associated with chromatin in the nucleus (7, 58). A Ran-specific GTPase-activating protein, Ran-GAP, which converts Ran to the GDP-bound state, is excluded from the nucleus (5,13,30). This asymmetric arrangement of regulatory factors ensures that a steep Ran:GTP gradient exists across the nuclear pores, and the collapse of this gradient inhibits many forms of nuclear traffic (32, 52).

Importin-β Is a GDP-to-GTP Exchange Factor of Ran

“…The requirement for RanBP1 in nuclear import has been demonstrated by many researchers; however, its role in this part of the pathway has remained obscure (15,20,34,35,39,40). In our model the biochemical function of RanBP1 in the nucleus is to co-activate the GDP exchange by importin-␤ in the import complex.…”

“…Subsequent to Ran-GTP formation, importin-␣ and NLS would dissociate from the complex, giving rise to a RanGTP⅐RanBP1⅐importin-␤ complex. Such a complex has been detected in vivo in the nucleus (40).…”

“…However, this mechanism alone does not explain the involvement of Ran-GDP, RanBP1, and NTF2 in the docking and translocation events of nuclear import (15)(16)(17)(18)(19)(20)(21)(33)(34)(35)(36)(37)(38)(39)(40). Here, we describe in vitro interactions between the yeast counterparts of these proteins, Kap95p, Kap60p, Gsp1p, Yrb1p, and Ntf2p, and the results suggest the possibility that in addition to the RCC1-dependent pathway, nuclear import could also be facilitated in at least some circumstances by Kap95p (importin-␤)-mediated nucleotide exchange.…”

RanBP1 downregulation sensitizes cancer cells to taxol in a caspase-3-dependent manner

“…RanBP1 regulates nucleotide turnover on Ran by stimulating the hydrolysis-activating protein RanGAP1, thereby facilitating RanGDP formation, and inhibiting the guanine exchange factor RCC1, thus preventing GTP exchange on Ran: both activities converge in reducing the concentration of available RanGTP (Bischoff et al, 1995). RanBP1 also exerts an important function in modulating the interaction of RanGTP with its effectors (Plafker and Macara, 2000;Goerlich et al, 2003).…”

Study of the SAGA deubiquitination module: identification of new modulators and its implication on Spinocerebellar Ataxia Type 7

“…Thus, the nuclear guanine nuclear exchange factor (GEF) named as Rcc1 in vertebrates (Prp20 in S. cerevisiae) catalyses GDP release and GTP-binding (Bischoff and Ponstingl, 1991). In contrast, the conversion of RanGTP to RanGDP occurs in the cytoplasm and it is mediated by a Ran GTPaseactivating protein (Ran GAP) known as Rna1 in S. cerevisiae and its coactivators Ran binding proteins such as Yrb1 which shuttles rapidly between the nucleus and cytoplasm although at steady-state it is localized in the cytoplasm (Künzler and Hurt, 2001;Plafker and Macara, 2000). The transport of RanGDP to the nucleus is mediated by the transport factor NTF2 .…”