Ran-GTP interacts strongly with importin-beta, and this interaction promotes the release of the importin-alpha-nuclear localization signal cargo from importin-beta. Ran-GDP also interacts with importin-beta, but this interaction is 4 orders of magnitude weaker than the Ran-GTP.importin-beta interaction. Here we use the yeast complement of nuclear import proteins to show that the interaction between Ran-GDP and importin-beta promotes the dissociation of GDP from Ran. The release of GDP from the Ran-GDP-importin-beta complex stabilizes the complex, which cannot be dissociated by importin-alpha. Although Ran has a higher affinity for GDP compared with GTP, Ran in complex with importin-beta has a higher affinity for GTP. This feature is responsible for the generation of Ran-GTP from Ran-GDP by importin-beta. Ran-binding protein-1 (RanBP1) activates this reaction by forming a trimeric complex with Ran-GDP and importin-beta. Importin-alpha inhibits the GDP exchange reaction by sequestering importin-beta, whereas RanBP1 restores the GDP nucleotide exchange by importin-beta by forming a tetrameric complex with importin-beta, Ran, and importin-alpha. The exchange is also inhibited by nuclear-transport factor-2 (NTF2). We suggest a mechanism for nuclear import, additional to the established RCC1 (Ran-guanine exchange factor)-dependent pathway that incorporates these results.
"This computational analysis suggests that RPAP2 may act as an importin-like protein during RNAP II nuclear import (Supplementary Figure S6). To this effect, stimulation of RPAP2–GPN1/RPAP4 binding by GDP is reminiscent of the case of other importins that form a complex with another GTPase, Ran, during import of various cargoes (36,42). We do not have evidence at this stage that the RPAPs participate in nuclear import of other proteins and may, therefore, be specific to RNAP subunits in mammals. "
[Show abstract][Hide abstract] ABSTRACT: The RNA polymerase II (RNAP II)-associated protein (RPAP) 2 has been discovered through its association with various subunits
of RNAP II in affinity purification coupled with mass spectrometry experiments. Here, we show that RPAP2 is a mainly cytoplasmic
protein that shuttles between the cytoplasm and the nucleus. RPAP2 shuttling is tightly coupled with nuclear import of RNAP
II, as RPAP2 silencing provokes abnormal accumulation of RNAP II in the cytoplasmic space. Most notably, RPAP4/GPN1 silencing
provokes the retention of RPAP2 in the nucleus. Our results support a model in which RPAP2 enters the nucleus in association
with RNAP II and returns to the cytoplasm in association with the GTPase GPN1/RPAP4. Although binding of RNAP II to RPAP2
is mediated by an N-terminal domain (amino acids 1–170) that contains a nuclear retention domain, and binding of RPAP4/GPN1
to RPAP2 occurs through a C-terminal domain (amino acids 156–612) that has a dominant cytoplasmic localization domain. In
conjunction with previously published data, our results have important implications, as they indicate that RPAP2 controls
gene expression by two distinct mechanisms, one that targets RNAP II activity during transcription and the other that controls
availability of RNAP II in the nucleus.
Nucleic Acids Research 05/2013; 41(14). DOI:10.1093/nar/gkt455 · 9.11 Impact Factor
"Similarly, direct interactions between GPCRs and the switch regions of Gα are problematic on stereochemical grounds . It is also noteworthy in this context that inportin-β, a protein involved in the transport of protein cargo into the nucleus, and also a presumptive structural analog of Ric-8A, has been shown to act as a GEF for Ran1•GDP . Crystallographic analysis demonstrates that importin-β induces conformational changes in the switch regions of Ran1•GDP . "
[Show abstract][Hide abstract] ABSTRACT: Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα•GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1•GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1•GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1•GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1•GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.
PLoS ONE 08/2011; 6(8):e23197. DOI:10.1371/journal.pone.0023197 · 3.23 Impact Factor
"Nuclear abnormalities produced by RanBP1 persistence are therefore unlikely to be solely caused through direct alterations in RanGTP levels. These observations draw attention to another established function of RanBP1, i.e. its ability to regulate RanGTP interactions with importin beta (Chi et al. 1996; Bischoff and Görlich 1997; Lounsbury and Macara 1997; Floer et al. 1997; Gorlich et al. 2003; Lonhienne et al. 2009). RanBP1 facilitates the dissociation of RanGTP/importin beta complexes ; physiologically, this is thought to provide a critical switch after termination of nuclear import, when RanGTP exits the nucleus with either importin beta or with importin alpha and CAS (the export factor for importin alpha). "
[Show abstract][Hide abstract] ABSTRACT: The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.
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