Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Ran

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_641

Synonyms

 Gsp1;  Spi1;  TC4

Historical Background

Ran is an abundant member of the Ras superfamily of small GTPases that is highly conserved across eukaryotes. Originally cloned from a human teratocarcinoma cell line as one of four novel genes with sequence homology to the GTP-binding domain of Ras (Drivas et al. 1990), the gene was initially named TC4 (teratocarcinoma clone 4) and found to encode a protein of 216 amino acid residues. TC4 was markedly different from other members of the Ras superfamily in two respects: it lacked the sites required for post-translational lipid modification and it was primarily localized to the nucleus. Accordingly, it was renamed Ran for Ras-related nuclear protein. Ran was subsequently purified as an essential cofactor for nuclear protein import (Moore and Blobel 1993) and over the following years was extensively characterized for its role in regulating nucleocytoplasmic transport. Ran was later discovered to be critical for mitotic spindle assembly (Carazo-Salas et al. 1999; Ohba et al. 1999) and for the post-mitotic assembly of the nuclear envelope (Hetzer et al. 2000; Zhang and Clarke 2000) and of nuclear pore complexes (Walther et al. 2003). Ran has more recently been implicated in diverse processes, including centrosome duplication (Budhu and Wang 2005), apoptosis (Wong et al. 2009), injury response signaling in neurons (Yudin and Fainzilber 2009), and ciliary trafficking (Dishinger et al. 2010).

Regulation of Guanine Nucleotide State and Subcellular RanGTP Distribution

Like other GTPases, Ran undergoes cycles of GTP exchange and subsequent hydrolysis to GDP. The rates of nucleotide exchange and hydrolysis by Ran are intrinsically low, and in vivo these reactions require accessory factors to proceed at physiological rates. GTP hydrolysis is stimulated by a GTPase-activating protein, RanGAP, and further enhanced by the RanGTP-binding proteins RanBP1 and RanBP2, whereas the replacement of GDP with GTP is accelerated by a guanine nucleotide exchange factor, RanGEF, which is called Regulator of Chromosome Condensation 1 (RCC1) in vertebrates (Binding partners of Ran are summarized in Table 1). These proteins act together to define an enzymatic cycle whereby Ran hydrolyzes GTP to GDP, releases the GDP, and accepts a new molecule of GTP (Fig. 1). Importantly, the accessory factors that modulate this cycle are distributed asymmetrically in the cell. RanGEF has a high affinity for chromatin and is restricted to the nucleus during interphase, whereas RanGAP, RanBP1, and RanBP2 localize to the cytosol or to the cytosolic face of the nuclear envelope. This gives rise to an asymmetric distribution of Ran, with the GTP- and GDP-bound forms prevailing in the nucleus and cytosol, respectively.
Ran, Table 1

Ran point mutants and binding partners

Protein

Function

Ran G19 V, L43E, Q69L

Ran point mutants locked in the GTP-bound form due to lack of GTPase activity

Ran T24 N

Ran point mutant that binds to RanGEF and inhibits its exchange activity, thereby preventing RanGTP generation

RanGAP

GTPase-activating protein for Ran. Localizes to the cytoplasm and nuclear pores during interphase and to mitotic spindle during mitosis

RanGEF/RCC1

Guanine nucleotide exchange factor for Ran. Associates with chromatin throughout the cell cycle

RanBP1

RanGTP-binding protein that localizes to the cytoplasm of nondividing cells. Acts as a cofactor for RanGAP, enhancing the rate of GTP hydrolysis on Ran

RanBP2/Nup358

Nucleoporin that localizes to the cytosolic face of the nuclear pore complex (NPC). Acts as a cofactor for RanGAP, enhancing the rate of GTP hydrolysis on Ran

RanBP3

Cofactor for CRM1-mediated nuclear export

RanBPM/RanBP9

Centrosomal protein that interacts with the GTP-bound form of Ran and is required for correct nucleation of microtubules

RanBP10

Tubulin-binding protein and cytoplasmic guanine nucleotide exchange factor for Ran

NTF2/p10

Import carrier for RanGDP

Mog1

Stimulates release of GTP from Ran. In combination with RanBP1 promotes GDP release and the selective binding of GTP to Ran

Dis3

Exoribonuclease subunit of the RNA-processing exosome complex that enhances the nucleotide-releasing activity of RCC1

Karyopherin-ß family members

Importin ß/Importin ß1

Associates with Importin a to mediate the nuclear import of proteins with a basic nuclear localization signal (NLS). Associates with Snurportin1 to mediate the import of UsnRNPs

Transportin 1/Importin ß2

Nuclear import receptor for diverse RNA-binding proteins

Transports the Kif17 motor protein and retinitis pigmentosa 2 protein to the primary cilium

Transportin SR

Nuclear import receptor for serine/arginine-rich (SR) proteins

Transportin SR2/Transportin 3

Nuclear import receptor for SR proteins, stem-loop binding protein (SLBP), HIV integrase

Importin 4

Nuclear import receptor for histones, ribosomal proteins, vitamin D receptor, transition protein 2

Importin 5/Importin ß3

Nuclear import receptor for histones, ribosomal proteins, recombinase protein RAG-2

Importin 7

Nuclear import receptor for ribosomal proteins, glucocorticoid receptor, HIV reverse transcriptase complex. Heterodimerizes with importin beta to import histone H1

Importin 8

Nuclear import receptor for Smad4 and signal recognition particle protein 19 (SRP19)

Importin 9

Nuclear import receptor for core histones and ribosomal proteins

Importin 11

Nuclear import receptor for ribosomal protein L12 and for the class III ubiquitin conjugating enzymes UbcM2, UbcH6, and UBE2E2

Importin 13

Nuclear import receptor for Ubc9, Rbm8, Mago-Y14, Pax6

Export receptor for translation initiation factor eIF1A

CAS

Export receptor for Importin a

CRM1/Exportin1

Export receptor for proteins bearing a leucine-rich nuclear export signal (NES). Cooperates with Ran to recruit nucleophosmin to centrosomes

Exportin-t/Xpot

Export receptor for tRNAs

Exportin 4

Export receptor for eukaryotic translation initiation factor 5A (eIF5A) and Smad3. Import receptor for transcription factors Sox2 and SRY

Exportin 5

Export receptor for microRNA precursors and 60S ribosomal subunit

Exportin 6

Export receptor for profilin/actin complexes

Exportin 7

Export receptor for p50RhoGAP, 14-3-3s

Ran, Fig. 1

Regulation of Ran’s guanine nucleotide state. The guanine nucleotide exchange factor RanGEF localizes to the nucleus, while the GTPase-activating protein RanGAP and the RanGTP-binding protein RanBP1 localize to the cytosol. This gives rise to an asymmetric subcellular distribution of RanGTP, which is at high concentration in the nucleus and at low concentration in the cytosol. NPC nuclear pore complex

The function of Ran depends on its conformation, which in turn is determined by the state of the guanine nucleotide bound to it (Fig. 2). As with other GTPases, the conformational changes in Ran involve two regions, called switch I and II, that are sensitive to the presence of the γ-phosphate of GTP (Wittinghofer and Vetter 2011). In addition, Ran possesses a 40-residue C-terminal extension which also displays a nucleotide-dependent conformation. In the GDP-bound state, the C-terminal extension folds intimately against the core domain of Ran; in the GTP-bound state, this region detaches from the core and becomes highly solvent accessible. Thus, the GDP- and GTP-bound conformations of Ran are strikingly different, allowing Ran-interacting factors to discriminate between RanGDP and RanGTP with high selectivity. Point mutations in Ran that either block GTPase activity or prevent RanGTP generation by inhibiting RanGEF-mediated nucleotide exchange have been instrumental in elucidating the role of Ran in diverse cellular pathways. These mutations are listed in Table 1.
Ran, Fig. 2

Structure of Ran in the GDP- and GTP-bound states. The conformations of the switch I and II regions and of the C-terminal extension depend on the bound nucleotide. GDP and GTP are shown as stick models, with the a, ß, and? phosphate groups labeled. Structures shown are those of pdb entries 1BYU (RanGDP) and 1IBR (RanGTP)

Role in Nucleocytoplasmic Transport

Ran regulates nuclear transport during interphase by acting as a molecular switch for the karyopherin-ß family of nuclear transport receptors, also known as importins and exportins (Fried and Kutay 2003). Karyopherins are responsible for delivering various classes of macromolecular cargo through the nuclear pore complex (NPC). Representative members include Importin ß, which cooperates with the adaptor protein Importin α to deliver proteins bearing a basic nuclear localization signal (NLS) from the cytosol to the nucleus; Transportin 1, which mediates the nuclear import of ribosomal proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs); CRM1/Exportin 1, which mediates the nuclear export of proteins bearing a leucine-rich nuclear export signal (NES); and Xpot, which exports tRNA. Additional family members are listed in Table 1.

Crystal structures determined for a number of transport receptors, both in isolation and in complex with binding partners, have greatly elucidated how these proteins function (Cook and Conti 2010; Stewart 2007). Karyopherins are superhelical structures made of tandem HEAT repeats that interact with the GTP-bound form of Ran. RanGTP binds to the N-terminal half of these proteins, while the cargo generally binds to the C-terminal half (Fig. 3a). RanGTP and cargo bind to importins in a mutually exclusive manner, whereas they bind to exportins cooperatively. This difference in binding mode, combined with the asymmetric distribution of RanGTP across the nuclear envelope, ensures the directionality of nuclear transport. Thus, importins bind their cargo in the cytosol, translocate through the NPC, and release the cargo in the nucleus upon encountering RanGTP (Fig. 3b). In contrast, exportins associate with their cargo in the nucleus together with RanGTP, forming a ternary complex that traverses the NPC and subsequently dissociates in the cytosol. In the cytosol, the binding of RanBP1 releases RanGTP from importins and exportins, and rebinding is prevented by RanGAP-mediated hydrolysis of Ran to the GDP-bound state. Nuclear transport factor 2 (NTF2) recycles RanGDP to the nucleus, where RanGEF mediates conversion to the GTP-bound form. RanGTP, thus, acts as a positional cue that defines the nuclear compartment and directs the disassembly and assembly of import and export complexes, respectively.
Ran, Fig. 3

Role of Ran in nucleocytoplasmic transport. (a) Mode of interaction with importins and exportins. Whereas RanGTP and cargo associate with importins in a mutually exclusive manner, they associate with exportins cooperatively. (b) Generic import (left) and export (right) pathways mediated by karyopherin ß members. Nucleotide exchange in the nucleus is mediated by RanGEF/RCC1, while GTP hydrolysis in the cytosol is promoted by RanGAP and RanBP1 or RanBP2

Role in Mitotic Spindle Organization

In addition to regulating nucleocytoplasmic transport during interphase, Ran plays an important role during mitosis, regulating several aspects of mitotic spindle assembly (Clarke and Zhang 2008; Gruss and Vernos 2004). These include microtubule nucleation, microtubule stability, production of antiparallel microtubule arrays, and the focusing of spindle poles. Many of these functions for Ran were discovered and characterized in studies using Xenopus laevis egg extracts. The importance of Ran during mitosis has been confirmed in mammalian somatic cells, where, for example, Ran regulates microtubule attachment to kinetochores, is required for microtubule nucleation by both centrosomes and kinetochores, and regulates centrosome cohesion during spindle pole formation (Arnaoutov and Dasso 2005; Roscioli et al. 2010).

The molecular mechanism by which RanGTP affects mitotic spindle assembly is closely related to that by which it regulates nucleocytoplasmic transport. After nuclear envelope breakdown at the onset of mitosis, RanGTP is concentrated near chromosomes by chromatin-bound RanGEF, while RanGAP and RanBP1 promote GTP hydrolysis by Ran distal to chromatin. This gives rise to a concentration gradient of RanGTP that is centered on chromosomes (Fig. 4). RanGTP influences the organization of the mitotic apparatus by activating spindle assembly factors (SAFs), which mediate microtubule stabilization and spindle assembly. One example of a RanGTP-activated SAF is TPX2 (Targeting Protein for Xklp2). TPX2 promotes spindle formation by targeting the kinesin-like motor protein Xklp2 to microtubule minus ends and by activating Aurora A kinase, which is involved in centrosome function. Another SAF activated by RanGTP is NuMA (Nuclear Mitotic Apparatus protein). NuMA associates with the motor protein dynein and its motility-activating complex dynactin, and translocates along microtubules to the spindle poles where it organizes and tethers microtubules to spindle poles. TPX2 and NuMA are inhibited by the Importin α/ß heterodimer, which bind to the NLS motifs of these SAFs. Near chromatin, RanGTP binds and displaces Importin ß, releasing and activating the SAFs. RanGTP, thus, acts as a positional marker that ensures the correct spatial regulation of spindle assembly in the vicinity of chromosomes.
Ran, Fig. 4

Role of Ran in mitotic spindle assembly. The binding of RanGTP to Importin ß in the vicinity of chromosomes causes the release of spindle assembly factors such as TPX2 and NuMA. The gradient of RanGTP established by chromatin-bound RanGEF is indicated by the magenta shading. SAF spindle assembly factor, Impa importin a, Impß importin ß

Role in Nuclear Envelope Assembly

In higher eukaryotes, the nuclear envelope is reconstituted around the segregated DNA at the end of mitosis. This process occurs in three steps: first, membrane vesicles are recruited to the vicinity of chromatin; next, these vesicles fuse into a continuous nuclear membrane; and finally, nucleoporins assemble to form NPCs that insert into the nuclear envelope. Although the mechanisms underlying these events are not fully understood, Ran clearly plays an important role. RanGTP stimulates membrane fusion and nuclear pore assembly, while Importin ß negatively regulates these events. More specifically, artificial beads coated with Ran and added to X. laevis egg extracts or other cell-free systems accumulate membrane vesicles that fuse into a continuous lipid layer, incorporate nucleoporins, and form NPCs in the absence of chromatin (Zhang and Clarke 2000). Both the generation of RanGTP by RanGEF/RCC1 and GTP hydrolysis by Ran are required for membrane fusion to occur (Hetzer et al. 2000). The generation of RanGTP is also required to release specific nucleoporins from Importin ß, to target these proteins to chromatin and to allow the association of NPC subcomplexes (Walther et al. 2003).

Additional Functions of Ran

  1. 1.

    Centrosome duplication. Centrosomes, the major microtubule organizing center of mammalian cells, are duplicated once and only once during the G1/S transition of the cell cycle. Ran and the nuclear export receptor CRM1 help orchestrate this event through their effect on nucleophosmin (NPM), which has been implicated as a licensing factor that regulates centrosome synthesis (Budhu and Wang 2005). A fraction of Ran and CRM1 localizes to centrosomes, where they recruit NPM through the latter’s leucine-rich NES motif to form a centrosomal CRM1/Ran/NPM complex. Mutation of the NES motif or inactivation of CRM1 leads to dissociation of NPM from centrosomes and to the premature initiation of centrosome duplication. Viral oncoproteins that cause abnormal centrosome duplication (e.g., adenovirus E1A and human papillomavirus E7 proteins) also interact physically with Ran and disrupt its centrosomal regulatory functions (Lavia et al. 2003).

     
  2. 2.

    Apoptosis. Apoptosis triggered by DNA damage leads to the redistribution of Ran from the nucleus to the cytosol and to an overall reduction in RanGTP levels (Wong et al. 2009). This redistribution has been linked to the action of Mst1, a kinase localized primarily to the cytosol but which accumulates in the nucleus during apoptosis following caspase-mediated cleavage of its nuclear export signal. Nuclear Mst1 phosphorylates serine residue S14 on histone H2B. This leads to RanGEF becoming more tightly bound to chromosomes and to inhibition of its guanine nucleotide exchange activity toward Ran. The resulting dissipation of nuclear RanGTP blocks entry into the nucleus of  NF-κB, a transcription factor with an important role in rescuing cells from apoptosis. Thus, dissipation of the RanGTP gradient due to histone phosphorylation prevents the initiation of an anti-apoptotic program.

     
  3. 3.

    Ciliary trafficking. The primary cilium is a microtubule-based organelle that projects from the cell surface and transduces environmental stimuli into intracellular signals. Entry of proteins into cilia appears to be regulated at the base of the cilia at a region known as the transition zone, where a structure analogous to the NPC has been proposed to exist. Studies of the microtubule motor Kif17 revealed a role for Ran in regulating protein entry into primary cilia which is strikingly similar to Ran’s role in nucleocytoplasmic transport (Dishinger et al. 2010). Targeting of Kif17 to primary cilia depends on a short carboxy-terminal sequence that contains several basic residues and shares similarities with NLSs. This “ciliary localization signal” (CLS) mediates binding to the nuclear import receptor Transportin 1. The Kif17/Transportin 1 complex is then targeted to the intraciliary compartment, where high RanGTP concentrations release the motor protein from the importin. Ciliary import of Kif17 thus mirrors the nuclear import of an importin/cargo complex and its dissociation by nuclear RanGTP.

     
  4. 4.

    Neuronal processes. Ran appears to play an important role in neuron development, as RNAi knockdown of Ran revealed defects in neuron development in both drosophila and mouse neurons, while the Ran-binding protein RanBPM has been implicated in cytoplasmic signaling in neuronal processes and in the regulation of neuronal outgrowth (Yudin and Fainzilber 2009). A role for Ran has also been identified in the regulation of retrograde injury signaling in peripheral sensory neurons. In axons, RanGTP forms part of a multimeric complex that includes the nuclear export receptor CAS, Importin α, and the microtubule-associated motor protein dynein. Nerve injury causes an increase in the cytoplasmic levels of RanBP1, RanGAP, and Importin ß. RanBP1 and RanGAP induce the release of RanGTP and CAS from the Importin α/dynein complex and promote the hydrolysis of RanGTP to RanGDP. This allows the newly translated Importin ß to associate with Importin α/dynein, thereby creating a retrograde injury-signaling complex capable of binding (via Importin a) NLS-bearing signaling cargos. As with the ciliary trafficking example above, these findings show that Ran can act as a regulator of importin-dependent transport and signaling at sites that are distant from the nucleus.

     

Summary

Ran regulates several fundamental processes throughout the cell cycle, including nucleocytoplasmic transport during interphase, the organization of the mitotic apparatus after nuclear envelope breakdown, the reassembly of the nuclear envelope after mitosis, and duplication of the centrosome. Ran also plays a role in more specialized processes, such as ciliary trafficking, the apoptotic response to a variety of conditions, and neuronal development and injury signaling. In many of these processes, Ran functions as a spatial marker describing where the chromatin or nucleus is. A commonly observed feature is the role played by transport receptors of the karyopherin ß family, which sequester different activators and inhibit their function until relieved by RanGTP. A challenge for the future will be to understand these Ran-regulated processes in detail, defining all the players involved and the molecular mechanisms by which these events are precisely orchestrated during the cell cycle.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Institut de Biologie Structurale Jean-Pierre Ebel, UMR 5075 (CEA/CNRS/Université Joseph Fourier)GrenobleFrance