Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.

Small GTP-Binding Proteins

▪ Abstract The compartmentation of eukaryotic cells requires all nuclear proteins to be imported from the cytoplasm, whereas, for example, transfer RNAs, messenger RNAs, and ribosomes are made in the nucleus and need to be exported to the cytoplasm. Nuclear import and export proceed through nuclear pore complexes and can occur along a great number of distinct pathways, many of which are mediated by importin β-related nuclear transport receptors. These receptors shuttle between nucleus and cytoplasm, and they bind transport substrates either directly or via adapter molecules. They all cooperate with the RanGTPase system to regulate the interactions with their cargoes. Another focus of our review is nuclear export of messenger RNA, which apparently largely relies on export mediators distinct from importin β-related factors. We discuss mechanistic aspects and the energetics of transport receptor function and describe a number of pathways in detail.

Transport between the cell nucleus and the cytoplasm.

▪ Abstract Small ubiquitin-related modifier (SUMO) family proteins function by becoming covalently attached to other proteins as post-translational modifications. SUMO modifies many proteins that participate in diverse cellular processes, including transcriptional regulation, nuclear transport, maintenance of genome integrity, and signal transduction. Reversible attachment of SUMO is controlled by an enzyme pathway that is analogous to the ubiquitin pathway. The functional consequences of SUMO attachment vary greatly from substrate to substrate, and in many cases are not understood at the molecular level. Frequently SUMO alters interactions of substrates with other proteins or with DNA, but SUMO can also act by blocking ubiquitin attachment sites. An unusual feature of SUMO modification is that, for most substrates, only a small fraction of the substrate is sumoylated at any given time. This review discusses our current understanding of how SUMO conjugation is controlled, as well as the roles of SUMO in a...

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Protein Modification by SUMO

A Small Ubiquitin-Related Polypeptide Involved in Targeting RanGAP1 to Nuclear Pore Complex Protein RanBP2

Active transport between the nucleus and cytoplasm involves primarily three classes of macromolecules: substrates, adaptors, and receptors. Some transport substrates bind directly to an import or an export receptor while others require one or more adaptors to mediate formation of a receptor-substrate complex. Once assembled, these transport complexes are transferred in one direction across the nuclear envelope through aqueous channels that are part of the nuclear pore complexes (NPCs). Dissociation of the transport complex must then take place, and both adaptors and receptors must be recycled through the NPC to allow another round of transport to occur. Directionality of either import or export therefore depends on association between a substrate and its receptor on one side of the nuclear envelope and dissociation on the other. The Ran GTPase is critical in generating this asymmetry. Regulation of nucleocytoplasmic transport generally involves specific inhibition of the formation of a transport complex; however, more global forms of regulation also occur.

Nucleocytoplasmic Transport: The Soluble Phase

RAN/TC4 is a small nuclear G protein1 that forms a complex with the chromatin-bound guanine nucleotide release factor RCC1 (ref. 2). Loss of RCC1 causes defects in cell cycle progression3,4, RNA export5-7 and nuclear protein import8. Some of these can be suppressed by overexpression of Ran/TC4 (ref. 1), suggesting that Ran/TC4 functions downstream of RCC1. We have searched for proteins that bind Ran/TC4 by using a two-hybrid screen, and here we report the identification of RanBP2, a novel protein of 3,224 residues. This giant protein comprises an amino-terminal 700-residue leucine-rich region, four RanBPl-homologous (refs 9, 10) domains, eight zinc-finger motifs similar to those of NUP153 (refs 11, 12), and a carboxy terminus with high homology to cyclophilin13. The molecule contains the XFXFG pentapeptide motif characteristic of nuclear pore complex (NPC) proteins14, and immunolocalization suggests that RanBP2 is a constituent of the NPC. The fact that NLS-mediated nuclear import can be inhibited by an antibody directed against RanBP2 supports a functional role in protein import through the NPC.

A giant nucleopore protein that binds Ran/TC4.

The nucleotide exchange activity of RCC1, the only known nucleotide exchange factor for Ran, a Ras-like small guanosine triphosphatase, was required for microtubule aster formation with or without demembranated sperm in Xenopus egg extracts arrested in meiosis II. Consistently, in the RCC1-depleted egg extracts, Ran guanosine triphosphate (RanGTP), but not Ran guanosine diphosphate (RanGDP), induced self-organization of microtubule asters, and the process required the activity of dynein. Thus, Ran was shown to regulate formation of the microtubule network.

https://science.sciencemag.org/content/sci/284/5418/1356.full.pdf

Self-Organization of Microtubule Asters Induced in Xenopus Egg Extracts by GTP-Bound Ran

A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with γ-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to γ-tubulin was faded by the addition of GTPγS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

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When Overexpressed, a Novel Centrosomal Protein, RanBPM, Causes Ectopic Microtubule Nucleation Similar to γ-Tubulin

Dis3p, a subunit of the exosome, interacts directly with Ran. To clarify the relationship between the exosome and the RanGTPase cycle, a series of temperature-sensitive Saccharomyces cerevisiae dis3 mutants were isolated and their 5.8S rRNA processing was compared with processing in strains with mutations in a S. cerevisiae Ran homologue, Gsp1p. In both dis3 and gsp1 mutants, 3' processing of 7S-to-5.8S rRNA was blocked at three identical sites in an allele-specific manner. In contrast, the 5' end of 5.8S rRNA was terminated normally in gsp1 and in dis3. Inhibition of 5.8S rRNA maturation in gsp1 was rescued by overexpression of nuclear exosome components Dis3p, Rrp4p, and Mtr4p, but not by a cytoplasmic exosome component, Ski2p. Furthermore, gsp1 and dis3 accumulated the 5'-A0 fragment of 35S pre-rRNA, which is also degraded by the exosome, and the level of 27S rRNA was reduced. Neither 5.8S rRNA intermediates nor 5'-A0 fragments were observed in mutants defective in the nucleocytoplasmic transport, indicating that Gsp1p regulates rRNA processing through Dis3p, independent of nucleocytoplasmic transport.

https://www.genetics.org/content/genetics/158/2/613.full.pdf

The Saccharomyces cerevisiae Small GTPase, Gsp1p/Ran, Is Involved in 3′ Processing of 7S-to-5.8S rRNA and in Degradation of the Excised 5′-A0 Fragment of 35S Pre-rRNA, Both of Which Are Carried Out by the Exosome

Charged amino acid residues of human RCC1 were converted to alanine and mutants which were unable to complement tsBN2 cells (a temperature-sensitive rcc1- mutant of the hamster BHK21 cell line) were selected. These RCC1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC1 mutants were unstable in tsBN2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC1 repeat showed an increased K(m), while their kcat values were comparable to that of wild-type RCC1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC1 repeat showed a value of K(m) similar to that of wild-type RCC1, while the kcat values of these mutants were reduced, depending upon the RCC1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcat among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction.

Tomoyuki Ohba

Papers

A giant nucleopore protein that binds Ran/TC4.

Self-Organization of Microtubule Asters Induced in Xenopus Egg Extracts by GTP-Bound Ran

When Overexpressed, a Novel Centrosomal Protein, RanBPM, Causes Ectopic Microtubule Nucleation Similar to γ-Tubulin

The Saccharomyces cerevisiae Small GTPase, Gsp1p/Ran, Is Involved in 3′ Processing of 7S-to-5.8S rRNA and in Degradation of the Excised 5′-A0 Fragment of 35S Pre-rRNA, Both of Which Are Carried Out by the Exosome

Conserved Histidine Residues of RCC1 Are Essential for Nucleotide Exchange on Ran