Saccharomyces cerevisiae putative G protein, Gtr1p, which forms complexes with itself and a novel protein designated as Gtr2p, negatively regulates the Ran/Gsp1p G protein cycle through Gtr2p.

Prp20p and Rna1p are GDP/GTP exchanging and GTPase-activating factors of Gsp1p, respectively, and their mutations, prp20-1 and rna1-1, can both be suppressed by Saccharomyces cerevisiae gtr1-11. We found that gtr1-11 caused a single amino acid substitution in Gtr1p, forming S20L, which is a putative GDP-bound mutant protein, while Gtr1p has been reported to bind to GTP alone. Consistently, gtr1-S20N, another putative GDP-bound mutant, suppressed both prp20-1 and rna1-1. On the other hand, gtr1-Q65L, a putative GTP-bound mutant, was inhibitory to prp20-1 and rna1-1. Thus, the role that Gtr1p plays in vivo appears to depend upon the nucleotide bound to it. Our data suggested that the GTP-bound Gtr1p, but not the GDP-bound Gtr1p, interacts with itself through its C-terminal tail. S. cerevisiae possesses a novel gene, GTR2, which is homologous to GTR1. Gtr2p interacts with itself in the presence of Gtr1p. The disruption of GTR2 suppressed prp20-1 and abolished the inhibitory effect of gtr1-Q65L on prp20-1. This finding, taken together with the fact that Gtr1p-S20L is a putative, inactive GDP-bound mutant, implies that Gtr1p negatively regulates the Ran/Gsp1p GTPase cycle through Gtr2p.     

Cex1p facilitates Rna1p-mediated dissociation of the Los1p-tRNA-Gsp1p-GTP export complex

Nuclear tRNA export plays an essential role in key cellular processes such as regulation of protein synthesis, cell cycle progression, response to nutrient availability and DNA damage and development. Like other nuclear export processes, assembly of the nuclear tRNA export complex in the nucleus is dependent on Ran-GTP/Gsp1p-GTP, and dissociation of the export receptor-tRNA-Ran-GTP/Gsp1p-GTP complex in the cytoplasm requires RanBP1/Yrb1p and RanGAP/Rna1p to activate the GTPase activity of Ran-GTP/Gsp1p-GTP. The Saccharomyces cerevisiae Cex1p and Human Scyl1 have also been proposed to participate in unloading of the tRNA export receptors at the cytoplasmic face of the nuclear pore complex (NPC). Here, we provide evidence suggesting that Cex1p is required for activation of the GTPase activity of Gsp1p and dissociation of the receptor-tRNA-Gsp1p export complex in S. cerevisiae. The data suggest that Cex1p recruits Rna1p from the cytoplasm to the NPC and facilitates Rna1p activation of the GTPase activity of Gsp1p by enabling Rna1p to gain access to Gsp1p-GTP bound to the export receptor tRNA complex. It is possible that this tRNA unloading mechanism is conserved in evolutionarily diverse organisms and that other Gsp1p-GTP-dependent export processes use a pathway-specific component to recruit Rna1p to the NPC.     

Yrb2p, a Nup2p-related yeast protein, has a functional overlap with Rna1p, a yeast Ran-GTPase-activating protein.

The Ran-GTPase cycle is important for nucleus-cytosol exchange of macromolecules and other nuclear processes. We employed the two-hybrid method to identify proteins interacting with Ran and the Ran GTP/GDP exchange factor. Using PRP20, encoding the Ran GTP/GDP exchange factor, we identified YRB1, previously identified as a protein able to interact with human Ran GTP/GDP exchange factor RCC1 in the two-hybrid system. Using GSP1, encoding the yeast Ran, as bait, we isolated YRB2. YRB2 encodes a protein containing a Ran-binding motif similar to that found in Yrb1p and Nup2p. Yrb1p is located in the cytosol whereas Nup2p is nuclear. Similar to Yrb1p, Yrb2p bound to GTP-Gsp1p but not to GDP-Gsp1p and enhanced the GTPase-activating activity of Rna1p. However, unlike Yrb1p, Yrb2p did not inhibit the nucleotide-releasing activity of Prp20p. While overproduction of Yrb1p inhibited the growth of a mutant possessing a PRP20 mutation (srm1-1) and suppressed the rna1-1 mutation, overproduction of Yrb2p showed no effect on the growth of these mutants. Disruption of YRB2 made yeast cold sensitive and was synthetically lethal with rna1-1 but not with nup2delta. Nuclear protein import and the mRNA export were normal in strains possessing mutations of YRB2. We propose that Yrb2p is involved in the nuclear processes of the Ran-GTPase cycle which are not related to nucleus-cytosol exchange of macromolecules.     

Identification and characterization of a novel RanGTP-binding protein in the yeast Saccharomyces cerevisiae

The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1,2 ) and nuclear envelope formation (3,4 ) during mitosis in higher eukaryotes. We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p. The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins. In contrast to Yrb1p, Yrb30p does not coactivate but inhibits RanGAP1(Rna1p)-mediated GTP hydrolysis on Ran, like the karyopherins. At steady state, Yrb30p localizes exclusively to the cytoplasm, but the presence of a functional nuclear export signal and the localization of truncated forms of Yrb30p suggest that the protein shuttles between nucleus and cytoplasm and is exported via two alternative pathways, dependent on the nuclear export receptor Xpo1p/Crm1p and on RanGTP binding. Whereas overproduction of the full-length protein and complete deletion of the open reading frame reveal no obvious phenotype, overproduction of C-terminally truncated forms of the protein inhibits yeast vegetative growth. Based on these results and the exclusive conservation of the protein in the fungal kingdom, we hypothesize that Yrb30p represents a novel modulator of the Ran GTPase switch related to fungal lifestyle.     

Reciprocal conversion of Gtr1 and Gtr2 nucleotide-binding states by Npr2-Npr3 inactivates TORC1 and induces autophagy

Autophagy is an intracellular degradation process that delivers cytosolic material to lysosomes and vacuoles. To investigate the mechanisms that regulate autophagy, we performed a genome-wide screen using a yeast deletion-mutant collection, and found that Npr2 and Npr3 mutants were defective in autophagy. Their mammalian homologs, NPRL2 and NPRL3, were also involved in regulation of autophagy. Npr2-Npr3 function upstream of Gtr1-Gtr2, homologs of the mammalian RRAG GTPase complex, which is crucial for TORC1 regulation. Both npr2∆ mutants and a GTP-bound Gtr1 mutant suppressed autophagy and increased Tor1 vacuole localization. Furthermore, Gtr2 binds to the TORC1 subunit Kog1. A GDP-bound Gtr1 mutant induced autophagy even under nutrient-rich conditions, and this effect was dependent on the direct binding of Gtr2 to Kog1. These results revealed that 2 molecular mechanisms, Npr2-Npr3-dependent GTP hydrolysis of Gtr1 and direct binding of Gtr2 to Kog1, are involved in TORC1 inactivation and autophagic induction.     

Association of the GTP-binding protein Gtr1p with Rpc19p, a shared subunit of RNA polymerase I and III in yeast Saccharomyces cerevisiae

Yeast Gtr1p and its human homolog RRAG A belong to the Ras-like small G-protein superfamily and genetically interact with RCC1, a guanine nucleotide exchange factor for Ran GTPase. Little is known regarding the function of Gtr1p. We performed yeast two-hybrid screening using Gtr1p as the bait to find interacting proteins. Rpc19p, a shared subunit of RNA polymerases I and III, associated with Gtr1p. The association of Gtr1p with Rpc19p occurred in a GTP-form-specific manner. RRAG A associated with RPA16 (human Rpc19p homolog) in a GTP-form-specific manner, suggesting that the association is conserved during evolution. Ribosomal RNA and tRNA synthesis were reduced in the gtr1Delta strain expressing the GDP form of Gtr1p, but not the GTP form of Gtr1p. Gel-filtration studies revealed an accumulation of the smaller Rpc19p-containing complex, but not of A135, in the gtr1Delta strain. Here, we propose that Gtr1p is involved in RNA polymerase I and III assembly by its association with Rpc19p and could be a mediator that links growth regulatory signals with ribosome biogenesis.     

The nuclear export receptor Xpo1p forms distinct complexes with NES transport substrates and the yeast Ran binding protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin beta-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.     

Yrb1p interaction with the gsp1p C terminus blocks Mog1p stimulation of GTP release from Gsp1p

Mog1p, a multicopy suppressor of gsp1, the temperature-sensitive mutant of the Saccharomyces cerevisiae Ran homologue, binds to GTP-Gsp1p but not to GDP-Gsp1p. The function of Mog1p in the Ran cycle is as yet unknown. This study found that Mog1p releases a nucleotide from GTP-Gsp1p but not from GDP-Gsp1p. Yrb1p, the S. cerevisiae homologue of RanBP1, which is a strong inhibitor of RCC1-stimulated nucleotide release, also inhibited the Mog1p-stimulated nucleotide release from GTP-Gsp1p. At a concentration corresponding to the molar concentration of GTP-Gsp1p, Yrb1p completely inhibited the Mog1p-stimulated nucleotide release. Consistently, the Yrb1p.GTP-Gsp1p complex was more stable than the Mog1p.GTP-Gsp1p complex. Yrb1p did not inhibit the Mog1p-stimulated nucleotide release from GTP-Gsp1DeltaC. The Gsp1DeltaC protein lacks the final eight amino acids of the C terminus, and for this reason, the interaction between GTP-Gsp1DeltaC and Yrb1p was strongly reduced. On the other hand, Mog1p binds to GTP-Gsp1DeltaC more efficiently than to GTP-Gsp1p.     

Disruption of the YRB2 gene retards nuclear protein export, causing a profound mitotic delay, and can be rescued by overexpression of XPO1/CRM1

Disruption of the YRB2 gene encoding a nuclear Ran-binding protein homologous to Yrb1p/RanBP1 makes Saccharomyces cerevisiae cold sensitive for colony-formation, but not for growth in liquid medium. Schizosaccharomyces pombe Hba1p, which is homologous to Saccharomyces cerevisiae Yrb2p, rescued the cold sensitivity of Deltayrb2 cells. When released from an alpha factor block, Deltayrb2 cells underwent a prolonged delay at the short spindle stage of mitosis with a normal level of Clb/p34(CDC28) kinase activity, but there was no chromosome loss, this being consistent with the finding that Deltayrb2 was synthetic lethal with neither Deltamad1 nor Deltamad3. The cold sensitive colony-formation of Deltayrb2 cells was rescued by both XPO1/CRM1 and GSP1, but not CDC5, carried on a multicopy vector. XPO1/CRM1 rescued Deltayrb2 even in a single copy. Consistent with such a tight functional interaction, Xpo1p/Crm1p directly bound to Yrb2p, but not Yrb1p, and Deltayrb2 cells were found to have a defect in nuclear export signal (NES)-dependent nuclear protein export. From these results together, the ability of Xpo1/Crm1p to export NES-proteins is suggested to be enhanced by both Yrb2p and Gsp1p, and thereby disruption of YRB2 retards nuclear protein export, resulting in the mitotic delay.     

Yeast Ran-binding protein Yrb1p is required for efficient proteolysis of cell cycle regulatory proteins Pds1p and Sic1p

Ubiquitin-dependent proteolysis of specific target proteins is required for several important steps during the cell cycle. Degradation of such proteins is strictly cell cycle-regulated and triggered by two large ubiquitin ligases, termed anaphase-promoting complex (APC) and Skp1/Cullin/F-box complex (SCF). Here we show that yeast Ran-binding protein 1 (Yrb1p), a predominantly cytoplasmic protein implicated in nucleocytoplasmic transport, is required for cell cycle regulated protein degradation. Depletion of Yrb1p results in the accumulation of unbudded G(1) cells and of cells arrested in mitosis implying a function of Yrb1p in the G(1)/S transition and in the progression through mitosis. Temperature-sensitive yrb1-51 mutants are defective in APC-mediated degradation of the anaphase inhibitor protein Pds1p and in degradation of the cyclin-dependent kinase inhibitor Sic1p, a target of SCF. Thus, Yrb1p is crucial for efficient APC- and SCF-mediated proteolysis of important cell cycle regulatory proteins. We have identified the UBS1 gene as a multicopy suppressor of yrb1-51 mutants. Ubs1p is a nuclear protein, and its deletion is synthetic lethal with a yrb1-51 mutation. Interestingly, UBS1 was previously identified as a multicopy suppressor of cdc34-2 mutants, which are defective in SCF activity. We suggest that Ubs1p may represent a link between nucleocytoplasmic transport and ubiquitin ligase activity.     

Mutants in a yeast Ran binding protein are defective in nuclear transport.

Ran, a Ras-like GTPase, has been implicated in controlling the movement of proteins and RNAs in and out of the nucleus. We have constructed strains of Saccharomyces cerevisiae which produce fusion proteins containing glutathione-S-transferase (GST) fused to Gsp1p, which encodes the essential yeast Ran homolog, and a mutant form of Gsp1p that mimics the GTP-bound state. A major protein with the apparent size of 34 kDa co-purifies with the GTP-bound form of Gsp1p. This protein was identified as Yrb1p (Yeast Ran Binding Protein) and stimulates GTP hydrolysis by Gsp1p in the presence of Rna1p, the Gsp1 GTPase activating protein. Yrb1p is located in the cytoplasm with some concentration at the nuclear periphery. Temperature-sensitive yrb1 mutants are defective in nuclear protein import and RNA export. A mutation in the highly conserved Ran binding region of Yrb1p reduces its ability to interact with Gsp1p. These data indicate that Yrb1p functions with Gsp1p and suggest that together they can control transport of macromolecules across the nuclear envelope.     

Random mutagenesis and functional analysis of the Ran-binding protein, RanBP1

Ran GTPase is required for nucleocytoplasmic transport of many types of cargo. Several proteins that recognize Ran in its GTP-bound state (Ran x GTP) possess a conserved Ran-binding domain (RanBD). Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins). In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells. We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51(ts)) in vivo. In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs. However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1. These mutants bound to Ran x GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51(ts) yeast cells. Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1(ts) mutant and did not bind detectably to Ran in vitro. However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to co-activate RanGAP. A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1(ts) mutation. When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1(ts) cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin-beta and Ran-GTP. The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran x GTP x importin-5 complex. Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran x GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate RanGAP activity toward GTP-bound Ran in these complexes.     

A new role of ran GTPase.

Ran is a G protein similar to Ras, but it has no membrane binding site. RanGEF, RCC1, is on chromatin and RanGAP, RanGAP1/Rna1p is in cytoplasm. Ran, thus, shuttles between the nucleus and the cytoplasm to complete its GTPase cycle, carrying out nucleocytoplasmic transport of macromolecules. A majority of Ran binding proteins, thus far found, are required for this process. A recently found novel Ran-binding protein, RanBPM, however, is localized in the centrosome. Subsequently, four groups reported that RanGTP, but not RanGDP, can induce microtubule self-organization in Xenopus egg extracts where no nuclear membrane is present. Thus, Ran is suggested to have a new role beyond the nucleocytoplasmic transport of macromolecules. In both microtubule assembly and nucleocytoplasmic transport, chromosomal localization of RCC1 is important to carry out the functions of RanGTPase. In this regard, a future intriguing question is how RCC1 interacts with chromatin DNA.     

Isolation of an extragenic suppressor of the rna1-1 mutation in Saccharomyces cerevisiae

The small GTPase Ran is essential for nucleocytoplasmic transport of macromolecules. In the yeast Saccharomyces cerevisiae, Rna1p functions as a Ran-GTPase activating protein (RanGAP1). Strains carrying the rna1-1 mutation exhibit defects in nuclear transport and, as a consequence, accumulate precursor tRNAs. We have isolated two recessive suppressors of the rna1-1 mutation. Further characterization of one of the suppressor mutations, srn10-1, reveals that the mutation (i) can not bypass the need for Rna1p function and (ii) suppresses the accumulation of unspliced pre-tRNA caused by rna1-1. The SRN10 gene is not essential for cell viability and encodes an acidic protein (pI?=?5.27) of 24.8?kDa. Srn10p is located in the cytoplasm, as determined by indirect immunofluorescence microscopy. Two-hybrid analysis reveals that there is a physical interaction between Srn10p and Rna1p in vivo. Our results identify a protein that interacts with the yeast RanGAP1.     

The Dcr2p phosphatase destabilizes Sic1p in Saccharomyces cerevisiae

Initiation of cell division is controlled by an irreversible switch. In Saccharomyces cerevisiae degradation of the Sic1p protein, an inhibitor of mitotic cyclin/cyclin-dependent kinase complexes, takes place before initiation of DNA replication, at a point called START. Sic1p is phosphorylated by multiple kinases, which can differentially affect the stability of Sic1p. How phosphorylations that stabilize Sic1p are reversed is unknown. Here we show that the Dcr2p phosphatase functionally and physically interacts with Sic1p. Over-expression of Dcr2p destabilizes Sic1p and leads to phenotypes associated with destabilized Sic1p, such as genome instability. Our results identify a novel factor that affects the stability of Sic1p, possibly contributing to mechanisms that trigger initiation of cell division.